Recombinant Human Erythropoietin (rhEPO) in Clinical Oncology: Scientific and Clinical Aspects of Anemia in Cancer, 2nd Edition

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SpringerWienNewYork

Prof. Dr. med. M. R. Nowrousian Department of Internal Medicine (Cancer Research), West German Cancer Center, University Hospital of Essen, Essen, Germany

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. Product Liability: The publisher can give no guarantee for all the information contained in this book. This does also refer to information about drug dosage and application thereof. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

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ISBN 978-3-211-25223-9 SpringerWienNewYork ISBN 3-211-83661-6 1st edition SpringerWienNewYork

Preface Since the introduction of rhEPO in clinical oncology in 1993, considerable insight has been gained into the pathogenesis, prevalence and incidence of anemia in cancer and its impact on the life of cancer patients and the course of their disease. Anemia is not only a frequent complication of cancer and its treatment but also a major factor deteriorating patients’ physical well-being and quality of life (QOL). In addition, it may be involved in the development of tumor resistance against radiotherapy and chemotherapy. A number of studies indicates a close relationship between anemia and tumor hypoxia, and shows that the latter negatively and significantly determines the outcome of radiotherapy. Also, there is evidence suggesting that hypoxia stimulates angiogenesis and contributes to the selection of a more malignant phenotype of tumor cells with a reduced sensitivity to irradiation and chemotherapeutic agents. These findings and the impact of anemia on metabolic and organ function, as well as QOL, identify anemia as a much more serious problem for cancer patients than previously recognized. Over many decades, treatment of anemia in cancer patients has consisted of red blood cell (RBC) transfusions. However, these are of limited value in achieving a sustained and sufficient increase in Hb level. Furthermore, fluctuating Hb levels resulting from repeated RBC transfusions may produce intermittent hypoxia, which is considered an important factor for tumor progression. Acute, cyclic hypoxia has been shown to be even more deleterious than chronic hypoxia in selecting aggressive, apoptosis-resistant tumor cells and promoting metastasis. RBC transfusions are also associated with a number of side effects and risks, such as febrile and allergic reactions, alloimmunization, transmission of infection, iron overload and suppression of cellular immunity, which may be of particular concern in cancer patients. Furthermore, RBC transfusions, because of their risks and limitations as well as shortness of supply, are usually given at Hb levels below 8 g/dl. Numerous studies, including large prospective randomized and nonrandomized trials and meta-analyses of data from these trials have shown that patients with cancer are already seriously affected by the impact of anemia on metabolic and organ functions, exercise capacity and QOL long before such low levels of Hb are present. In addition, they have indicated that by using erythropoiesis-stimulating agents (ESAs), it is possible to achieve sustained physiological and much more effective Hb levels than with RBC transfusions. Furthermore, they have shown that such an increase in Hb level not only reduces or eliminates the need for RBC transfusions, but it is also associated

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Preface

with significant improvement in exercise capacity, physical well-being and QOL of patients. The majority of these studies was carried out in anemic patients receiving chemotherapy or chemoradiotherapy and the results have identified ESAs not only as significantly effective but also comparatively safe drugs. However, some studies have reported on a slight but significantly increased risk of thromboembolic events associated with the use of ESAs. This has to be considered in patients with a history of such events and patients who are receiving thrombogenic treatments. Some other studies have raised the question on the safety of ESAs with regard to the outcome of anticancer treatment. These studies, however, were performed in settings other than currently approved for the use of ESAs in cancer patients and the results have been critically reviewed because of a number of methodological problems, which have been present in these studies, e.g. high initial and/or target Hb levels used and imbalances in the distribution of patients and disease-related factors determining survival between patients receiving ESAs and control patients (Chapters 17–19, 31). Another issue of concern exclusively arising from in vitro studies has been a possible stimulatory effect of ESAs on tumor cells expressing EPO receptors (EPO-R). The results of these studies are in part controversial and have become a subject of critical reviews because of methodological problems associated with the determination of EPO-R and their functionality. In many of these studies, the stimulatory effects of ESAs on tumor cells were marginal and typically achieved with extremely high concentrations of ESAs, which do not occur in the treatment of cancer patients receiving ESAs. In some studies, in-vitro concentrations used were at several magnitudes higher than the peak concentrations observed in serum of patients treated with ESAs (Chapters 3, 4, 17, 18, Addendum 1). Furthermore, numerous animal experiments have failed to show tumor growth promotion by ESAs, even when the tumor cells were expressing EPO-R. In many of these studies, ESA treatment of anemic animals significantly improved the results of radiotherapy or chemotherapy. Nevertheless, further preclinical and clinical studies are required to better understand the benefits and risks of treatment with ESAs and to use these drugs with the highest possible benefit and safety for our patients. Like its first edition, the second edition of this book aims to be a comprehensive source of information on clinical and scientific aspects of anemia in cancer and its treatment with ESAs. All chapters were updated and some new chapters were added to achieve a greater spectrum of topics and to include future developments of ESAs in other fields of clinical medicine, such as neurology and cardiology. Under certain circumstances, e.g. in preventing adverse effects of radiotherapy and chemotherapy, neuroprotective and cardioprotective effects of ESAs may be of value for patients with malignant diseases. It was again a great pleasure and honor for me, and highly appreciated, that outstanding authors, all experts on their topics, agreed to contribute to

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this edition and to comprehensively present the state of knowledge in their fields. My sincere gratitude is also extended to Mrs. Ch. Wartchow for her help in proof-reading and Mrs. I. Demirer and Mrs. U. Senkel-Nicklaus for their excellent organizational assistance in preparing the book. Prof. Dr. M. R. Nowrousian

Contents

Contributors

xiii

1.

Physiology of erythropoiesis U. Testa

2.

Biology of EPO and EPO-receptor C. Lacombe, and P. Mayeux

67

3.

The role of erythropoietin receptor expression on tumor cells J. Fandrey

81

4.

Problems associated with erythropoietin receptor determination on tumor cells A. Österborg

103

Definition, classification and characterization of anemia in cancer M. R. Nowrousian

117

5.

6.

Pathophysiology of anemia in cancer M. R. Nowrousian

7.

Prevalence and incidence of anemia and risk factors for anemia in patients with cancer H. Ludwig

1

149

189

8.

Significance of anemia in cancer chemotherapy M. R. Nowrousian

207

9.

Incidence and impact of anemia in radiation oncology J. Dunst and M. Molls

249

10. Relationship between hemoglobin levels and tumor oxygenation P. Vaupel, A. Mayer and M. Höckel

265

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Contents

11. Tumor hypoxia and therapeutic resistance P. Vaupel and M. Höckel

283

12. Symptoms of anemia R. Pirker

307

13. Impact of anemia and red blood cell transfusion on organ function M. R. Nowrousian

317

14. Relationship of hemoglobin, fatigue, and quality of life in anemic cancer patients Z. Butt and D. Cella

369

15. When to use red blood cell transfusions in cancer patients with solid tumours? J. K. Jacob and P. J. Barrett-Lee

393

16. Pharmacology, pharmacokinetics and safety of recombinant human erythropoietin preparations W. Jelkmann

407

17. Epoetin treatment of anemia associated with multiple myeloma and non-Hodgkin’s lymphoma A. Österborg

433

18. rhEPO in anemic patients with solid tumors and chemotherapy – efficacy and safety M. R. Nowrousian

449

19. Early intervention with recombinant human erythropoietin for chemotherapy-induced anemia G. H. Lyman and J. Glaspy

509

20. Recombinant human erythropoietin (rhEPO) therapy in myelodysplasia E. Hellström-Lindberg

531

21. Prediction of response to rhEPO in the anemia of cancer Y. Beguin and G. Van Straelen

541

22. rhEPO in hematopoietic stem cell transplantation G. Van Straelen and Y. Beguin

583

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23. Treatment of anemia with rhEPO in radiation oncology J. Dunst

615

24. Recombinant human erythropoietin in pediatric oncology C. Hastings and J. Feusner

635

25. rhEPO in surgical oncology M. J. Fontaine and L. T. Goodnough

663

26. Erythropoiesis, iron metabolism and iron supplementation during erythropoietin therapy L. T. Goodnough 27. Are there risks for use of iron in cancer patients? P. Gascón 28. Metabolic and physiologic effects of rhEPO in anemic cancer patients K. Lundholm and P. Daneryd 29. Effects of rhEPO on quality of life in anemic cancer patients S. Chowdhury, J. F. Spicer, and P. G. Harper 30. Thrombosis during therapy with erythropoiesis stimulating agents in cancer J. Glaspy 31. The effect of rhEPO on survival in anemic cancer patients T. J. Littlewood 32. From bench to bedside: Neuroprotective effects of erythropoietin H. Ehrenreich and C. Bartels

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703

713

729

745

759

771

33. rhEPO in patients with anemia and congestive heart failure D. S. Silverberg, D. Wexler, A. Iaina, S. Steinbruch, Y. Wollman, and D. Schwartz

793

34. Cost-effectiveness of treating cancer anaemia P. Cornes

813

Addendum

851

Index

853

Contributors Barrett-Lee Peter J., Dr. Velindre Cancer Centre, Velindre NHS Trust, Whitchurch, Cardiff, Wales CF14 2TL, UK E-mail: [email protected] Bartels Claudia, Dipl.-Psych. Division of Clinical Neuroscience, Max-Planck-Institute of Experimental Medicine, Hermann-Rein Strasse 3, 37075 Göttingen, Germany E-mail: [email protected] Beguin Yves, MD, Prof. University of Liège, Department of Hematology, CHU Sart-Tilman, 4000 Liège, Belgium E-mail:[email protected] Tel. +32-4-366-7690 Fax +32-4-366-8855 Butt Zeeshan, PhD Center on Outcomes, Research and Education, Evanston Northwestern Healthcare, 1000 University Place, Suite 100, Evanston, Illinois 60201, USA E-mail: [email protected] Cella David, Dr. Center on Outcomes, Research and Education, Evanston Northwestern Healthcare, 1000 University Place, Suite 100, Evanston, Illinois 60201, USA E-mail: [email protected] Tel. +1-847-570-7370 +1-847-570-8033 Chowdhury Simon, MA, MRCP, PhD Medical Oncology, 3rd Floor Thomas Guy House, Guy’s Hospital, St Thomas Street, London SE1 9RT, UK E-mail: [email protected] Tel. +44-207-955-2933 Fax +44-207-955-4939

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Contributors

Cornes Paul, Dr. Bristol Haematology & Oncology Centre, 9 Royal Victoria Park, Bristol, BS2 8ED, United Kingdom E-mail: [email protected] Tel. +44-117-928-3008 Fax +44-117-928-4409 Daneryd Peter, MD, PhD, Prof Department of Surgery, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden Dunst Jürgen, Prof. Dr. Department of Radiation Oncology, University Clinic Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany E-mail: [email protected] Tel. +49-451-500-6660 Fax +49-451-500-33324 Ehrenreich Hannelore, MD, DVM, Prof. Dr. Head, Division of Clinical Neuroscience, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany E-mail: [email protected] Tel. +49-551-3899628 Fax +49-551-3899670 Fandrey Joachim, Prof. Dr. med. Institut für Physiologie, Universitätsklinikum Essen, Universität DuisburgEssen, Hufelandstrasse 55, 45147 Essen, Germany E-mail: [email protected] Tel. +49-201-723-4600 Fax +49-201-723-4648 Feusner James, MD, Prof. Director, Pediatric Oncology, Department of Hematology and Oncology, Children’s Hospital and Research Center of Oakland, 747 52nd Street, Oakland, CA 94609, USA E-mail: [email protected] Tel. +1-510-428-3689 Fax +1-510-601-3916 Fontaine Magali J., MD, PhD Assistant Professor of Pathology, Associate Director of Transfusion Services, 300 Pasteur Drive L235, Stanford CA 94305, USA E-mail: [email protected] Tel. +1-650-450-1459 Fax +1-650-723-9178

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xv

Gascón Pere, Prof. Dr. Division of Medical Oncology, Hematology-Oncology Department (ICMHO), Hospital Clínic, Villarroel 170, escalera 2, planta 5, Barcelona, Spain E-mail: [email protected] Tel. 34-93-2275402 Fax 34-93-4546520 Glaspy John, MD, Prof. Professor of Medicine, Division of Hematology-Oncology, UCLA School of Medicine, Los Angeles, California 90095, USA E-mail: [email protected] Goodnough Lawrence T., Prof. Dr. Washington University School of Medicine, Transfusion Services, Barnes – Jewish Hospital, 660 South Euclid Avenue, Box 8118, St. Louis, MO 63110, USA E-mail: [email protected] Tel. +1-314-362-3186 +1-314-362-1461 Harper, Peter G., Dr. Medical Oncology, 3rd Floor Thomas Guy House, Guy’s Hospital, St Thomas Street, London SE1 9RT, UK E-mail: [email protected] Tel. +44-207-955-2933 Fax +44-207-955-4939 Hastings Caroline, MD Department of Pediatric Hematology and Oncology, Children’s Hospital and Research Center of Oakland, 747 52nd Street, Oakland, CA 94609, USA E-mail: [email protected] Tel. +1-510-428-3631 Fax +1-510-601-3916 Hellström-Lindberg Eva, MD, PhD Karolinska Institutet, Department of Medicine, Division of Hematology, Karolinska University Hospital, Huddinge, Ihn 86, 141 86 Stockholm, Sweden E-mail: [email protected] Höckel Michael, Prof. Dr. Department of Obstetrics and Gynecology, University of Leipzig, PhilippRosenthal-Strasse 55, 04103 Leipzig, Germany E-mail: [email protected] Tel. +49-341-9723-400 Fax +49-341-9723-419

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Contributors

Iaina Adrian, MD Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel Jacob Jayin K., Dr. Velindre Cancer Centre, Velindre NHS Trust, Whitchurch, Cardiff, Wales CF14 2TL, UK E-mail: [email protected] Jelkmann Wolfgang, Prof. Dr. Director, Professor of Physiology, Institute of Physiology, Medical University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany E-mail: [email protected] Tel. +49-451-500-4152 Fax +49-451-500-4151 Lacombe Catherine, MD, PhD, Prof. Unité 363, Université René Descartes Hematology Department, Hôpital Cochin, 27 rue du Faubourg St. Jacques, 75014 Paris, France E-mail: [email protected] Tex. +33-1-4633-1409 Fax +33-1-4633-9297 Littlewood Tim, Dr. Department of Haematology, John Radcliffe Hospital, Oxford OX3 9DU, UK E-mail: [email protected] Tel. +44-1865-220364 and +44-1865-741166 Fax +44-1865-221778 Ludwig Heinz, Prof. Dr. Department of Medicine I, Center for Oncology and Hematology, Montleartstrasse 37, 1171 Vienna, Austria E-mail: [email protected] Tel. +43-1-49150-2101 Fax +43-1-49150-2109 Lundholm Kent, MD, PhD, Prof. Department of Surgery, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden E-mail: [email protected] Tel. +46-31-342-2239 Fax +46-31-413-892

Contributors

xvii

Lyman Gary H., MD, MPH, FRCP (Edin) Duke University School of Medicine, Duke Comprehensive Cancer Center, Durham, North Carolina, USA E-mail: [email protected] Tel. +1-585-275-3335 Fax +1-585-276-1885 Mayer Arnulf, Dr. Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, 55099 Mainz, Germany E-mail: [email protected] Tel. +49-6131-392-5203 Fax +49-6131-392-5774 Mayeux P., Dr. Université Paris-Descartes, Faculté de Médecine, Unité 363, Service d’Hématologie, INSERM, CNRS, Hôspital Cochin (U567-UMR 8104), Paris, France E-mail: [email protected] Tel. +33-1-0140516514 Fax +33-1-0140516510 Molls M., Univ.-Prof. Dr. med. Department of Radiation Oncology, Technical Ismaninger Straße 22, 81675 München, Germany E-mail: [email protected] Tel. +49-89-414-04502 Fax +49-89-414-04477

University

Munich,

Nowrousian M. R., Prof. Dr. Department of Internal Medicine (Cancer Research), West German Cancer Center, University Hospital of Essen, Hufelandstrasse 55, 45122 Essen, Germany E-mail: [email protected] Tel. +49-201-723-3127 Fax +49-201-723-5984 Österborg Anders, Dr. Department of Oncology (Radiumhemmet), Karolinska University Hospital, 171 76 Stockholm, Sweden E-mail: [email protected] Tel. +46-85-177-3385 Pirker Robert, MD, Prof. Department of Internal Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria E-mail: [email protected] Tel. +43-1-40400-4422 Fax +43-1-40400-4461

xviii

Contributors

Schwartz Doron Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel Silverberg Donald S., MD Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel E-mail: [email protected] Tel. Office 972-3-6973270 Tel. Home 972-9-8666013 Tel. Cellular 972-0522-555412 Fax: 972-9-8665715 Spicer James F., MA, MRCP, PhD Medical Oncology, 3rd Floor Thomas Guy House, Guy’s Hospital, St Thomas Street, London SE1 9RT, UK Tel. +44-20-7188-7188 Steinbruch S. Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel Testa Ugo, Dr. Department of Hematology and Oncology, Istituto Superiore di Sanitá, Viale Regina Elena 299, 00161 Roma, Italy E-mail: [email protected] Van Straelen Gaetan University of Liège, Department of Hematology, CHU Sart-Tilman, 4000 Liège, Belgium Vaupel Peter, Prof. Dr. Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, 55099 Mainz, Germany E-mail: [email protected] Tel. +49-6131-392-5929 Fax +49-6131-392-5774 Wexler Dov, MD Department of Cardiology and Heart Failure Clinic, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel E-mail: [email protected] Wollman Y. Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv 64239, Israel

Chapter 1

Physiology of erythropoiesis U. Testa Department of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy

Ontogenesis of erythropoiesis The hematopoietic system in vertebrates requires the presence of cells that ensure a continuous production of new cells needed to replace mature blood elements endowed with only a limited life span. This continual blood cell production is ensured by pluripotent stem cells which have the unique property of both self-renewal and differentiation through progressive commitment to multipotent progenitors, then to committed progenitors and, finally, to progressively maturing precursors of the different hemopoietic lineages. Commitment of stem cells to the specific cell lineages appears not to be regulated by exogenous growth factors. Rather, stem cells develop into differentiated cell types through incompletely defined molecular events that are intrinsic to the stem cell itself. Following lineage commitment, hematopoietic progenitors and precursors come increasingly under the regulatory influence of growth factors and hormones. The hematopoietic cells and, particularly, the primitive embryonic erythroid cells originate from the ventral mesoderm through a differentiation process under the control of bone morphogenetic proteins (BMP). Particularly, the effects of BMP seem to be related to the induction of the expression of hematopoietic transcription factors, such as GATA-1, GATA-2, Tal-1, LMO2 and EKLF in mesodermic progenitors (Adelman et al. 2002; Schmerer and Evans 2003). The differentiation of embryonic stem cells to primitive erythroid elements is promoted by vascular endothelial growth factor (VEGF) (Cerdan et al. 2004). In the mouse (and also in humans) during embryonic/fetal development three distinct stages of erythropoiesis have been defined. The first stage corresponds to primitive embryonic erythropoiesis and occurs at the level of blood islands developing within the yolk sac around day 7 of murine embryonic life; the distinctive feature of erythropoiesis at this stage corresponds to the erythropoietin (EPO)-independency (Wu et al. 1995). The initial erythroid differentiation from pluripotent embryonic stem cells requires a mesoderm patterning factor, such as bone morphogenetic factor 4 and an

2

U. Testa

angiogenic factor, like VEGF. Primitive erythroblasts differentiate within the bloodstream, remain predominantly nucleated at the end of their maturation and have a very large size (accordingly, they are called “megaloblasts”). The second stage of erythropoietic development corresponding to definitive erythropoiesis, occurs around day 10 of gestation and consists in a rapid and marked proliferation of erythroid elements in the fetal liver; the distinctive feature of this erythropoietic stage consists in the development of EPO, EPO receptor (EPO-R) and JAK2 dependency (Neubauer et al. 1998; Parganas et al. 1998). The third stage of erythropoietic development is characterized by the migration of hematopoietic stem cells at the level of bone marrow, where adult hemopoiesis and, particularly, adult erythropoiesis develops. A distinctive feature of adult erythropoiesis consists in its negative regulation mediated by SHP-1 phosphatase (Klingmuller et al. 1995). The human hematopoiesis begins in the second-to-third embryonic weeks with formation of mesoderm-derived blood islands in the extraembryonic mesoderm of the developing secondary yolk sac. Blood islands develop foci of nucleated erythroblasts (known for their large size as “megaloblasts”) intimately associated with and surrounded by endothelium. Yolk sac blood cells consist of nucleated primitive erythrocytes synthesizing exclusively embryonic hemoglobins (like ζ2ε2). After the onset of circulation occurring around the day 21 of development, yolk sac cells are found in embryonic blood. The fetal liver subsequently replaces the yolk sac as the main hematopoietic tissue with the appearance of definitive enucleate, macrocytic erythrocytes synthesizing fetal hemoglobin (α2γ2). Recent in vitro studies have provided evidence that hematopoiesis and particularly primitive erythropoiesis originates from embryonic bodies-derived endothelial progenitors endowed with both endothelial and hemopoietic differentiation capacities (hemangioblasts) (Wang V et al. 2004; Zambidis et al. 2005). Erythropoiesis involves the progressive differentiation starting from hemopoietic stem cells to mature erythrocytes. The differentiative steps involved in this complex differentiation process are numerous and involve first the differentiation to multipotent hemopoietic progenitors generating mixed colonies in vitro (CFU-Mix), then to committed erythroid progenitors subdivided in early erythroid progenitors (burst forming unit-erythroid, BFU-E) generating in vitro large erythroid colonies and late erythroid progenitors (colony forming unit-erythroid, CFU-E), generating small erythroid colonies and, finally, to morphologically recognizable erythroid precursors. During this differentiation process the cells become progressively sensitive to EPO due to the appearance on these cells of the EPO-Rs. The terminal stages of bone marrow erythropoiesis occur in peculiar cellular associations named erythroblastic islands consisting of a centrally located macrophage surrounded by maturing erythroid precursors. The erythroblastic islands are considered as morpho-functional units of erythropoiesis where differentiation of CFU-E takes place (Bernard 1991). The formation of erythroblastic islands occurs in a region away from the

Physiology of erythropoiesis

3

sinusoidal endothelium and the erythroblastic islands migrate toward the sinusoids as erythroid maturation proceeds (Yokoyama et al. 2003). It was suggested that macrophages/stromal cells present in the erythroblastic islands provide an essential microenvironment for maturing erythroblasts, releasing various cytokines, and maintaining intimate contacts with erythroblasts until enucleation and release of reticulocytes. The adhesion of erythroblasts to the extracellular matrix and to the central macrophage has been shown to be important for erythroid cell differentiation and involves several adhesion and extracellular matrix molecules, such as the erythroblast membrane receptor, integrin α4β1 and its ligand VCAM-1, integrin-associated protein, fibrinonectin and erythroid-specific adhesion receptor membrane protein. In this context, particularly relevant seems to be the role of the erythroid-specific intercellular adhesion molecule-4 (ICAM-4). As a binding partner of both α4β1 and αV integrins, ICAM-4 could play a multifunctional role within the erythroblastic islands: ICAM-4 – α4β1 association may mediate adhesion between adjacent erythroblasts, while ICAM-4 – αV interaction may affect binding of erythroblasts to the central macrophage (Lee et al. 2003). The compartment of bone marrow erythroid precursors is well characterized and involves a proliferative-maturative compartment, involving the maturation from proerythroblasts to polychromatophilic erythroblasts through the basophilic stage, and a maturative non-proliferative compartment involving the maturation from polychromatophilic erythroblasts to mature red blood cells through the reticulocyte stage. Most red blood cells (RBC) are released into circulation as reticulocytes and mature further over the next days to become erythrocytes. During this maturation process they lose their mitochondria and ribosomes. Consequently, they lose the ability to synthesize hemoglobin and to carry out oxidative metabolism. The end cells, erythrocytes, are uniform biconcave disks lacking a nucleus and organelles and composed in large part by a highly specialized protein, hemoglobin. The mature RBC rely on glucose and the glycolytic pathway for their metabolic needs, including the production of large amounts of 2,3-diphosphoglycerate (2,3-DPG) that reduced the affinity of the hemoglobin for oxygen, thereby facilitating the release of oxygen at the tissues. Mature RBC have a relatively long life span (about 120 days), but a continuous bone marrow erythropoiesis is required to replace the constant destruction of senescent RBC. The bone marrow erythropoiesis is a highly efficient system that tunes the rate of erythropoietic production to physiologic needs. In this system the physiologic stimulus is represented by hypoxia that represents the signal inducing the production of EPO, the main cytokine involved in the control of erythroid production. In spite of the efficiency of the erythropoietic system, a small, but significant level of inefficient erythropoiesis due to premature intramedullary death of erythroid cells occurs. Recent studies indicate that this inefficient erythropoiesis is due to apoptotic death of erythroblasts. The main biochemical and molecular events occurring during the process of erythroid differentiation and maturation are briefly outlined in Fig. 1.

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Physiology of erythropoiesis

5

Fig. 1. Outline of the process of erythroid differentiation. In the top panel the main steps of erythroid cell differentiation are schematically represented. Initially, through a series of complex differentiation events hemopoietic stem cells (HSC) differentiate to early erythroid progenitors, called burst forming units-erythroid (BFU-E). BFU-Es generate in vitro within two weeks of culture large colonies composed by mature erythroid elements. BFU-Es in turn differentiate into late erythroid progenitors, colony forming unit-erythroid (CFU-Es), able to generate in vitro small erythroid colonies after one week of culture. CFU-Es undergo mitotic divisions to differentiate into mature erythroid cells through several maturation steps represented by proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and othochromatic erythroblasts. Finally, othrochromatic erythroblasts undergo terminal maturation by extruding their nuclei and progressively loosing their organelles, thus generating reticulocytes and then erythrocytes. In the other four panels the kinetics of expression of several molecules during erythroid differentiation is shown. These molecules are divided, according to their function, in three main groups represented by early differentiation antigens, transcription factors, growth factor receptors and proteins important for erythroid cell structure and/or function

䉳

BFU-E progenitors take about 14 days to differentiate in vitro forming large colonies; CFU-E progenitors take about 7 days to form colonies in vitro. CFU-E progenitors undergo 3–5 mitotic divisions to differentiate to terminal erythroid elements through different maturation steps: proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and orthochromatic erythroblasts. Finally, these cells undergo terminal maturation by extruding their nuclei and progressively losing all organelles: as a consequence of these processes young erythrocytes, called reticulocytes, are formed. As erythroid precursors progress in their maturation, they undergo a progressive decrease in their size, increase in hemoglobin concentration and chromatin density. In vitro studies have shown that the erythroid differentiation process from BFU-E to mature RBC takes place in about 2 weeks.

Basic mechanisms in the control of erythropoiesis In mammals, oxygen (O2) is transported to tissues bound to the hemoglobin contained within erythrocytes. The mature RBC is an anucleated cell, discoid in shape, 8 μM in diameter and extremely pliable and therefore capable to traverse the microcirculation. Normal RBC production results in the daily replacement of 0.8 to 1% of all circulating RBC in the body. The rate of RBC production is tuned to the physiologic requirement for these cells. The ensemble of the machinery responsible for RBC production is called the erythron. The erythron is composed by a pool of proliferating elements represented by bone marrow erythroid precursors and by a large mass of

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nonproliferating cellular elements represented by mature RBC. The size of RBC mass represents the balance of RBC production and destruction. The rate of RBC production is directly related to the mass of bone marrow erythroid precursors. The rate of RBC production is regulated through a feedback mechanism, where the triggering mechanism is represented by hypoxia. When the RBC volume decreases below normal, the erythropoietic activity of bone marrow increases, and conversely, when the RBC volume increases above normal, the erythropoietic activity of the bone marrow decreases. These adjustments are brought about by changes in the circulating level of EPO, a circulating cytokine that contains 165 amino acid residues and four oligosaccharide chains, required for its activity in vivo. The existence of this cytokine was postulated since 1906 by Carnot and DeFlandre who, based on their experiments of induction of reticulocytosis by injection of plasma derived from a bled animal to a normal recipient rabbit, hypothesized the existence of a humoral factor, “hemopoietine”, regulating RBC production (Carnot and DeFlandre 1906). The biological activity of EPO was subsequently characterized and in 1977 human EPO was purified at homogeneity and its structure determined in detail (Miyake et al. 1985). The availability of the primary sequence of the protein allowed the cloning of the gene for EPO and the development of a transfected cell line in Chinese hamster ovary cells that provided recombinant EPO for therapeutic purposes (Jacobs et al. 1985; Lin et al. 1985). EPO is a member of the family of class I cytokines which fold into a compact globular structure consisting of 4 α-helical bundles. Its molecular mass is 30.4 kDa, although it migrates with an apparent molecular weight of 34–38 kDa on SDS-polyacrylamide gels. The peptide core of the protein is formed by 165 amino acids and is sufficient for receptor binding and for the in vitro capacity to stimulate erythropoiesis, while the carbohydrate moiety (about 40% of the total molecule) is required for the in vivo survival of the cytokine. The function of the four carbohydrate chains of EPO was studied in detail: the 3 complex-type N-linked oligosaccharides at asparagines 24, 38 and 83 play a key role in the stability of the protein in circulation, while the small O-linked oligosaccharide at serine 126 seems to be devoid of any functional role. The development of specific and sensitive immunologic methods to measure EPO levels in biologic fluids clearly showed that the plasmatic levels of this cytokine are inversely related to the RBC mass (i.e. hematocrit) (Erslev 1991). The normal plasmatic level of EPO ranges from 10 to 25 mU/mL. When hemoglobin levels fall below 100 to 120 g/L plasma EPO levels increase in proportion to the severity of the anemia (Fig. 2). In adults the large majority (about 85%) of the EPO is synthesized at the level of the kidneys and a minority (about 15%) in the liver. EPO can be extracted also from the salivary glands and the spleen; however, since both

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Fig. 2. Feedback mechanism regulating the rate of EPO production. This feedback mechanism is based on the levels of circulating hemoglobin which determine the blood oxygen tension. The level of blood oxygen is sensed by the kidney through a molecular oxygen sensing mechanism. A decrease in blood oxygen level determines an increased rate of kidney EPO production. EPO, in turn, stimulates bone marrow erythropoiesis, with a consequent increased production of red blood cells

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these tissues do not possess the mRNA encoding for EPO and therefore they do not appear to synthesize this cytokine. During fetal and neonatal life the main site of erythropoiesis and EPO production is the liver. When erythropoiesis, during the fetal development, is taken on by the bone marrow the major site of EPO production is switched at the level of kidneys. When renal mass is reduced in adults by chronic renal diseases or nephrectomy, the liver cannot compensate for EPO production and anemia develops. In adults, EPO is produced by interstitial cells in the peritubular capillary bed of the kidneys (Koury et al. 1988; Fisher et al. 1996) and by perivenous hepatocytes in the liver (Zanjani et al. 1977; Koury et al. 1991). EPO is also produced in the brain where it exerts a cytoprotective effect against oxidative damage caused by hypoxic conditions. Finally, in the uterus and oviducts EPO production is induced by estrogens and contributes together with other growth factors to the development of estrogen-induced angiogenesis. The fundamental stimulus for EPO production is the availability of O2 for metabolic needs. Impaired O2 delivery to the kidney can result from a decreased RBC mass (anemia), impaired O2 loading of the hemoglobin molecule (hypoxemia) or, rarely, impaired blood flow to the kidney (renal artery stenosis). The molecular mechanism involved in the control of EPO production has been in part elucidated. The main stimulus for EPO production is hypoxia. The regulation of EPO expression by hypoxia is a complex phenomenon that implies several molecular steps (Fig. 3): in a first step hypoxia factor 1α (HIF-1α) is induced and stabilized; in a second step HIF-1α translocates from the cytoplasm to the nucleus, where it dimerizes with the arylhydrocarbon receptor nuclear translocator (HIF-1β); in a third step the HIF-1α/HIF-1β complex binds the hypoxia-responsive enhancer located in the 3′ region of the EPO gene (Michiels et al. 2002). This enhancer element is a 50-bp hypoxia-inducible enhancer that is located approximately 120 bp 3′ to the polyadenylation site (Huang and Bunn 2003). This enhancer contains a nucleotide sequence required for the binding of HIF. Many studies have been devoted to define the molecular mechanisms involved in HIF-1α activation/stabilization. On the basis of the experimental evidence so far accumulated, two models of HIF-1α activation have been proposed. The first model is based on the observation that under normoxic conditions, HIF-1α protein is hydroxylated at its prolyl residues located in the oxygen-dependent domain by a family of prolyl hydroxylase enzymes (Ivan et al. 2001; Jaakkola et al. 2001). Hydroxylation of HIF-1α initiates binding of the Von Hippel-Lindau (VHL) protein that acts as an E3 ubiquitin ligase that then promotes the degradation of HIF-1α by proteosomes. The activity of these prolyl hydroxylases requires oxygen, ascorbic acid and iron. Therefore, prolyl hydroxylases are inhibited by hypoxia, cobaltous ions and iron chelators, explaining why HIF-1α is activated by hypoxia, transition metals, and by iron chelation (Epstein et al. 2001).

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Fig. 3. Mechanism of oxygen sensing through the hydroxylation of the transcription factor HIF-1α. HIF-1α protein domain structures are shown schematically: basic helix-loop-helix (bHLH), Per/Arnt/Sim (PAS), N-terminal transactivation (NAD) and C-terminal transactivation (CAD). The NAD domain is also called the oxygen dependent degradation domain (ODD) and represents the site of binding of pVHL. Under low oxygen conditions HIF activates transcription acting in combination with coactivators p300 and CBP. At these oxygen concentrations, von Hippel-Lindau tumor suppressor protein (pVHL) cannot bind to HIF. Under normal or high oxygen conditions, and Fe2+ and 2-oxoglutarate, HIF-1α is hydroxylated by FIH (Factor Inhibiting HIF) at the level of aminoacid residue Asn 803, thus preventing coactivator recruitment at the level of CAD and reducing gene activation. At high oxygen concentrations, the NAD is hydroxylated at the level of two prolyl sites: this event determines the binding of pVHL to HIF with its subsequent proteolytic degradation

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VHL recognizes HIF-α subunits through two independent sites that contain the common motif LXXLAP: the hydroxylation of specific proline residues into this conserved box is required for the interaction of VHL with HIF. In addition to prolyl hydroxylases, also an asparaginyl hydroxylase seems to play a key role in the mechanism of control of HIF activity, via hydroxylation of a saparaginyl residue present in the C-terminal transactivation domain. Factor-inhibiting HIF (FIH) has been identified as the asparaginyl hydroxylase involved in this hydroxylation (Lando et al. 2002). Asparagine hydroxylation prevents the interaction of HIF-1α with the coactivators CREB-binding protein (CBP) and p300. These co-activators of the transcription, CBP and p300, are recruited by the activated HIF-1α protein and are transported by HIF at the level of the EPO hypoxia enhanxcer element, cooperating in the stimulation of the expression of this gene. The second model is based on the observation that hypoxia leads to a decreased production of reactive oxygen species, due to inhibition of nicotinamide adenine nucleotide phosphate oxidase activity. The fast diffusing reactive oxygen species oxidize and destabilize HIF-1α protein resulting in a decreased expression of hypoxia-inducible genes, including EPO. The decrease in superoxide production after hypoxia leads to HIF-1α stabilization and activation (Ehleben et al. 1997). In line with this model, extracellular superoxide dismutase acts as a repressor of hypoxia-induced EPO gene expression, thus implicating superoxide as a signaling intermediate in HIF-1α activation (Zelko and Folz 2005). There are three HIFα family members (HIF-1α, HIF-2α, HIF-3α) and three β family members (HIF-1β/ARNT1, HIF-2β/ARNT2, HIF3β/ARNT3). The structure of a HIFα protein implies the presence of four domains: basic helix-loop-helix (bHLH), Per/ARNT/Sim(PAS), N-terminal transactivation (NTAD) and C-terminal transactivation (CTAD). The NTAD and CTAD domains are involved in activation of transcription when bound to DNA in complex with a β subunit. Over 100 HIF target genes have been identified and, typically, they contain a canonical HIF-binding site 5′-RCGTG-3′. These target genes include genes that regulate erythropoiesis, angiogenesis, apoptosis and glucose uptake and metabolism. HIFβ proteins are not affected by changes in oxygen level. In contrast, HIFα proteins are highly unstable in the presence of high oxygen level owing to polyubiquitinylation and proteosomal destruction. Under low oxygen levels, HIFα subunits stabilize, translocate to the nucleus and activate transcription acting in concert with a β family member. In addition to HIF members, another protein, the von Hippel-Lindau tumor suppressor protein (pVHL), plays an essential role in oxygendependent regulation of EPO. The mutations of the gene encoding pVHL are responsible for a hereditary cancer syndrome: tumor cells lacking pVHL do not degrade HIFα subunits in the presence of O2 and then they overexpress a series of HIF-responsive genes, including VEGF and EPO (Gnarra et al. 1996). Interestingly, in two types of tumors (hemangioblastoma and

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renal cell carcinoma), associated with Von Hippel-Lindau disease, the coexpression of EPO and EPO-R was observed (Vortmeyer et al. 2003; Lee et al. 2005). pVHL forms a molecular complex with elongins, cullin and Ring-box1: this complex possesses ubiquitin ligase activity and polyubiquitinylates HIFα subunits in the presence of oxygen. pVHL binds to the NTAD region (also called the oxygen dependent degradation domain, ODD) of HIFα, when this region, at high O2 level, is hydroxylated in correspondence of proline residues 402 and 564. Germline mutations of the pVHL gene are associated in some rare patients to congenital polycythemias (Gordeuk et al. 2005). Targeted disruption of HIF members provided important information on their biologic role in hematopoiesis. Embryos lacking HIF-1β have defects in yolk sac vascularization and a global defect in hematopoiesis: the hematopoietic defect seems to be dependent upon absent VEGF production and cannot be corrected by exogenous EPO (Adelman et al. 1999). Mice lacking completely the HIF-1α expression die due to multiorgan malformations and developmental arrest. Examination of HIF-1α−/− mice showed the incapacity to stimulate EPO production following a chronic hypoxic stimulus, thus suggesting a role for HIF-1α in EPO production. Analysis of the in vitro differentiation of embryonic stem cells HIF-1α−/− showed a defective erythropoiesis. Finally, mice lacking HIF-2α exhibit pancytopenia. The defective hematopoietic differentiation is related to an altered bone marrow microenvironment. This defect in hematopoiesis, and particularly in erythropoiesis, cannot be rescued by addition of VEGF, but is related to a defective EPO production (Scortegnagna et al. 2005). In addition to its key role in the control of EPO production, HIF-1 plays an essential role also in the modulation of iron metabolism. In fact, among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, erythroid 5-aminolevulinate synthase and ceruloplasmin which coordinately regulate iron metabolism (Lee PJ et al. 1997; Rolfs et al. 1997; Lok and Ponka 1999; Tacchini et al. 1999; Mukhopadhyay et al. 2000). Increased iron uptake, release of iron from the liver, plasma transport and uptake in the bone marrow are essential to sustain the erythropoietic function of EPO. Iron deficiency is known to induce EPO gene expression and HIF-1α protein stabilization (Wang and Semenza 1993), a phenomenon seemingly related to inactivation of the iron-dependent protein hydroxylases PHD1 to 3 and FIH. On the other hand, copper stabilizes nuclear HIF-1α under normoxic conditions, through a mechanism independent on the iron concentration (Martin et al. 2005). According to these observations it was proposed that HIFdependent gene regulation plays a key role in the regulatory network for oxygen, iron and copper metabolism, regulating the oxygen-, iron- and copperbinding transport proteins hemoglobin, transferrin and ceruloplasmin. In addition to HIF-1α other transcription factors play an important role in the modulation of EPO gene expression. Thus, the transcription factor GATA-2 lowers EPO gene transcription by binding to the EPO gene promoter under normoxic conditions (Imagawa et al. 1994; Imagawa et al. 1997).

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The GATA-2 mediated inhibition of EPO gene expression could be responsible for the reduced EPO synthesis observed in patients with chronic renal failure: in fact the NO synthase inhibitor, NG-monomethyl-L-arginine (LNMMA), one of the candidate molecules responsible for the suppression of EPO synthesis observed in patients with chronic renal failure, lowers EPO synthesis by stimulating GATA-2 DNA binding (Tarumoto et al. 2000). The EPO gene 5′ flanking sequences contain also binding sites for the kB nuclear factor (NFkB). Both GATA-2 and NFkB are involved in the inhibition of EPO gene expression observed in inflammatory conditions. The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) activate both GATA-2 and NFkB and, through this mechanism, inhibit EPO synthesis (La Ferla et al. 2002). Interestingly, the GATA-specific inhibitor K-7174 restores EPO production in IL-1, TNF-α or L-NMMA treated human hepatoma cell cultures and experimental mice (Imagawa et al. 2003). The oral administration of the GATA inhibitors K-7174 and K-11706 were efficacious in restoring normal EPO production in animal models of anemia related to inflammatory diseases (Nakano et al. 2004). Interestingly, another GATA transcription factor, GATA-4, is responsible for the high EPO expression in fetal liver. In fact, the expression of this transcription factor is high in fetal liver, but low in adult liver (Dame et al. 2004). It is of interest to note that erythroid cells grown under particular cell culture conditions (i.e. in the presence of stem cell factor and IL-6/sIL-6R) are able to produce EPO according to an autocrine pattern (Sato et al. 2000). This finding explains the capacity of cord blood hemopoietic progenitors to undergo terminal erythroid maturation when grown in the presence of stem cell factor (SCF) and IL-6, but in the absence of exogenous EPO (Sui 1996).

Role of EPO, EPO-R, Kit Ligand and c-kit in the control of erythropoiesis EPO is the main cytokine involved in the control of erythropoiesis. RBC production is strictly dependent on the interaction of EPO with its single transmembrane receptor, EPO-R. EPO is secreted from kidney and fetal liver according to a molecular mechanism triggered by hypoxia (Ebert and Bunn 1991). Bone marrow erythroid cells expressing EPO-R are the main target of EPO: particularly, the late erythroid progenitors, CFU-E and proerythroblasts, where the EPO-R is maximally expressed (about 1,000 receptors per cell). The EPO-R pertains to the super family of cytokine receptors, characterized by the presence of regions of homology at the level of four conserved cysteine residues and the WSXWS motif located near to the transmembrane region. The EPO-R has a large extracellular ligand binding domain, a single transmembrane helix composed by 22 hydrophobic amino acids and a large cytoplasmic domain (Fig. 4). It is synthesized as a 62 kDa

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Fig. 4. Schematic representation of the EPO receptor. A single receptor chain is shown in the figure. The extracellular, transmembrane and cytoplasmic (intracellular) domains of the receptor are outlined (sizes drawn to scale). The receptor is synthesized as precursor of 508 aminoacids, while the mature receptor is composed by 484 amino acids. In the extracellular domain several important structural features are outlined: four conserved cysteine residues, a site of N-glycosylation, a fibronectin-3like sub-domain and a WSXWS motif. In the intracellular, cytoplasmic domain are evidenced two proximal conserved boxes 1 and 2, one of them being involved in JAK2 binding and activation and 9 tyrosine residues playing a major role in the mechanism of EPO receptor signaling. The molecules binding to each of these Tyr (Y) residues are indicated on the right

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precursor that is quickly glycosylated through a high-mannose glycosylation process. The mature EPO-R exhibits a 66 kDa molecular mass. The unliganded EPO-R exhibits an open scissor-like conformation and the dimerization induced by the transmembrane domain maintains this receptor in an inactivated state. The receptor exists as a preformed homodimer, each binding one EPO molecule. Therefore, each EPO molecule shows two binding sites, where the binding site1 has a high affinity (in the nanomolar range) for one EPO-R subunit, while the binding site2 exhibits a relatively low-affinity (in the micromolar range) for the other EPO-R subunit. The two binding interactions determine a high-affinity interaction of EPO with the EPO-R homodimer. The interaction of EPO with EPO-R homodimer determines a conformational change of the EPO-R subunits, with consequent activation of the signal transduction machinery (Remi et al. 1999). Although the primary role of EPO and its receptor is the regulation of RBC production, EPO and its receptor have been localized to several nonhematopoietic tissues and cells, including the central nervous system, endothelial cells, the liver, the uterus and several solid tumors (Farrell and Lee 2004). The biology of EPO and EPO-R, as well as the role of EPO-R expression on tissues other than the erythron and on tumor cells are subjects of separate chapters in this book (see chapter II and III).

Molecular mechanisms of control of erythropoiesis: role of transcription factors The process of erythropoietic differentiation, as well as the whole process of hemopoietic differentiation, is orchestrated at molecular level by a complex network of transcription factors that act regulating the expression of a set of target genes. Particularly, evidence has been accumulated showing that lineage-specific transcription factors, acting together with general transcription factors, play an essential role in the process of erythroid differentiation. Among these different transcription factors GATA-1 seems to play a key role in erythroid development. This zinc-finger transcription factor, expressed in erythroid and megakaryocytic cells, binds to GATA-binding motifs present in the promoters and/or enhancers of all erythroid-specific genes (reviewed in Cantor and Orkin 2002). Three functional domains have been identified within the GATA-1 protein: an N-terminal activation domain, the N-terminal zinc finger (N-finger) and the C-terminal zinc finger (Cfinger). The C-finger is essential for GATA-1 function, since it is responsible for recognition of the GATA-consensus sequence and consequent binding to DNA. The N-finger plays a crucial role in GATA-1 capacity to induce erythroid differentiation. Furthermore, the N-finger mediates the formation of complexes between GATA-1 and other cofactors, such as FOG-1 (Friend of GATA-1) (Ferreira et al. 2005).

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GATA-1 is scarcely expressed in quiescent erythroid progenitors, but is rapidly induced when EPO induces these cells to erythroid differentiation and then progressively accumulates during erythroid maturation being abundantly expressed during all stages of erythroid maturation (Sposi et al. 1992; Labbaye et al. 1995). The highest levels of GATA-1 are observed in CFU-Es and proerythroblasts, while low GATA-1 levels are observed in BFU-Es (Suzuchi et al. 2003). GATA-1 levels decrease during the maturation from proerythroblasts to orthochromatic erythroblasts and this seems to be required for terminal erythroid maturation (Ferreira et al. 2005). The essential role of GATA-1 in the control of erythropoiesis is directly supported by the study of the hemopoietic phenotype of GATA-1 knock-out mice exhibiting a severe anemia with production of an erythroid progeny resulting in a maturational arrest at the level of proerythroblasts. Studies on GATA-1−/− embryonic stem cells induced in vitro to erythroid maturation showed that these cells failed to mature beyond the proerythroblast stage and undergo rapid apoptosis, thus suggesting an essential role for GATA-1 in erythroid survival, in addition to erythroid maturation (Weiss et al. 1994). Proerythroblast-like cells derived from GATA-1 knockdown ES cells have the ability to proliferate vigorously, but a GATA-1 level of 5% cannot sustain the gene expression required for maturation of proerythroblasts (Suwabe et al. 1998). These findings have been also confirmed in a model of GATA-1 knock-down using a promoter-specific disruption of the GATA-1 gene. Therefore, high levels of GATA-1 are required in immature erythroblasts to ensure their maturation. Using a promoter interference approach it was possible to demonstrate that 5% expression of GATA-1 is insufficient to support erythropoiesis during embryonic development, while 20% expression of GATA-1 is sufficient to support erythroid cell maturation (Takahashi et al. 1997). Low levels of GATA-1 expression are sufficient to allow the proliferation of definitive erythroblasts and to protect them from apoptosis, while they are unable to sustain their maturation (Pan et al. 2005). On the other hand, over expression of GATA-1 in erythroid cells inhibit erythroid differentiation both in vitro and in vivo (Wyatt et al. 1997; Wyatt et al. 2000). However, erythroid cells overexpressing GATA-1 differentiate normally in vivo when in the presence of wild-type cells. This intriguing phenomenon was explained by assuming that normal erythroid cells generate within the erythroblastic island a RBC differentiation signal able to overcome the intrinsic defect in GATA-1-overexpressing erythroid cells (Gutierrez et al. 2004). These findings demonstrate the importance of intercellular signaling in regulating GATA-1 activity and indicate that this homotypic signaling between erythroid cells is crucial to normal erythroid differentiation. GATA-1 is required for both primitive and definitive erythropoiesis. In primitive erythroid cells GATA-1 expression is controlled by a 5′ enhancer element called the GATA-1 hematopoietic enhancer, while in definitive

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erythroid cells GATA-1 expression requires a regulatory element in the first intron, in addition to the GATA-1 hematopoietic enhancer. According to these observations it seemed logical to assume that one or more GATA-1 target genes, which normally exert their physiological function at the proerythroblast/basophilic erythroblast stages, are strictly necessary for the survival and terminal maturation of erythroid precursors. However, surprisingly, an examination of the expression of several important potential target genes showed that they continued to be expressed at approximately normal levels in the absence of GATA-1 (Tsang et al. 1998). Among these potential target genes, one could be represented by Bcl-XL, an anti-apoptotic protein, from which the expression is strongly induced by GATA-1 (Weiss and Orkin 1995). An elegant approach for the identification of GATA-1 target genes consisted in the micro array transcriptosome analysis of GATA-1- rescued erythroblasts. This analysis provided evidence that GATA-1 induced a complex genetic program of cell cycle control consisting in the coordinate upregulation of cell cycle inhibitors (such as p18 and p27) and repression of mitogenic genes, such as cyclin D2 and cyclin dependent kinase 6, events mainly related to GATA-1 mediated inhibition of c-myc (Rylski et al. 2003). In a second analysis, carried out on a similar cellular model, clear evidence was provided about a stimulatory effect of GATA-1 on the expression of a set of genes including known GATA-1 gene targets, but also a repressor effect on another set of genes. Notable examples of genes induced by GATA-1 are represented by FOG-1, globins, glycophorin-A, Bcl-XL: the inductive effect on FOG-1 was rapid, while the stimulatory effect on the other genes was delayed. Two remarkable inhibitory effects of GATA-1 are exerted on GATA-2 and c-myc genes. In addition to its key role in the control of erythropoiesis GATA-1 affects also the differentiation capacities of early hemopoietic progenitors/stem cells, as shown by experiments involving the enforced expression of this transcription factor in murine stem cells or in committed hemopoietic progenitors (Iwasaki et al. 2003). On the other hand, the differentiation to the granulo-monocytic lineage required mandatorily the inhibition of GATA-1 activity via binding of the transcription factor PU.1 at the level of GATA-1 binding sites of GATA target genes (Rekhtman et al. 2003). In addition to PU.1, also other transcription factors such as Ski (Ueki et al. 2004) and HERP2 (Elagib et al. 2004) are able to interact with GATA-1 and to inhibit its transcriptional activity. The loss of GATA-1 transforms primitive blood precursors into myeloid cells, resulting in a massive expansion of granulocytic neutrophils and macrophages (Galloway et al. 2005). The ensemble of these observations clearly indicates that GATA-1 acts as a master regulator of erythropoiesis. It is therefore evident that GATA-1 acts as an activator and repressor of different target genes: it represses cell proliferation and early hemopoietic genes (such as GATA-2), while it

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activates later during differentiation erythroid genes. These different tasks are accomplished by GATA-1 through the formation of different molecular complexes with other transcription factors (Rodriguez et al. 2005): the GATA-1/Gfi-1B acts early during differentiation and suppresses genes involved in cell proliferation, such as c-myc and c-myb; the GATA-1/FOG-1/MeCP1 complex also acts early during differentiation to suppress the expression of genes, such as GATA-2, involved to maintain multipotency of progenitors; finally, the GATA-1/FOG-1 and the GATA-1/Tal-1/Lbb1 complexes act at later stages of differentiation promoting erythroid-specific gene expression. In addition to bind DNA, other transcription factors exert their activity through protein-protein interactions. A prototype of this category of factors is represented by FOG-1, a zinc finger transcription factor that binds to the amino zinc finger of GATA-1 (reviewed in Cantor and Orkin 2002). FOG-1 is a polypeptide of 998 amino acids that contains nine zinc fingers, from which four interact independently with GATA-1. FOG-1, abundantly expressed in erythroid and megakaryocytic cells, is co-expressed with GATA-1 during erythroid development and exerts a key role in the control of erythropoiesis: in fact, FOG-1 mice die of severe anemia caused by an erythroid maturation block similar to that observed in GATA-1−/− mice (Gregory et al. 1999). Genetic evidences based on the study of in vitro obtained or spontaneously occurring GATA-1 mutants proved that the physical interaction between GATA-1 and FOG-1 is required for the erythropoietic and megakaryocytopoietic effects of these transcription factors (Nichols et al. 2000). At the moment, the mechanisms by which FOG-1 influences the biologic activity of GATA-1 are largely unknown, but the majority of the studies suggest that the simple interaction between GATA-1 and FOG-1 is sufficient to activate GATA-1 (Cantor and Orkin 2002). In vitro differentiation studies of mouse stem cells and conditional gene expression assays allowed to better define the role of FOG-1 in erythroid and megakaryocytic cell differentiation: FOG-1 exerts an inhibitory effect on the proliferation of erythroid cells, while it is required for late stages of megakaryopoiesis (Tanaka et al. 2004). Several mutually occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity (Liew et al. 2005). Interestingly, excess GATA-1 mutant protein lacking FOG-1 binding capacity abrogates lethal anemia that is owing to GATA-1 deficiency, but it cannot rescue megakaryocyte differentiation (Shimizu et al. 2004). Another transcription factor pertaining to the GATA family, GATA-2 exerts also effects on erythropoiesis. In the early mouse embryo, loss of GATA-1 leads to a qualitative defect in yolk sac erythropoiesis, while loss of GATA-2 determines only a moderate quantitative effect at the yolk sac stage. At later times of development early erythroid progenitors, as well as other hemopoietic progenitors, strictly require GATA-2 for their development

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(Tsai et al. 1994). The deficiency of both GATA-1 and GATA-2 determines a complete blockade of erythropoiesis, as well as of other hemopoietic lineages (Fujiwara et al. 2004). GATA-2 expression is down-regulated and GATA-1 expression increases as hemopoietic precursor cells differentiate into erythroid cells. Although major questions remained unanswered regarding the exact consequences of GATA-2 down-regulation, this appears to be important for differentiation, as sustained expression of GATA-2 alters erythroid differentiation. The repression of GATA-2 expression during normal erythropoiesis seems to be dependent upon a transcriptional repressor activity exerted by GATA-1, via binding to GATA sites present in a regulatory region of the GATA-2 locus (Martowicz et al. 2005). Erythroid differentiation is influenced also by other transcription factors acting either at early or late stages of the differentiation process. In this context, among the transcription factors regulating the early events of hematopoietic differentiation, an important role in the control of erythropoiesis is certainly played by Tal-1, a member of the basic helix-loop-helix (bHLH) family of transcription factors, initially identified for its involvement in some chromosomal translocations occurring in patients with T-cell acute lymphoblastic leukemia. Tal-1 expression is essential for the early developments of the primitive and definitive hematopoietic systems. Experiments on embryonic stem cells Ta1−/− clearly indicate that this transcription factor is required for the development of competence of mesodermal cells to become hematopoietic (Endoh et al. 2002). However, studies of conditional Tal-1 gene targeting have shown that Tal-1 expression, although essential for the genesis of hematopoietic stem cells, is not essential for the function of these cells (self-renewal, proliferation and commitment). Finally, Tal-1 expression is strictly required for proper erythroid and megakaryocytic differentiation (i.e. in the absence of Tal-1 no erythroid and megakaryocytic differentiation is observed) (Mikkola et al. 2003). Like other tissue-restricted bHLH transcription factors, Tal-1 binds DNA as a heterodimer with the ubiquitously expressed E protein, which recognizes the hexanucleotide sequence CANNTG found in a wide variety of eukaryotic transcriptional enhancers, particularly at the level of erythroid and megakaryocytic genes. In normal adult tissues Tal-1 expression is restricted to hematopoietic and endothelial cells and, particularly in erythroid cells, Tal-1 is initially expressed at low levels in quiescent erythroid progenitors, but becomes highly expressed throughout all the differentiative and maturative process and forms active heterodimers with E2A, participating in the activation of erythroid-specific genes (Gabbianelli et al. 2000; Gabbianelli et al. 2003). Thus, it was shown that Tal-1 is strictly required for glycophorin-A expression (Lahil et al. 2004). In fact, antisense oligomers to Tal-1 inhibit proliferation and self-renewal of erythroleukemia cells (Green et al. 1991), and forced Tal-1 expression exerts a stimulatory effect on erythroid development of normal hemopoietic progenitors (Valtieri et al. 1998).

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It is of interest to note that several studies suggest that the capacity of Tal-1 to influence the hemopoietic differentiation depends more on the capacity to form transcriptional complexes with other transcription factors than on its DNA-binding properties. This intriguing conclusion is directly supported by the observation that Tal-1 mutants that lack a DNA-binding domain are able of rescuing primitive erythropoiesis from in vitro differentiated Tal-1−/− embryonic stem cells (Porcher et al. 1999). However, a recent study clearly indicates that some of the effects of Tal-1 on erythropoiesis require its DNA-binding activity (Ravet et al. 2004). Furthermore, Tal-1 was shown to form transcriptional complexes with E47, GATA-1, LMO2 and Ldb1: these complexes bind to E boxes present in the promoters of some erythroid genes, thus stimulating their transcription (Xu et al. 2003). On the other hand, other transcription factors, such as Erythroid Kupperlike Factor (EKLF) and NF-E2, act late during erythroid maturation. EKLF is a zinc finger transcription factor which binds to a binding site consensus sequence 5′-NCNCNCCN-3′ present within the adult β-globin gene promoter. Binding sites for EKLF are found in close proximity of GATA-1 binding sites in the regulatory regions of several erythroid-specific genes. Mice with genetic deletion of the EKLF genes die from severe anemia at the fetal liver stage due to failure of adult β-globin gene activation. Recent studies provided evidence that EKLF could act coordinating erythroid cell proliferation and hemoglobinization (Coghill et al. 2001). In addition to its role in the control of globin synthesis, EKLF plays also an important role in the control of the expression of several genes involved in heme synthesis, such as ALA2S and PBGD (Drissen et al. 2005). Expression of the p45 subunit of transcription factor NF-E2 is restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia and a number of RBC defects, including anosocytosis and hypochromia (Levin et al. 1999). NF-E2 level is low in early erythroid progenitors and progressively increases during erythroid differentiation (Labbaye et al. 1995). The low expression of NF-E2 during the early stages of erythroid differentiation seems to be related to an inhibitory effect exerted by NF-κB (Liu et al. 2003), whose expression is high in erythroid progenitors (Zhang, MY et al. 1998). In addition to these lineage-specific transcription factors, other ubiquitously expressed transcription factors play an important role in the control of erythropoiesis. Among them a peculiar role is played by the CBP, a ubiquitously expressed histone acetyltransferase, able to interact with a large number of proteins. Particularly, CBP is able to interact with GATA-1 and to acetylate it at the level of lysine residues: as a consequence of this acetylation, the transcriptional activity of GATA-1 is stimulated (Hung et al. 1999). The functional role of GATA-1 acetylation is directly supported by the observation that mutation of the acetylated lysine residues to arginine markedly reduces the GATA-1 ability to rescue erythroid maturation after transfection

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in a GATA-1−/− cell line. CBP is also able to acetylate Tal-1: this acetylation destabilizes the interaction of Tal-1 with mSin3A repressor, while enhancing binding to DNA and transcriptional activity (Huang et al. 2000). Recent studies provided evidence that the transcription factor Gfi-B could play an important role in the control of erythropoiesis. Gfi-1B is a highly related zinc finger protein with a selective pattern of expression restricted to hemopoietic cells. This transcription factor possesses a typical SNAG domain and binds to the consensus DNA sequence TAAATCAC (A/T) GC (A/T). Some of the target genes of Gfi-1B have been identified (p21; Socs1, Socs3); in this case Gfi-1B acts as a transcriptional repressor. The Gfi-1B transcriptional factor is expressed in the bone marrow and spleen in mice, selectively in erythroid cells in chicken, while in man its expression is restricted to erythroid and megakaryocytic cells (Osawa et al. 2002). The expression of Gfi-1B gene in erythroid cells is under the control of GATA-1 that, in cooperation with NF-Y, acts as a transcription activator of the Gfi-1B gene (Huang et al. 2004). Studies of gene targeting in mice showed that Gfi-1B embryos exhibit delayed maturation of primitive erythroid cells and subsequently die with a failure to produce definitive erythroid cells. The fetal liver of Gfi-1B−/− mice contains erythroid and megakaryocytic precursors arrested in their development, while myelopoiesis is normal (Saleque et al. 2002). The role of Gfi-1B in erythroid differentiation was also confirmed by studies of enforced expression of Gfi-1B gene in normal human hemopoietic progenitors, showing a marked proliferation of erythroid cells in an EPO-independent manner, associated with an inhibition of myeloid cell differentiation. Deletion of the SNAG repressor domain abolished Gfi-1Binduced erythroid maturation, strongly suggesting that Gfi-1B acts in the late stages of erythroid differentiation as a transcription repressor (Garçon et al. 2005). Although the role of Gfi-1B on erythroid differentiation is clear, its position in the hierarchy of hemopoietic transcription factors, as well as its functional relationship to GATA-1 and FOG-1, remains to be evaluated. Transcription factors of the AP-1 family are activated by EPO in erythroid cells and play a role in the control of apoptosis, cell proliferation and differentiation. The AP-1 family of transcription factors consists of the Fos and Jun proteins, which are known as “early response” proteins due to their up-regulation in response to extracellular stimuli regulating cell growth, differentiation and survival. C-Jun and particularly JunB is induced during early and late stages of erythroid differentiation: the late JunB expression is required for erythroid maturation (Jacobs-Helber et al. 2002). There is also evidence that c-jun may play a role in the protection from apoptosis elicited by EPO. In addition to these transcription factors that act on erythropoiesis modulating the expression of sets of target genes, other transcription factors are also essential for normal erythropoiesis, but they act through a different mechanism. In this context particularly relevant is the retinoblastoma (Rb)

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gene product (pRb), a key regulator of cell proliferation and differentiation. A large set of data indicates that pRb is an important controller of erythroid differentiation. In fact, Rb−/− mice die of ineffective erythropoiesis during embryonic development, with decreased numbers of BFU-E, CFU-E and definitive erythrocytes. Although subsequent studies have cast doubt as to whether these defects in erythropoiesis are intrinsic to the erythroid lineage (Jacks et al. 1992), more recent studies have largely clarified the role of pRb in erythropoiesis (Whyatt and Grosfeld 2002). Thus, it was shown that pRb is greatly accumulated during in vitro human erythropoiesis and it’s silencing by RNA antisense oligonucleotides resulted in a marked inhibition of erythroid colony formation (Condorelli et al. 1995). Culture experiments using hemopoietic progenitors derived from Rb−/− mice showed that these cells have an impaired terminal maturation (Clark et al. 2004) because pRb seems to be required for erythroblast enucleation (Spike et al. 2004). Particularly, pRb is also required for the development of cells, such as macrophages that support erythropoiesis. In fact, pRb promotes the differentiation of macrophages by opposing the inhibitory function of the transcription factor Id2 on PU.1, a master regulator of macrophage differentiation (Iavarone et al. 2004). Recent studies provided evidence that the octamer-binding protein-1 (Oct-1) is required for normal erythropoiesis. In fact, Oct-1−/− mice are anemic and suffer from a lack of erythroid precursor cells. Oct-1−/− embryonic stem cells display a markedly reduced capacity to differentiate to erythroid progenitors and to mature erythroid elements (Wang et al. 2004). Gene targeting studies in mice have shown that the loss of VEGF or VEGF-RI or VEGF-RII or c-myc resulted in a blockade of primitive erythropoiesis, but this phenomenon is not specific to erythropoiesis and seems to be related to a more generalized defect of hematopoiesis. Although the inactivation of c-myb determines a generalized defect in hemopoiesis, this transcription factor seems particularly relevant for erythropoiesis. In fact, using a gene targeting approach allowing only a partial inactivation of c-myb gene expression, it was possible to show that c-myb activity is strictly required for the transition of CFU-Es to immature erythroblasts (Emambokus et al. 2003). After this stage of differentiation, c-myb expression is downmodulated through a repressive mechanism mediated by the binding of GATA-1 at the level of the c-myb promoter (Bartunck et al. 2003). In addition to transcription factors that cooperate and enhance the activity of erythroid transcription factors, other transcription factors antagonize their activity. An important example is provided by the myeloid and Blymphoid transcription factor PU.1: PU.1, when overexpressed in erythroid cells, inhibits their maturation through a molecular mechanism related to a physical interaction and a functional antagonism with GATA-1 (Zhang et al. 2000). However, at earlier stages of erythroid differentiation PU.1 promotes the self-renewal of erythroid progenitors (Back et al. 2004). In contrast, other

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transcription factors, such as c-jun, antagonize the transcriptional activity of GATA-1 through induction of the expression of the basic helix-loop-helix protein HERP2: this protein acts as a repressor of GATA-1 transcription (Elagib et al. 2004). A key event during terminal differentiation of erythroid cells is the activation of globin gene transcription, leading to hemoglobin synthesis. The high expression of globin genes in erythroid cells is dependent on the presence of peculiar gene regions, locus control region (LCR), which consists of five nuclease hypersensitive sites spread over 20–30 kilobases of DNA and located upstream the β-globin like genes. The β-globin LCR contains numerous binding sites for both erythroid-specific and ubiquitous transcription factors, including E-boxes (recognized by Tal-1), CACC motifs (recognized by Kruppel-like proteins and by the Sp family of proteins), GATA motifs and MAREs (Maf recognition elements, recognized by the transcriptional activation complex NF-E2, which comprises the hematopoietic-specific p45 subunit and the ubiquitous Maf protein 18). Using quantitative proteomics the transcriptional complexes binding to the β-globin LCR have been characterized (Brand et al. 2004). Interestingly, the transcriptional molecular complexes formed at the level of MAREs sites change during erythroid differentiation: in immature erythroid precursors the p18 subunits forms heterodimer with Bach1, recruiting co-repressor complexes (including NuRD, SIN3) that generate a transcriptionally repressive chromatin structure; in maturing erythroid precursors the p18 subunit forms heterodimers with p45, recruiting a co-activating complex (including Tal-1 and CBP) that generates a transcriptionally permissive structure (Brand et al. 2004).

Anti-apoptotic mechanisms operating in erythroid cells Stat5 The signal transducer and activator of transcription (Stat) proteins have a dual role as signal transducers and activators of transcription. These proteins are latent in the cytoplasm and are activated by extracellular signaling cytokines or growth factors that bind to specific cell surface receptors (Levy and Darnell 2002). Following the interaction of these ligands with their receptors, various tyrosine kinases are activated in the cell that phosphorylate Stat proteins; phosporylated Stat proteins become active and accumulate in the nucleus to drive transcription. Stat 5 is a transcription factor present in the cytoplasm in a latent form and activated by the EPO-R, as well as many other cytokine receptors. As a consequence of EPO-R activation Stat5 binds to phosphorylated tyrosines present on the cytoplasmic tail of the EPO-R, and itself becomes phosphorylated at the level of tyrosine residues. The activated Stat5 dimerizes and

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translocates from the cytoplasm to the nucleus where it modulates the expression of several genes involved in the control of cell proliferation and differentiation. In addition to these genes, Stat5 activates in erythroid cells the expression of several anti-apoptotic genes and, notably, of Bcl-XL through a direct binding at the level of Stat5 binding consensus sequences present in the promoter of the Bcl-XL gene (Dumon et al. 1999; Nosaka et al. 1999; Ariyoshi et al. 2000; Levy and Darnell 2002). These effects of Stat5 on Bcl-XL gene expression represent the molecular basis to explain the antiapoptotic effects elicited by Stat5 in erythroid cell lines (Levy and Darnell 2002), while dominant-negative Stat5 induces a growth arrest and apoptosis of erythroid precursors (Chida et al. 1999). Stat5 was reported to induce also Bcl-2 mRNA, but its induction seems to be indirect. Studies on mice that lack the expression of both Stat5a and Stat5b isoforms have provided definitive evidence about the protective role of Stat5 in erythroid cell survival. Stat5a−/−/Stat5b−/− mice embryos are severely anemic as a consequence of an impaired survival of liver erythroid progenitors. Particularly, fetal liver cells derived from Stat5a−/−Stat5b−/− animals generated a low number of erythroid colonies in vitro, are less sensitive to EPO and showed a three-time increase in the frequency of apoptotic cells and a pronounced increase in the percentage of apoptotic cells when grown in vitro in the presence of EPO (Levy and Darnell 2002). In spite of the marked anemia during the embryonic life, Stat5a−/−Stat5b−/− mice exhibited at birth a moderate condition of anemia, which was progressively attenuated in animals of adult age (at adult age only about 50% of Stat5a−/−Stat5b−/− mice were anemic). However, the adult Stat5a−/−Stat5b−/− mice, in spite of their nearnormal hematocrit levels, are deficient in generating high erythropoietic responses following stress stimulation (i.e. the induction of a chemically induced hemolytic anemia). The analysis of erythropoiesis in Stat5a−/−5b−/− animals anemic during adult life under steady-state conditions showed the existence of an increased pool of immature erythroblasts exhibiting reduced Bcl-XL levels and undergoing apoptosis at a high rate with respect to the corresponding cells of wt animals (Socolovky et al. 2001). In conclusion, the analysis of Stat5 knockout mice strongly suggests that the anti-apoptotic effect of this transcription factor in erythroid cells is exerted at the level of immature erythroid cells and is mediated mainly via modulation of Bcl-XL. In line with this observation, Stat5a and Stat5b and Bcl-XL exhibit a similar pattern of expression during normal erythroid maturation, with a progressive increase of expression during the differentiation from BFU-E to CFU-E and to immature erythroid precursors, followed by rapid and marked decline at later stages of maturation (polychromatophilic and orthochromatic erythroblasts) (Fig. 5). It is of interest to note that Stat5a5b deficiency induces also a premature death of myeloid precursors, which seems to be related to a decrease of Bcl-2 and Bcl-XL (Kieslinger et al. 2000).

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Fig. 5. Kinetics of Stat5a, Stat5b, JAK-2, Bcl-XL and Bcl-2 expression during erythroid differentiation. Purified hemopoietic progenitor cells have been induced to erythroid differentiation under unilineage cell culture conditions, either in the presence of EPO alone or EPO plus kit ligand. Cell aliquots were recovered at different days of culture and Stat5a, Stat5b, JAK-2, Bcl-XL and Bcl-2 levels have been evaluated by Western Blotting analysis on samples normalized according to β-actin content

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Bcl-XL The protective role exerted by Stat5 on apoptosis of erythroid precursors is mainly related to the induction of Bcl-XL. Bcl-XL pertains to the family of Bcl-2-related proteins, important regulators of cell death. The Bcl-2 family members can be classified into three different groups on the basis of comparative analysis of their structure-function: (i) multidomain death antagonists (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1); multidomain death agonists (Bax, Box and Bok); (iii) BH3-only pro-apoptotic proteins (Bim, Bid, Bik and Bak). The first two groups of Bcl-2 members function to protect or disrupt the integrity of mitochondrial membranes, respectively, while BH3-only proteins trigger cell death through binding to the receptor domain of multidomain Bcl-2 members, thereby mediating the inactivation of the antiapoptotic members or the activation of the pro-apoptotic members (Joza et al. 2002; Kaufmann et al. 2001). In normal cells, inactive BAX is located in the cytosol, but following an apoptotic stress, BAX is inserted into mitochondria as homooligomerized multimer, resulting in downstream mitochondrial function (Danial and Korsmeyer 2004). Bcl-XL, as well as Bcl-2, is targeted to mitochondria through its interaction with a protein, FKBP38, pertaining to the family of immunophilins. Bcl-XL and Bcl-2 reside on the outer mitochondrial membrane. The main function of the anti-apoptotic Bcl-2 family members consists in promoting adaptation and maintaining vitality of mitochondria to various types of perturbations of cellular metabolism (Vander Heiden and Thompson 2002). Particularly, the anti-apoptotic mechanism of Bcl-XL, as well as of other Bcl-2 family members, consists in the inhibition of the activity of Bid and Bax which cooperate in the formation of pores in the membrane of mitochondria, allowing the release of components of these organelles in the cytoplasm, with subsequent loss of their function (Kuwana et al. 2002). Recent studies, however, suggest a broader role of Bcl-2 in the control of apoptosis, consisting in a general control of caspase activation program independently of the cytochrome c/Apaf-1/caspase-9 apoptosome (Marsden et al. 2002). Therefore, two models have been proposed: in the first model, BH3-only proteins can directly bind and activate Bak/Bax, and this phenomenon may be inhibited by Bcl-2/Bcl-XL-mediated sequestration of BH3-only proteins; in the second model, the binding of BH3-only proteins to their primary targets Bcl-2/Bcl-XL leads to a neutralization of these anti-apoptotic factors, resulting in the activation of Bax/Bak (Cory and Adams 2002). Gene targeting studies of anti-apoptotic Bcl-2 family members provided clear evidence that they exhibit a unique physiologic role: Bcl-2 is required for the survival of kidney, melanocyte stem cells and mature lymphocytes; Bcl-XL for neuronal and erythroid cells; Bcl-w for sperm progenitors; A1 for neutrophils and Mcl1 for zygote implantation (reviewed in Cory and Adams 2002). Recent studies indicate that Mcl1 is strictly required for the survival

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of hemopoietic stem cells and hemopoietic progenitor cells and its function cannot be replaced by other Bcl-2 members (Opferman et al. 2005). In this context, particularly interesting were the phenotypic features observed in the Bcl-X gene knockout: in fact Bcl-X−/− mice die at E13, exhibiting massive cell death at the level of neuronal elements of the central nervous system and of erythroid elements present in fetal liver (Motoyama et al. 1995). The level of action of Bcl-XL in the protection of erythroid cells from apoptosis was finely investigated using mouse embryonic stem cells in which both alleles of the Bcl-XL gene were disrupted. Bcl-XL−/− embryonic stem cells were unable to contribute to the generation of definitive erythroid cells, thus indicating that the expression and function of this anti-apoptotic protein is essential for the production of definitive erythroid cells. Bcl-XL−/− embryonic stem cells generated a number of erythroid colonies similar to that originated from wt embryonic stem cells; however, a significant proportion of erythroid precursors present in the erythroid colonies of Bcl-XL−/− cells undergo apoptosis during their maturation and do not reach the terminal stage (Motoyama et al. 1999). These findings strongly suggest that Bcl-XL is a critical anti-apoptotic regulator of erythropoiesis. The role of Bcl-XL in adult erythropoiesis was explored through the study of transgenic mice conditionally deficient in Bcl-XL gene. These animals exhibited at three months of age a condition of severe hemolytic anemia, associated with platelet deficiency (Wagner et al. 2000). The analysis of the bone marrow of these animals showed a hyperplasia of both megakaryocytic and erythroid precursors; the rate of apoptosis in erythroid precursors of Bcl-XL deficient animals was only slightly increased as compared to that observed for the corresponding cells of normal animals. This conditional knockout model was recently re-evaluated providing clear evidence that Bcl-XL plays a key role in the survival of the late-stage erythroblasts in all phases of the cell cycle, but not in early stage erythroblasts (Rhodes et al. 2005). Studies on an immortal line of phenotypically normal mouse erythroblasts provided further details about the anti-apoptotic role of Bcl-XL in erythroid maturation. These cells were maintained in an undifferentiated state by agents promoting self-renewal, such as SCF and glucocorticoids, while they were induced to differentiate by EPO. Bcl-XL overexpression allowed these cells to undergo terminal differentiation to mature erythrocytes in the absence of EPO (Dolznig et al. 2002). The molecular basis responsible for the elevated expression of Bcl-XL in erythroid cells is at the moment unknown. However, studies on the Bcl-XL gene promoter have in part helped to understand the regulation of the expression of this gene in erythroid cells. The promoter of the Bcl-XL gene used for the start of the transcription in erythroid cells is localized in 5′ in close proximity to the start initiation codon ATG. This promoter region contains several putative regulatory sites recognized by some transcription factors, including

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GATA-1, NF-E2 and Ets-1 and other transcription factors pertaining to the Ets family (Grillot et al. 1997). This promoter region contains also a Statbinding element, able to bind Stat-5 (Silva et al. 1999). Studies carried out on embryonic stem cells isolated from GATA-1−/− mice provided evidence that GATA-1 plays an important positive role in the regulation of Bcl-X gene expression. Particularly, it was shown through the analysis of conditional knockout models that the expression of GATA-1 in embryonic stem cells strongly induces Bcl-XL, but not Bcl-2, expression in erythroid cells and this may represent one of the mechanisms responsible for the anti-apoptotic effects exerted by GATA-1 in erythroid cells (Gregory et al. 1999). Finally, more recent studies have led to the identification of the major site of start of Bcl-XL gene transcription in erythroid cells. This site corresponds for the human Bcl-X gene at −654 relative to the ATG initiation codon; furthermore, an enhancer element was identified at the level of the position −1804 through −1734 (Tian et al. 2003). EPO deprivation induces the activation of caspase-3, leading to apoptosis of erythroblasts (Gregoli and Bondurant 1999). Since activated caspase3 was reported to cleave Bcl-XL (Negoro et al. 2001), it is conceivable that EPO protects erythroid cells from apoptosis in part via blockage of caspase3-dependent cleavage of the Bcl-XL protein. Furthermore, it was shown that the activation of ERK1 and ERK2 by EPO up-regulates Bcl-XL expression via inhibition of caspase activities, thus resulting in the protection of erythroid cells from apoptosis (Mori et al. 2003). The kinetics of Bcl-XL expression during the process of erythroid differentiation (from early BFU-Es to CFU-Es) and maturation (from proerythroblasts to reticulocytes) was explored in detail. Few studies have explored the expression of Bcl-XL, as well as of Bcl-2, in hemopoietic progenitors (i.e. in total CD34+ cells or in fractions of CD34+ cells, such as 34+/38− or 34+/38+). Studies on human CD34+ cells have shown that both Bcl-XL and Bcl-2 are widely expressed in these cells; in contrast, in CD34+/38− cells (corresponding to early, immature hemopoietic progenitors) Bcl-XL is preferentially expressed as compared to Bcl-2 (Park et al. 1995; Peters et al. 1998). The kinetics of Bcl-XL expression during erythroid differentiation and maturation was explored using different cellular models of erythroid differentiation. Using human CD34+ cells grown either in erythroid (SCF + EPO) or in granulocytic (SCF + G-CSF) cell culture medium, it was shown that at day 4 and 8 of culture Bcl-XL expression was markedly more pronounced in erythroid than in granulocytic precursors (Dumon et al. 1999; Josefsen et al. 2000; Dolznig et al. 2001). Bcl-XL was expressed at very low levels in immature erythroblasts and its expression progressively and markedly increased during maturation up to terminal erythroblasts (Gregoli and Bondurant 1997). Using a cell culture system allowing the study of all stages of erythroid differentiation from BFU-E to mature erythroblasts it was possible to carefully study Bcl-XL expression during erythroid differentiation and

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maturation (Testa 2004). In quiescent CD34+ human cells a low Bcl-XL expression was observed (Fig. 5). A marked increase in Bcl-XL expression was observed during early stages of the differentiation of CD34+ to the erythroid lineage (day 4 and day 7, when the cells correspond to a stage intermediate from a BFU-E to a CFU-E and to CFU-E/proerythroblasts, respectively); Bcl-XL levels continued to increase during early and intermediate stages of erythroid maturation, reaching peak level of expression when the majority of erythroid cells have reached the stage of polychromatophilic erythroblasts; during terminal stages of erythroid maturation (day 14–16 of culture) a marked decline in Bcl-XL expression was observed (Fig. 5). The administration of SCF to the erythroid cultures moderately potentiated BclXL expression, associated with terminal erythroid maturation (Fig. 5). In contrast, Bcl-2 expression was limited only to the early stages of erythroid differentiation and completely declined to very low or undetectable levels during the maturation of erythroid cells. Bcl-XL could exert, in addition to its role as an anti-apoptotic factor in erythroid cells, other biologic functions. Inhibition of Bcl-XL expression (by antisense transcripts) in Friend erythroleukemia cells elicited a marked inhibition of hemoglobin synthesis after DMSO induction, as well as an increased rate of apoptosis (Hafid-Medhab et al. 2003). Importantly, this inhibitory effect of Bcl-XL deficiency on hemoglobin synthesis was observed also in cells over expressing Bcl-2 (Hafid-Medhab et al. 2003). The inhibitory effect on hemoglobin synthesis caused by Bcl-XL deficiency was due to an inhibition of heme synthesis and not to a reduction of globin mRNA expression. Since in differentiating erythroleukemic cells Bcl-XL remains localized to mitochondria, it was suggested that it could exert in this organelle a dual function being involved as an anti-apoptotic factor in mitochondria integrity and in heme synthesis (Medhab et al. 2003). However, it remains to prove whether Bcl-XL exerts this dual function also in normal erythroid cells. Recent studies on conditional Bcl-XL knockout have shown that in the absence of Bcl-XL there is only a slight decrease during late stages of erythroid maturation (Rhodes et al. 2005). Furthermore, studies of enforced expression in the FDCP-Mix multipotent progenitor cell line have indicated a novel role for Bcl-XL in cell fate decision beyond cell survival. In fact, the erythroid expression of Bcl-XL in these cells was associated with induction of erythroid differentiation and prohibited generation of myeloid cells (Haughn et al. 2003). It is of interest to note that recent studies have indicated that Bcl-XL may represent a major target of some anti-tumor chemotherapeutic drugs. Therefore, Bcl-XL levels may represent a major determinant in the protection of the erythroblasts from apoptotic cell death induced by DNA damaging chemotherapeutic drugs. In spite of their structural heterogeneity and their different molecular targets, many chemotherapeutic agents kill cells by inducing apoptosis. The

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majority of these drugs mediate cell death through an activation of the intrinsic apoptosis pathway; this pathway is activated by different types of intracellular stresses, such as genetic damage or growth factor deprivation, and implicates the Apaf-1-dependent activation of caspase-9, following release of cytochrome C and Smac/Diablo from mitochondria (Johnstone et al. 2002). This pathway is regulated by the proapoptotic multidomain and BH3-only Bcl-2 proteins and their anti-apoptotic counterparts, including also Bcl-XL. In addition to represent one of the main molecules involved in the cell protection in the intrinsic apoptotic pathway, Bcl-XL may represent also a direct target of some chemotherapeutic drugs. In fact, it was shown that Bcl-XL could be modified by deamidation of asparagine residues, at the level of an unstructured loop separating the (1helix-BH4 domain and the α2helix-BH3 domain (Deverman et al. 2002). Bcl-XL deamidation was observed in tumor cells or in mouse embryonic fibroblasts p53−/− treated with different types of genotoxic agents, including cisplatin, etoposide, taxol and γ-irradiation (Deverman et al. 2002). The deamidation of Bcl-XL has as the consequence of its inactivation, with consequent loss of Bcl-XL binding to Bim and subsequent induction of apoptosis. The same authors also showed that the hypophosphorylated active form of Rb protein inhibits deamidation of BclXL, suppressing through this mechanism the apoptosis induced by DNA damaging agents. Anemia is a common complication of cancer, often resulting in a decrease of quality of life and influencing the outcomes of patient care. The myelosuppressive effects of chemotherapy are a major cause of anemia in cancer patients. Chemotherapy-induced anemia is the result of two different mechanisms acting at the level of: (a) the bone marrow progenitor/stem cell compartment, (b) the immature erythroid precursors (proerythroblasts and basophilic erythroblasts). This last mechanism was recently clarified, showing that immature normal erythroblasts are extremely sensitive to the cytotoxic effect of chemotherapeutic agents, such as cisplatin, etoposide or camptothecin, while mature erythroblasts (acidophilic erythroblasts) are almost completely resistant. Importantly, EPO also at high doses was unable to protect immature erythroblasts from chemotherapy-induced apoptosis. According to these observations it was concluded that the primary target of chemotherapy-induced apoptosis is represented by proerythroblasts and basophilic erythroblasts. Interestingly, preincubation of erythroid cells with SCF resulted in a marked protection of erythroid cells from chemotherapyinduced apoptosis of immature erythroid cells. The mechanism of this protection seems to be related to an SCF-mediated up-modulation of Bcl-2 and Bcl-XL expression, associated with a consequent inhibition of caspase activation (Zeuner et al. 2003). At the moment it is unknown whether the inhibition of chemotherapy-induced apoptosis could be related to an inhibition of Bcl-XL deamididation, possibly mediated by upregulation of hypophosphorylated Rb expression.

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Hydroxyurea, another myelosuppressive agent, elicited also apoptosis of erythroid cells through a mechanism involving a strong up-modulation of the TRAIL receptor 2 (Wang et al. 2002).

Role of apoptosis in the control of erythropoiesis The mitochondrial “intrinsic” and the transmembrane “extrinsic” pathways are the two principal pathways leading to apoptosis, both of which converge on caspases, a family of cysteine proteases. The “intrinsic” apoptosis pathway triggered by intracellular injury such as DNA damage or oxidative stress controls caspase activation through the Bcl-2 gene family. In this pathway damage sensors induce transcription of Bcl-2 homology 3 (BH3) domain proteins (i.e. Noxa, Bim, Bmf). These apical regulators activate downstream pro-apoptotic Bcl-2 members (i.e. Bax, Bak), overcoming inhibition by anti-apoptotic Bcl-2 family members (i.e. Bcl-XL, Bcl-2): activated Bax and Bak trigger mitochondrial release of factors that promote caspase activation in the cytosol. One of these factors is cytochrome c, which cooperates with Apaf-1 to activate caspase-9. This apical caspase activates the effector caspases 3, 6, and 7, inducing apoptotic cell death. During this process, two other mitochondrial factors, Smac/Diablo and Omi/HtrA2 prevent IAPs from inhibiting caspase activation. A key regulator of the intrinsic apoptotic pathway is the transcription factor p53, a potent tumor suppressor that acts as a stress sensor activated in response to DNA damage, hypoxia, nucleotide depletion, aberrant growth signals and chemotherapeutics drugs. Activated p53 induces a series of biological processes and, notably, acts as an activator of the intrinsic apoptotic pathway. Several evidences indicate that p53 triggers apoptosis by activating the intrinsic pathway: (i) by inducing over-expression of Bcl-2 or its pro-survival homologues; (ii) by stimulating the expression of the pro-apoptotic proteins BAX, BIM, BID, NOXA and PUMA; (iii) by directly binding to the BAX or BAK proteins and thereby inducing apoptosis through a transcription-independent mechanism (Michalak et al. 2005). The “extrinsic” pathway is triggered by extracellular death ligands such as TNF relatives FasL and TRAIL, which signal through specific membrane receptors. This apoptotic pathway seems to play an important role in the control of erythropoiesis.

Role of cell death receptors in the control of erythropoiesis Ligands and cell death receptors pertain to the tumor necrosis factor (TNF) and TNF receptor (TNF-R) superfamilies, respectively. Members of the TNF family of membrane-bound and secreted ligands pair off with one or more

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specific cell surface receptors that form a corresponding family of cognate receptors. Each ligand-receptor pair is considered a system, and actually more than 40 distinct ligand-receptor systems are currently recognized (Ware 2003). Members of both the TNF ligand and TNF-R superfamilies exhibit several remarkable structural similarities. The TNF ligands are type II transmembrane proteins, characterized by an extracellular C terminus domain, named the TNF homology domain: this domain contains a conserved framework of aromatic and hydrophobic residues, responsible for the association of single molecules to form a trimer (Bodmer et al. 2002). Most of the TNF ligands are synthesized as membrane-bound proteins, but soluble forms are originated by a process of limited proteolysis. The solubilization of some ligands is associated with a considerable loss of biological activity: this is the case of the Fas ligand (Fas L) and of the TNF-related apoptosis-inducing ligand (TRAIL). The main molecules pertaining to the TNF ligand superfamily are represented by TNF-α, FasL, TRAIL, TWEAK, RANKL, APRIL, lymphotoxin-α (LTα). More than 40 TNF-Rs have been identified. The majority of them are type I transmembrane proteins, with an extracellular domain N terminus and with an intracellular domain C terminus; a notable exception is represented by the TRAIL-R3, which is anchored to the cell membrane through a covalently linked C-terminal glycolipid (Bodmer et al. 2002). Many of these receptors possess a stretch of amino acids, the death domain (DD), as part of their sequence (Bhardway and Aggarwal 2003). The DDs are required for apoptotic signaling. For some of these receptors, soluble forms may be generated through a process of limited proteolysis (TNF-R1 and TNF-R2) or of alternative splicing (Fas). The different members of the TNF-R superfamily exhibit a typical structural feature represented by the presence of cysteinerich domains, which are usually repeats containing each six cysteine residues involved in the formation of three disulfide bonds (Bodmer et al. 2002). The TNF-Rs exist as preformed trimers on the cell membrane before the interaction with their respective ligands. Each member of the TNF ligand superfamily binds at least one receptor of the TNF-R superfamily: FasL binds selectively to Fas; TRAIL may bind to four different membrane receptors, called TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4; TNF-α binds two membrane receptors, TNF-R1 and TNF-R2. The majority of the TNF-R superfamily members function as membrane receptors transducing a signal following interaction with their ligands. These signaling receptors may be subdivided into two groups on the basis of the structure and function of their cytoplasmic region: one of the two receptor groups possesses at the level of this cytoplasmic region, a death domain which mediates the association between the receptor and a death adaptor protein; the other group does not possess this death cytoplasmic domain. The TNF-Rs, TRAIL-R1 and TRAILR2, as well as Fas are typical examples of death receptors. Two examples of death domain adaptors are represented by FADD (also known as MORT1)

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and TRADD: FADD possesses two interfaces, one interacting with the death domain (DD) of Fas and the other one (death effector domain, DED) interacting with intracellular effector enzymes (caspases that initiate the apoptotic process) (Ashkenazi 2002). The induction of death by FADD depends on the DED rather than the DD domain. The DED of FADD recruits two DEDcontaining caspases, caspase-8 and caspase-10, to form the death-inducing signal complex (DISC), thereby initiating apoptosis. The DEDs have no enzymatic function but link participants in a signaling chain through homotypic interactions, containing other proteins with DEDs (Tibbetts et al. 2003). The principle structural feature of the DEDs in various proteins is the conserved backbone of six α-helices, a conserved negatively charged residue at position 19 and the Arg-X-Asp-Leu (RXDL) motif at positions 78–81 (Tibbetts et al. 2003). The other adaptor called TRADD (TNFR-associated death domain) can initiate apoptosis through FADD or may stimulate protein kinases involved in the control of phosphorylation cascades to induce the transcription of some immune-system modulation genes. TRADD seems to be involved in the apoptotic signaling of the TNF-R, but not of Fas and TRAILRs (Dempsey et al. 2003). Some of the receptors of the TNF receptor superfamily are decoy receptors able to compete with signaling receptors for ligand binding, thereby inhibiting their function. Examples of decoy receptors are given by DcR1 and DcR2 (also known as TRAIL-R3 and TRAIL-R4), competing with TRAILR1 and TRAIL-R2, respectively, for binding of TRAIL and DcR3 (sFas) competing with Fas for binding of FasL (Ashkenazi 2002). The physiologic role of these receptors is still unclear, but it has been suggested that they could protect some normal cells by their cytotoxic ligands. The series of biochemical events elicited by the interaction of a death ligand with its receptor has been in part elucidated and can be summarized briefly as follows (Fig. 6). The engagement of death receptors by their ligands activates the extrinsic apoptotic pathway. This pathway leads, as the intrinsic apoptotic pathway, to the activation of caspases independently on p53. The activation of a death receptor by its ligand leads to the recruitment at the level of the receptor of a death adaptor, which in turn determines the rapid assembly of a death-inducing signaling complex (abbreviated as DISC), with consequent activation of the apoptosis-initiating caspases 8 and 10. Caspase-10 is an initiator caspase found in primates but not in rodents. The role of this caspase in initiating the apoptotic process is supported by two observations: a) lymphocytes from ALPS II patients bearing caspase-10 mutation are resistant to TRAIL-mediated killing (Wang et al. 1999); b) endogenous caspase-10 is activated and recruited at the level of the DISC and is capable of transmitting an apoptotic signal in the absence of caspase8 (Kischkel et al. 2001). These caspases, in turn, activate caspase-9; in turn, the activated caspase9 activates “executioner” caspases-3, -6 and -7. Activated caspases-8 and -10

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Fig. 6. Signal transduction through the extrinsic apoptotic pathway. Engagement of TRAIL or FasL with its cognate receptor TRAIL-R2 results in the activation of two different signaling pathways involving the activation of the apoptotic cascade (right) or a two signaling pathways, MAPK and NFκB. The activation of the apoptotic cascade involves the formation of a receptor proximal complex containing the adaptor proteins FADD. These adaptor proteins in turn recruit additional key pathway-specific enzymes, such as caspase-8, they became activated and initiate downstream events leading to apoptosis

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are also able to activate by proteolytic cleavage Bid, which is responsible for the activation of the pro-apoptotic arm of the Bcl-2 gene superfamily, with consequent release of apoptogenic factors from mitochondria. Particularly, SMAC/DIABLO released from mitochondria promotes apoptosis through its capacity to bind and neutralize the inhibitor of apoptosis proteins (IAP), thus preventing the activity of these proteins as attenuators of the caspase activation. Depending on the cell type, activated caspase-8 formed at the level of the DISC induces apoptosis by two different signaling pathways. In type I cells, large amounts of active caspase-8 formed at the DISC induce direct cleavage/activation of pro-caspase-3 independently of mitochondria. In type II cells, small amounts of active caspase-8 are insufficient to activate procaspase-3 directly. Instead, caspase-8 cleaves the “BH3-only protein” Bid, generating a Bid fragment that activates the mitochondrial death pathway. The activation of caspase-8 and -10 is antagonized by cellular FLICE inhibitory protein (c-FLIP), an enzymatically-inactive relative of caspase-8 and -10, that binds to the DISC. Three c-FLIP proteins are present: c-FLIPL (55 kDa), c-FLIPS (26 kDa) and c-FLIPR (22 kDa) (Golks et al. 2005). Knockdown of c-FLIPL, as well as of c-FLIPS, augments DISC recruitment, activation and processing of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis (Sharp et al. 2005). These observations clearly indicate that c-FLIP functions as an inhibitor of death receptor-mediated apoptosis. In the last years, evidence was accumulated showing that death receptors and their ligands may act as negative regulators of erythropoiesis and that their action may play a physiologically relevant role in the control of the rate of erythropoiesis. Furthermore, abnormalities of these receptors may play an important role in the physiopathology of some anemic conditions.

Effects of TNF-a on normal erythropoiesis The binding of TNF-α to the TNF-R1 leads to a cascade of events, above outlined, that determines the induction of apoptosis, NF-κB and JNK activation. TNF-R1 signaling involves assembly of two molecularly and spatially distinct signaling complexes that sequentially activates NF-κB and caspases (Muppidi et al. 2004). Within the first minutes after binding of TNF-α to its receptor TNF-R1, a signaling complex termed complex I is formed. This complex is composed by TNF-R1 itself, TRADD, TRAF2 and RIP1. Complex I transduces signals that lead to NF-κB activation. Later (i.e. >2 hours) TRADD, TRAF2 and RIP1 dissociate from the receptor and recruit FADD and caspase-8 with subsequent formation of the complex II. In conditions where the complex I is able to induce a sufficient level of NF-κB activation, expression of anti-apoptotic proteins is induced and the activation of apical caspases in complex II is inhibited (Muppidi et al. 2004). In addition to activating

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antiapoptotic genes, NF-κB can suppress apoptosis by inhibiting sustained activation of the MAP-kinase family member Jun-kinase (JNK) (Varfolomeev and Ashkenazi 2004). Given the signal transduction pathways activated by this cytokine, it is not surprising that TNF-α is able to induce a variety of biological effects. Mouse TNF and TNF-R1 gene knockout studies have clearly shown that this cytokine plays a key role in protection against infection by bacterial, fungal and parasitic pathogens. One of the main functions of TNF-α consists in its capacity to induce the production and release of the pro-inflammatory cytokines, IL-1, IL-6 and IL-8. In initial studies carried out in vivo (Moldawer et al. 1989; Ulich et al. 1990) and in vitro (Akahane et al. 1987) evidence was provided that TNF-α exerted an inhibitory effect on erythropoiesis. In addition, in a phase I study of TNF-α in cancer patients a clear decrease in hemoglobin levels after 1 month of TNF-α treatment was observed. Subsequent in vitro studies, however, showed that the inhibitory effect of TNF-α on erythropoiesis at the CFU-E level was likely mediated by IFN-γ released by macrophages in response to TNF-α and not due to a direct effect of TNF-α on erythroid progenitors (Means et al. 1990; Rusten and Jacobsen 1995). However, a direct inhibitory effect of TNF-α on erythroid cells was supported by subsequent studies. In one study, a direct effect of TNF-α on BFU-E was suggested, mediated by the TNF-R1 (Rusten and Jacobsen 1995). In another study using preparations of virtually pure erythroid precursors derived from unilineage erythroid cultures it was shown that TNF-α exerts a moderate, but significant, inhibitory effect on the proliferation of immature erythroblasts (proerythroblasts and basophilic erythroblasts), associated with a slight induction of apoptosis, in the presence of EPO. Furthermore TNF-α, like other death receptor ligands, exerted an inhibitory effect on erythroid maturation (De Maria et al. 1999). A direct inhibitory effect of TNF-α on erythroid cells was also supported by a recent study showing that: (i) both murine and human CFU-E release TNF-α; (ii) the addition of neutralizing anti-TNF-α-antibody to cultures of human CD34+ cells stimulated with EPO increased the generation of erythroid cells; (iii) the number of BFU-E colonies was higher in the bone marrow of TNF-α−/− mice than in wt mice; (iv) the addition of TNF-α to TNF-α−/− mice elicited a significant inhibition of BFU-E colony formation, while TNF-α exerted only a slight inhibitory effect on wt mice BFU-Es (Jacobs-Helber et al. 2003). An inhibitory effect of TNF-α on the generation of erythroid cells from purified human CD34+ cells was also reported in another recent study (Xiao et al. 2002). In this study it was also shown that detectable expression of TNF-R1 was observed only during the initial stages of erythroid differentiation, while TNF-R2 expression was observed during all stages (Xiao et al. 2002). However, it is unlikely that the inhibitory effects of TNF-α on erythropoiesis could be mediated via TNF-R2 signaling, since this receptor lacks a death domain in its cytoplasmic tail. The inhibitory effect of TNF-α on erythroid maturation may also NF-κB induction (Xiao et al.

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2002). In fact, NF-κB activity is high in early erythroid progenitors and then declines at later stages of erythroid maturation. In fact, low NF-κB levels are required for the induction of the expression of erythroid-specific genes (Liu et al. 2003). Taken together, these observations suggest that TNF-α exerts an inhibitory effect on erythropoiesis and this effect could be physiologically relevant. An increased TNF-α production is involved in the inhibition of erythropoiesis observed in Fanconi anemia patients (Dufour et al. 2003). An increased TNF-α production seems to be involved also in the lack of response to EPO therapy observed in some patients with chronic kidney disease. In fact, patients with chronic kidney disease who persistently fail to respond to EPO therapy, express abnormally high levels of the pro-inflammatory cytokines TNF-α and IFN-γ, which are known to inhibit erythropoiesis (Macdougall and Cooper 2002; Cooper et al. 2003).

Effects of Fas-FasL pathway on normal erythropoiesis Activation-induced cell death (AICD) is the primary homeostatic mechanism used by the immune system to control T-cell responses, promote tolerance to self-antigens, and prevent autoimmunity. Following activation, T cells express Fas and FasL and become sensitive to Fas/FasL-mediated autocrine and paracrine apoptosis (Krammer 2000). Growing evidence indicates that the Fas/FasL system could also play a relevant role in the regulation of hematopoiesis and, particularly, of erythropoiesis. Fas is a 45-kDa, type I cell surface protein with an extracellular domain that binds to FasL and a cytoplasmic domain that transduces the death signal. Apoptosis is executed by the engagements and co-aggregation of Fasl with the Fas receptor on the cell surface followed by a series of intracellular molecular interactions that coordinate the hierarchical activation of caspases and cell death, as outlined above. FasL protein is expressed in three distinct molecular forms: (i) membranous form on the cell surface; (ii) membranous form stored in intracellular microvesicles, which are secreted into the intercellular milieu in response to various physiologic stimuli; (iii) soluble form (sFasL) generated from the cleavage of the membranous molecule by matrix metalloproteinases within minutes of cell surface expression. Membranous Fasl is the primary mediator of cell apoptosis through formation of trimers on the cell surface. In contrast, sFasL can have pro-apoptotic, anti-apoptotic or neutrophil chemiotactic functions. Initial studies on mice with mutations of either the Fas or FasL system failed to show significant modifications of the hematopoiesis. However, a re-evaluation of the hematopoiesis in Fas or FasL-deficient mice showed a striking extramedullary increase in hematopoietic progenitors, including both erythroid and nonerythroid progenitors. Furthermore, colony forming

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unit-spleen (CFU-S) and colony forming unit-culture (CFU-C) on peripheral blood and in the spleen clearly increased in these mice after birth (Schneider et al. 1999). These observations clearly indicate that the Fas/FasL system can affect hematopoiesis during both the fetal and adult life. These conclusions were supported by a recent study showing that mice with mutations of the Fas gene (lpr mice) and mostly of the FasL gene (gld mice) show a pronounced increase in the number of colony forming unit-granulocyte/ macrophage (CFU-GM) (Alenzi et al. 2002). The expression of Fas/FasL during erythroid differentiation, as well as the inhibitory effects of Fas activation, has been explored in detail. Initial studies were focused to evaluate Fas expression on immature hemopoietic progenitor cells and showed low Fas mRNA expression in CD34+ cells isolated from human bone marrow (Nagafuji et al. 1995; Takenaka et al. 1996). Immature primitive hematopoietic progenitors (CD34+/38−) from human fetal liver express significant amounts of Fas antigen, whereas the more mature progenitors (CD34+/38+) showed low Fas antigen expression (Barcena et al. 1996). Both TNF-α and interferon-γ (IFN-γ) induced a marked increase in Fas expression on CD34+ cells (Maciejewski et al. 1995; Nagafuji et al. 1995; Barcena et al. 1996). Interestingly, the expression of Fas antigen on CD34+ cells was greatly increased following the induction of the cycling of these cells with cytokines, such as SCF, IL-3 and granulocyte/ macrophage colony forming factor (GM-CSF) (Barcena et al. 1996). Other studies have explored the consequences of Fas activation in progenitor cells on the induction of apoptosis, proliferation and differentiation. Basically these studies have provided evidence that immature progenitor cells are resistant to Fas-mediated apoptosis. Particularly, studies carried out in mice have shown that Lin-/Sca+/cKit+ stem cells reveal little or no constitutive expression of Fas and are resistant to the apoptotic triggering by an anti-Fas agonistic antibody (Bryder et al. 2001). However, if these stem cells are induced to cycle by incubation with early-acting growth factors and are incubated in the presence of TNF-α, they exhibit a marked up modulation of Fas antigen expression and become sensitive to Fas-mediated apoptotic triggering (Bryder et al. 2001). These findings are also confirmed by studies carried out using human CD34+ cells derived from either peripheral blood or cord blood: these cells are found to be resistant to the apoptotic triggering elicited by either soluble FasL or anti-Fas agonistic antibody (Kim et al. 2002). However, if CD34+ cells are induced to proliferate in the presence of flt3 ligand, SCF and thrombopoietin, they become sensitive to anti-Fasmediated apoptosis. The resistance of quiescent CD34+ cells to Fas-mediated apoptosis seems to be related to the expression in these cells of elevated levels of c-FLIP (FLICE-inhibitory protein), a dominant negative inhibitor of caspase-8 (Kim et al. 2002). Other studies have directly evaluated Fas/FasL expression in erythroid cells at various steps of differentiation and maturation (Fig. 7). Initial studies

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Fig. 7. Kinetics of TRAIL, TRAIL-R1, TRAIL-R2, Fas and FasL expression during erythroid differentiation. Purified hemopoietic progenitors have been induced to unilineage erythroid differentiation; the developing erythroid cells have been harvested at different days of culture and processed for TRAIL, TRAIL-Rs, Fas and FasL by flow cytometry after labeling with specific monoclonal antibodies

based on a strategy focused on obtaining a population of CFU-E cells originated in vitro from BFU-E, showed that these cells constitutively express low levels of Fas, whose expression was potentiated by IFN-γ (Dai et al. 1998); interestingly, CFU-E-like cells possess low, but significant amounts of FasL, whose levels are potentiated by IFN-γ treatment and by differentiation (Dai et al. 1998). The expression of Fas and FasL was explored in detail by De Maria et al. (De Maria et al. 1999) using a selective system of unilineage culture of human erythroid cells starting from purified CD34+ peripheral blood cells. Using this cell culture system it was shown that Fas is clearly upregulated during initial steps of erythroid differentiation from BFU-E to CFU-E, reaching elevated levels of expression at the stage of immature erythroblasts (proerythroblasts and basophilic erythroblasts) and remaining expressed at significant levels up to terminal stages of erythroid maturation (orthochromatic erythroblasts) (De Maria et al. 1999) (Fig. 7). These findings

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were also confirmed through the analysis of erythroid cells present in normal bone marrow. Fas cross-linking was effective in inducing an apoptotic response only in immature erythroblasts, while mature erythroblasts are resistant to this triggering. In contrast, an opposite pattern of FasL expression was observed, this membrane-bound ligand being expressed in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL expressed on the membrane of mature erythroblasts was found to be functional in that it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-mediated cytotoxicity against immature Fas-positive erythroblasts, which was in part abrogated by high concentrations of EPO (De Maria et al. 1999). According to these findings, it was suggested that erythropoiesis is regulated according to a negative feedback mediated by mature and immature erythroblasts, whereby the former cells might exert a cytotoxic Fas-mediated effect on the latter cells within the erythroblastic island (Testa 2004). It is conceivable to assume that this feedback may operate in the presence of physiologic, low EPO concentrations, while it is inhibited when EPO concentrations are high and there is the need of high rate of erythroid cell production. As mentioned above, EPO was only in part able to protect immature erythroid cells from a strong triggering of the Fas-mediated apoptosis, as it is induced by Fas agonistic antibodies. There is, however, evidence that other growth factors, like SCF, may exert a more pronounced protective effect than EPO. In this context, an initial observation was derived from the studies of Lee et al. showing that anti-HLA-DR monoclonal antibody elicited a marked upregulation of Fas on primary human bone marrow cells, thereby increasing their susceptibility to Fas-mediated cell death. SCF partially antagonized Fas-mediated apoptosis of primary cells, which suggests that SCF protects hemopoietic precursor cells from Fas-mediated apoptosis (Lee JW et al. 1997). In a subsequent study it was shown that SCF might protect CFU-E from Fas-induced apoptosis: the protective effect of SCF was significantly more effective than the effect elicited by EPO. This protective effect of SCF involves the activation of Src-kinases (Nishio et al. 2001). In a recent study it was shown that the simultaneous addition of IFN-γ and FasL to CFU-E elicited a marked activation of caspase-8 and caspase-3 with a consequent apoptotic response; this phenomenon was greatly inhibited by SCF addition, via a mechanism involving the upmodulation of c-FLIP expression (Chung et al. 2003). In another set of studies the mechanisms through which Fas-mediated apoptosis exerts a negative control on erythropoiesis was explored in detail. These experiments take advantage on the observation that Fas triggering in the presence of high EPO concentrations elicited a low apoptotic response in immature erythroblasts, but induced a blockade of erythroid maturation. This last mechanism requires caspase activation. One of the major effects of

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caspase activation consisted in the degradation of the transcription factor GATA-1; the induction of GATA-1 degradation seems responsible for the Fas-mediated blockade of erythroid cell maturation since the transduction in erythroid progenitors of a caspase-resistant GATA-1 mutant resulted in a complete protection against Fas-mediated blockade of erythroid maturation. Interestingly, also the erythropoiesis blockade elicited by EPO deprivation was in part inhibited by the transduction of caspase-resistant GATA-1 in erythroid progenitors (De Maria et al. 1999). In a second study, evidence was provided that another transcription factor, Tal-1, is an additional target protein cleaved by activated caspases in erythroid cells. The transduction in hemopoietic progenitors of a Tal-1 mutant resistant to caspase cleavage protected erythroid cells from Fas-mediated inhibitory effect on erythroid maturation and cleavage of GATA-1. Interestingly, the expression of the Tal-1 caspase-resistant mutant in erythroid cells completely protects these cells from the apoptosis elicited by EPO deprivation (Zeuner et al. 2003). These observations indicate that the apoptotic effects and the inhibitory actions on cell differentiation elicited by EPO deprivation or by death receptor triggering involve as a necessary step the caspase-mediated cleavage of two transcription factors, Tal-1 and GATA-1, whose integrity and activity is essential for erythroid maturation and survival (Fig. 8).

Effects of TRAIL on normal erythropoiesis In addition to Fas and TNF-R1, erythroid cells express other death receptors, such as TRAIL-R1 and TRAIL-R2 (De Maria et al. 1999). The expression of these two membrane receptors was higher in immature than in mature erythroblasts: particularly, the highest TRAIL-R1 and TRAIL-R2 expression was observed in proerythroblasts and basophilic erythroblasts; in polychromatophylic erythroblasts the expression of these two receptors declined, and then almost completely disappeared in late orthochromatic erythroblasts (Fig. 7). TRAIL-R3 and TRAIL-R4 are not expressed at any stage of erythroid maturation (Secchiero et al. 2004). Furthermore, it was shown that the membrane-bound form of the TRAIL was also expressed in normal erythroblasts, during all stages of erythroid maturation. Therefore, the pattern of the TRAIL/TRAIL-R system in erythroid cells was highly comparable to that observed for the Fas/FasL system. The analysis of the biological effects of TRAIL on erythroid cells showed that immature erythroid cells are sensitive to the antidifferentiative (in the presence of EPO) or cytotoxic (at low EPO concentrations) effects induced by TRAIL following its interaction with the TRAIL-Rs expressed on these cells (De Maria et al. 1999). These findings were confirmed in a subsequent study, showing that hemopoietic progenitors as well as mature erythroblasts are resistant to the apoptotic effects induced by TRAIL (Zamai et al. 2000).

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Fig. 8. Negative regulation of erythropoiesis triggered by death receptor activation or by EPO deprivation. Both death receptor activation or growth factor deprivation induce the apoptotic cascade, with consequent activation of caspases that cleave the transcription factors GATA-1 and Tal-1. The cleavage of these transcription factors is responsible for either the maturation arrest or apoptosis of erythroid cells

The blockade of erythroid maturation induced by TRAIL-R stimulation involves the ERK1/2 activation, as suggested by two observations: TRAIL induced ERK1 and ERK2 phosphorylation; pharmacological inhibitors of the ERK pathway blocked the anti-differentiative effects induced by TRAIL (Secchiero et al. 2004). The mechanism of TRAIL-induced apoptosis of erythroid cells was also explored in experimental models of erythroid differentiation. In this context, studies in the erythroleukemia K562 cell line showed a sensitivity of these cells to TRAIL only after induction with hemin. The induction of TRAIL sensitivity was not related to the stimulation of expression of TRAIL receptors, but to a downmodulation of c-FLIP (Hetakugas et al. 2003). Interestingly, the expression of TRAIL-R1 may be upmodulated by ionizing radiations on erythroleukemic cells, but not on normal erythroblasts. According to this pattern of TRAIL-R1 modulation, TRAIL sensitizes the cytotoxic effects of ionizing radiations on erythroleukemia cells, but not on normal erythroid cells (Di Pietro et al. 2001).

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An increased production of TRAIL in the bone marrow represents one of the main pathogenetic mechanisms involved in the genesis of anemia observed in myeloma (Silvestris et al. 2002) and in myelodysplastic syndromes (Campioni et al. 2005).

Effects of IFN-g on normal erythropoiesis IFN-γ is the example of a cytokine that per se does not induce an apoptotic signaling, but may induce apoptosis of erythroid progenitors through an indirect mechanism involving upmodulation of both Fas and FasL, as mentioned above. Interestingly, recent studies clearly indicate that the inhibitory effect exerted by IFN-γ involves multiple members of the TNF superfamily, including FasL, TRAIL and TWEAK (Felli et al. 2005). In fact, only the combined neutralization of FasL, TWEAK and TRAIL was able to restore erythroid cell survival, proliferation and maturation in the presence of IFN-γ, indicating the simultaneous involvement of these three ligands as effectors of IFN-γ in erythropoietic inhibition. The inhibitory effect of IFN-γ on erythropoiesis is exerted at the level of early stages of erythroid differentiation: in in vitro assays, BFU-Es are inhibited by IFN-γ more than CFU-Es (Wang et al. 1995). In addition, IFN-γ upmodulates the level of expression and activates several caspases, including caspase-8 and -3, in erythroid progenitors (Dai and Krantz 1999). Paradoxically, IFN-γ may exert a protective effect on apoptosis on erythroid cells at later stages of differentiation (Choi et al. 2000). Ceramide, an intracellular second messenger produced by sphingommyelin hydrolysis has been involved as the mediator of apoptosis induced by a number of cytokines, including IFN-γ and TNF-α. Ceramide induces an inhibition of CFU-E colony formation. Interestingly, the ceramide antagonist sphingosine-1-phosphate significantly reversed the CFU-E colony inhibition induced by IFN-γ. These observations suggest that ceramide is one of the key mediators of the inhibition of CFU-E colony formation by IFN-γ (Dallalio et al. 1999).

Role of caspases in erythroid maturation The caspases are the central executioners of the apoptotic process. There is evidence, however, that these proteolytic enzymes may display, in addition to a role in the apoptotic process, also a function in cell differentiation/maturation. The activation of some caspases is required for the process of normal erythroid differentiation. Recent studies have provided evidence that the “spontaneous” activation of caspases was observed during late steps of differentiation in various hemopoietic lineages, including monocytic, megakaryocytic and erythroid lineages.

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During the process of normal maturation of monocytes to macrophages an activation of caspases 3 and 9 was observed, associated with release of cytochrome C from the mitochondria and cleavage of the protein acinus, but without induction of cell death. The blockade of the caspase activity when using synthetic cell-permeable inhibitors blocked the cell maturation process (Sordet et al. 2002). Similar observations have been made in megakaryocytic cells, where caspase activation occurred at two stages of maturation: (i) a first step of caspase activation (caspase 3 and 9 became spontaneously activated) occurred before proplatelet activation and was limited only to some cellular compartments; (ii) a second step of diffuse caspase activation occurred at the end of the maturation process after platelet release. The first process of caspase activation did not lead to cell death, but the second wave was associated with cell death (Botton et al. 2002). Importantly, the addition of caspase inhibitors to megakaryocytic cultures inhibited platelet release. In line with these observations transgenic mice overexpressing the antiapoptotic gene Bcl-XL exhibited impaired platelet fragmentation (Kaluzhny et al. 2002). Several lines of evidence indicate that caspase activation occurs also during the process of erythroid maturation and could play an important role in this process. In this context, an initial study showed that caspases 1, 2, 3, 5, 6, 7, 8 and 9 are expressed in erythroid cells. The level of procaspase 2, 3 and 8 were markedly higher in immature erythroblasts than in mature erythroblasts (Gregoli and Bondurant 1999). As it is expected, EPO deprivation elicited a marked increase in caspase activation. In a second study based on a peculiar cell culture system which implies a first amplification of CD34+ cells in the presence of IL-3, SCF and IL-6, after 7 days, erythroid progenitors selected according to the CD36 positivity were cultured or additional 7 days in the presence of SCF, IL-3, EPO and TGFβ1. The CD36+ erythroid progenitors underwent a progressive differentiation during the second week of culture. A transient caspase-3 and -7 activation occurred during erythroid maturation corresponding to proerythroblasts and basophilic erythroblasts; this phenomenon, however, was transient in that the caspase activation regressed at later stages of erythroid maturation. These caspases, transiently activated through the mitochondrial pathway, cleaved proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus) without inducing cell death. Inhibitors of caspases, such as z-VAD, added to erythroid cultures just before the moment of caspase activation elicited a block of erythroid maturation at the basophilic stage (Zermati et al. 2001). The selective blockade of caspase-3 by transfection of small interfering RNA (siRNA) directed to caspase-3 in differentiating erythroid precursors elicited the inhibition of erythroid maturation (Carlile et al. 2004). These findings have also been confirmed in murine erythroblasts. Furthermore, it was shown that the overexpression of Raf-1, which prevents caspase activation, impairs erythroid maturation by reducing differentiation-associated caspase activation. An opposite phenomenon was observed in Raf-1−/− mice (Kolbus et al. 2003).

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The terminal maturation of erythroblasts is characterized by enucleation, a process of nuclear extrusion, preceded by nuclear chromatin condensation, reduction of nuclear size and migration of the nucleus to the plasma membrane. In the two other cell types showing enucleation at the end of maturation, keratinocytes and lens epithelial cells, caspase activation is required for enucleation. In contrast, erythroblast enucleation does not require caspase activation. More particularly, erythroblast enucleation does not involve a marked reorganization of nuclear structures by activated caspases (Krauss et al. 2005). Therefore, erythroblast enucleation, the terminal step of erythroblast maturation, occurs without evidence of major activation of apoptotic proteins. A possible involvement of caspase in the control of erythroid cell production is also supported by the analysis of the phenotype of mice with the targeting of the genes encoding some of these caspases. In this context, the most interesting findings were originated from the analysis of caspase-8−/− mice (Varfolomeev et al. 1998). Caspase-8 deficiency resulted in embryonic lethality, associated with two salient features: impaired heart muscle development and congested accumulation of erythrocytes (Varfolomeev et al. 1998). These features resemble the phenotype reported in mice with targeted disruption of FADD gene (Yeh et al. 1998; Zhang J et al. 1998). Recent studies show a nonapoptotic role of caspase-8 in the control of hemopoietic differentiation (Kang et al. 2004). The analysis of the defects in the erythroid lineage observed in these animals, however, was limited to the analysis of erythrocytes and it is, therefore, impossible to know whether mice with caspase8 and FADD deficiency exhibit abnormalities of erythroid cell maturation, in addition to an increased expansion of the number of mature erythrocytes. It is of interest that in a recently described family exhibiting a complete deficiency of caspase-8, no hemopoietic abnormalities have been described (Chun et al. 1998). The discrepancy between the phenotype of caspase-8 deficiency in humans and mice could be related to the function in humans of caspase-10, the closest paralogue of caspase-8 (caspase-10 has no known orthologue in mice). In addition to caspases, recent studies suggest a possible role of p53 during late stages of erythroblast maturation. Particularly, an overexpression of the p53 protein was observed in late orthochromatic erythroblasts: this p53 activation may be related to the nuclear degradation occurring in these cells, without a fully executed apoptotic process because of exhaustion of caspase3/7 (Peller et al. 2003).

Apoptotic mechanisms in mature RBC The occurrence of apoptotic processes and, particularly, of a possible activation of caspases was explored also in mature RBC. These cells lack the

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nucleus and after a life-span of about 120 days undergo a process of senescence and are then removed from the circulation. During this process senescent RBC undergo typical morphological changes, such as shrinkage as the consequence of a progressive release of microvesicles from the cell membrane and shape transformation from a discocyte to a spherocyte form, enabling first their recognition and then their phagocytosis by macrophages. In addition to these changes, there is a series of modifications at the level of the cell membrane, such as a progressive loss of the structural membrane protein spectrin and the loss of membrane asymmetry associated with the externalization of phosphatidylserine residues, resembling those observed in the process of apoptotic cell death in nucleated cells. These observations prompted studies aimed to evaluate whether mature RBC possess an apoptotic machinery similar to that observed in the majority of nucleated cells. In this context, preliminary experiments carried out in human RBC showed that these cells do not undergo programmed cell death when either treated with staurosporine and cycloheximide or when cultured in the absence of serum, conditions that induce apoptosis in all types of nucleated cells. According to these observations it was concluded that mature RBC do not have an apoptotic machinery and therefore do not undergo an apoptotic programme. This issue was re-explored on chicken RBC showing that serum deprivation or treatment with staurosporine and cycloheximide induces the death of these cells; however, although these erythrocyte deaths displayed many features that are typical of apoptotic cells, they are not blocked or inhibited by different types of caspase inhibitors (Weil et al. 1998). According to these observations it was concluded that chicken RBC die without apparently activating caspases. More recently, the problem was reevaluated focusing the attention to human RBC. In this context, it was observed that mature erythrocytes contain considerable amounts of caspase-3 and caspase-8, while other main components of the apoptotic machinery such as caspase-9, Apaf-1 and cytochrome C are absent. Although present at relatively high levels, caspase3 and caspase-8 were not activable by various types of proapoptotic stimuli (Berg et al. 2001). However, recent studies suggest a possible role for caspase3 in erythrocyte aging. First, it was observed that activated caspase-3 can be detected in old, but not in young RBC; second, this activated caspase-3 was able to cleave cell membrane band 3, disrupting its interaction with the peripheral membrane protein 4.2 (Mandal et al. 2003). These observations suggested that some caspases activated during RBC aging could participate in the degradation of crucial erythrocyte membrane proteins involved in the maintenance of shape and function. In line with these findings it was observed that loss of band 3 was associated with premature erythroid cell death (dyserythropoiesis) (Berg et al. 2001). Since an increase in Ca2+ concentration was associated with the aging of RBC and was considered as one of the molecular mechanisms responsible

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for the senescence of these cells, additional studies explored whether the induction of an increase of Ca2+ concentration in RBC could trigger an apoptotic or apoptosis-like response. Following the induction of an increase of cytosolic Ca2+ concentration RBC underwent a rapid self-destruction process exhibiting several apoptotic features, such as plasma membrane microvesiculation, and phosphatidylserine externalization, a process which culminated either in RBC destruction or, in the presence of macrophages, in macrophage phagocytosis with subsequent destruction. This process was inhibited by cysteine protease inhibitors, but not by caspase inhibitors. Importantly, cysteine protease inhibitors allowed erythrocyte survival both in vitro and in vivo (Bratosin et al. 2001). According to these findings it was proposed that an increase in intracellular Ca2+ concentration in RBC determines the activation of the cysteine protease calpain that mediates spectrin cleavage and other morphological changes leading to cell shrinkage (Fig. 8).

Role of Deoxyribonuclease (DNase) IIa in erythroid maturation During terminal stages of erythroid maturation, erythroblasts exhibit several features typical of an apoptotic process, such as reduction of cell size, withdrawal from the cell cycle, nuclear condensation and nuclear expulsion. The progressive transition from the rapidly proliferating compartment of immature erythroblasts to the terminal slowly proliferating compartment of mature erythroblasts is regulated by growth inhibitory genes (Aerbajinai et al. 2004). Many endonucleases have been implicated in the apoptotic process and particularly in DNA digestion occurring during apoptosis, such as caspaseactivated DNase (CAD), DNaseI and DNaseII. Two types of DNase II exist in mammalians, designated as DNase IIα and DNase IIβ. DNase IIα is ubiquitously expressed, while DNase IIβ is expressed only in the salivary gland. Recent studies have shown a role for this enzyme in erythroid maturation, particularly in the process of enucleation occurring at very late stages. To evaluate this, mice deficient in DNase IIα expression were generated. These animals died of severe anemia and showed a marked decrease of circulating RBC (i.e. they have only 1/10 of the physiological number of circulating erythrocytes), associated with the presence in the circulation of definitive nucleated RBC, a cell type normally present only in bone marrow and not in peripheral blood. The reduced presence of anucleated RBC in the peripheral blood of DNase IIα−/− mice could not be related to a defect in erythroid commitment, since these animals exhibited in their fetal liver a normal number of both BFU-E and CFU-E (Kawane et al. 2001). However, mutant fetal liver erythroblasts showed a normal maturation after transfer into irradiated normal mice, thus suggesting that the defect of DNase II−/− erythroblasts was not intrinsic to these cells. In fact, histological experiment suggested that the source of DNase II responsible for the rescue of the maturation of

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DNase II−/− erythroblasts was represented by macrophages. It was in fact suggested that central macrophages present in erythroblastic islands might represent the source of DNase IIα (Kawane et al. 2001). According to these findings it was concluded that DNase II present in macrophages was responsible for digesting nuclear DNA expelled from maturing late erythroblasts. This conclusion was directly supported by the analysis of a second knockout of DNase IIα. The generated DNase II−/− mice died at birth exhibiting at the level of the liver macrophages large DNA bodies resulting from the engulfment of undigested nuclei extruded from erythroblasts (Krieser et al. 2002). According to these findings, macrophages appear to play an essential role in erythroid maturation through a biochemical mechanism involving DNase IIα. These observations indicate that a regulated process of DNA fragmentation is strictly required for normal tissue homeostasis (Zhang and Xu 2002), and that the enucleation event occurring during late erythroid maturation is a regulated process requiring extensive signaling between erythroblasts and macrophages.

Role of AKT pathway in the apoptotic control of the erythroid lineage Phosphorylation and activation of the serine/threonine kinase PKB controls fundamental processes such as cell cycle progression, survival, apoptosis and mRNA translation. The AKT/PKB pathway involves a cascade of activation/signaling steps (Brazil et al. 2002; Djordjevic and Driscoll 2002; Nicholson and Anderson 2002; Vivanco and Sawyers 2002): – the first step involves the activation and modulation of phosphatidylinositol-3-kinase (PI3K); PI3K may be activated through different mechanisms, involving either the binding of the complex p85/p110 (p85 is the regulatory subunit, while p110 is the catalytic subunit) to phosphorylated tyrosine residues present at the level of the cytoplasm domain of activated receptor tyrosine kinases or the interaction with members of the small GTPase family, such as Ras; – the second step consists in the generation of 3′-phosphorylated inositol lipids (PIP3) which function as classical second messengers by binding proteins that harbor PIP3-binding domains. PIP3 binds the binding domain of the serine/threonine PKB/AKT, with its consequent activation. The activated PKB/AKT phosphorylates several components of the apoptotic machinery and through this mechanism induces anti-apoptotic effects. Particularly, PKB/AKT promotes cell survival by multiple mechanisms: i) phosphorylation and inactivation of the proapoptotic protein Bad; ii) maintenance of mitochondrial integrity; iii) decrease of the expression of death genes, such as FasL and Bim, via the phosphorylation of forkhead transcription factor FKHR-L1: phosphorylated FKHR-L1 is exported from the nucleus to the cytoplasm where it is sequestered by 14-3-3 pro-

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teins; iv) increase of the transcription of survival genes through the activation of NF-κB and CREB transcription factors (Djordjevic and Driscoll 2002; Nicholson and Anderson 2002; Vivanco and Sawyers 2002). Furthermore, recent studies indicate that the PKB/AKT pathway is involved in the upregulation of surviving expression observed during cell cycle entry (Fukuda et al. 2002). Survivin pertains to the family of inhibitors of apoptosis protein (IAP) that inhibit apoptosis by inactivating several caspases (Altieri 2003). Finally, PI3K is involved in Gab2 phosphorylation. Both EPO and SCF activate phospatidylinositol-3.kinase (PI3K) and its target PKB. However, compared with EPO, SCF is much more potent in inducing PKB phosphorylation (von Lindern et al. 2004). Both the catalytic p110 and the regulatory p85 subunits are required for PI3K activity. P85−/− mice are pale with a marked reduction in RBC in their peripheral blood (Huddleston et al. 2003). Erythroid progenitors BFU-Es and CFU-Es are reduced in the fetal liver of these animals and display reduced proliferative response to both EPO and SCF (Huddleston et al. 2003). A molecular balance between the regulatory and catalytic subunits of PI3K is therefore required for optimal control of erythropoiesis. Initial studies on erythropoiesis and PKB/AKT signaling were based on the use of chemical inhibitors of PI3K. In a first set of studies, a PI3K inhibitor called wortmannin was used: basically these studies provided evidence that the inhibitor blocked the PI3K activation elicited by EPO and considerably decreased the proliferation of erythroid precursors expanded in vitro (Sui 1998). Using a more specific and potent PI3K inhibitor (LY294002), an inhibitory effect on erythroid cell proliferation was confirmed, but it was also shown that PKB/AKT inhibition elicited the apoptotic death of a significant proportion of erythroid precursors (Haseyama et al. 1999). In a subsequent study the effects of the PI3K inhibitor LY294002 at earlier stages of erythroid differentiation were explored, showing a marked inhibitory effect on the differentiation of CD34+ to erythroid precursors: particularly, the addition of the inhibitor to CD34+ cultures grown in the presence of EPO and SCF resulted in a marked inhibition of the generation of glycophorin-A+ cells (Myklebust et al. 2002). The essential role of the PI3K in the mechanism of induction of erythroid proliferation and survival was also confirmed in a recent study showning that three different mechanisms equally active in erythroid cells are responsible for PI3K activation: direct association of the PI3K to the EPO-R; phosphorylation of Gab via either Tyr 343 or Tyr 401 of the EPO-R; phosphorylation of the IRS2 adaptor protein (Bouscary et al. 2003). The activation of PKB/AKT following EPO stimulation requires a normal protein kinase C (PKC) activity, as suggested by the experiments carried out with PKC inhibitors (Von Lindern et al. 2000). The Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors is an important effector of PKB in regulating apoptosis and cell cycle

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progression. Members of this subfamily, FoxO4, FoxO1a and FoxO3a (FKHR-L1) are directly phosphorylated by PKB, leading to cytoplasmic retention and inhibition of their transcriptional activity required for the control of the expression of several genes involved in cell cycle control, cell death and oxidative stress (Burgering and Kops 2002). There is evidence that the activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal (Dijkers et al. 2002). FKHR-L1 is expressed in erythroid cells, and its expression progressively decreases during erythroid maturation (Kashii et al. 2000). In immature erythroid precursors, EPO rapidly phosphorylated FKHR-L1 through a PI3K dependent pathway (Kashii et al. 2000). In addition to FKHR-L1, also FoxO3a exhibits an important role in erythropoiesis. In fact, FoxO3a expression and nuclear accumulation increased during erythroid differentiation. Enforced expression of FoxO3a in erythroid progenitors accelerated differentiation of these cells to erythroblasts (Bakker et al. 2004). An additional target of PKB/AKT is the glycogen synthetase kinase-3 (GSK 3), which is a serine/threonine kinase involved in metabolic processes (glycogen metabolism) and also implicated in apoptosis regulation in primary human erythroid progenitors. The activity of GSK 3 is suppressed by EPO, as well as by SCT. Importantly, the inhibition of GSK3 using specific chemical inhibitors prevents apoptosis of erythroid progenitors by EPO deprivation (Somervaille et al. 2001). Taken together, these observations implicate at least two PKB/AKT targets, FKHR-L1 and GSK3, in the regulation of the survival of erythroid cells.

Further signaling pathways involved in the apoptotic regulation of erythroid cells In addition to the PKB/AKT, other signaling pathways seem to play a role in the apoptotic control of erythroid cells. In this context, it was shown that the transcription activator protein 1 (AP1) could be involved in the regulation of apoptosis mediated by EPO in erythroid cells. AP1 is a transcriptional complex comprising members of the Jun and of the Fos families of transcription factors. Evidence was provided both in primary human hemopoietic progenitors and in EPO-dependent cell lines that both EPO addition or deprivation induces AP-1 activity which seems to be required both for induction of erythroid proliferation or apoptosis. The analysis of the composition of AP-1 transcriptional complex in these two conditions showed an interesting finding: JunB is present in erythroid cells triggered to apoptosis by EPO deprivation, while c-Jun was present in erythroid cells induced to proliferate (Jacobs-Helber et al. 2000). Finally, a recent study showed that one of the two negative regulators of EPO-R signaling, the small cytokine-inducible SH2-domain (CIS) containing protein plays a role in the control of erythroid cell apoptosis. CIS belongs to the family of suppressor of cytokine signaling

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proteins and acts reducing the Stat-5 activation elicited by EPO. The enforced expression of this protein in erythroid progenitors not only reduced their proliferation, but also induced a significant level of apoptosis (Ketteler et al. 2003). Due to these findings it may be that the level of CIS protein is involved in the control of both the proliferation and survival of erythroid progenitors.

Summary Erythropoiesis is a complex multistep process encompassing the differentiation of hemopoietic stem cells to mature erythrocytes. The stem cells first differentiate to early erythoid progenitors, BFU-E, then to late erythroid progenitors, CFU-E, and finally to morphologically recognizable erythroid precursors. A key event in the the late stage of erythropoiesis is nuclear condensation, which is followed by extrusion of the nucleus to produce enucleated reticulocytes and finally mature erythrocytes. During the differentiation process, the cells became progressively sensitive to EPO, which controls both the survival and proliferation of erythroid cells. A normal homeostasis of the erythropoietic system requires an appropriate balance between the rate of erythroid cell production and RBC destruction. Anumber of apoptotic and antiapoptotic mechanisms play a major role in the control of erythropoiesis both under physiologic and pathologic conditions. Withdrawal of EPO or stimulation of death receptors such as Fas or TRAIL-Rs leads to activation of a subset of caspase-3, -7 and -8, which then cleave the transcription factors GATA-1 and TAL-1 and trigger apoptosis. In addition, there is evidence that a number of caspases are physiologically activated during erythroid differentiation and are functionally required for erythroid maturation. Several caspase substrates are cleaved in differentiating cells, including the protein acinus whose activation by cleavage is required for chromatin condensation. The studies on normal erythropoiesis clearly indicate that immature erythroid precursors are sensitive to apoptotic triggering mediated by activation of the intrinsic and extrinsic apoptotic pathways. These apoptotic mechanisms are frequently exacerbated in some pathologic conditions, associated with the development of anemia (i.e. thalassemias, multiple myeloma, myelodysplasia, aplastic anemia). The considerable progress in our understanding of the apoptotic mechanisms underlying normal and pathologic erythropoiesis may offer the way to improve the treatment of several pathologic conditions associated with the development of anemia.

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Chapter 2

Biology of EPO and EPO-receptor C. Lacombe1,2 and P. Mayeux1 1

Université Paris-Descartes, Faculté de Médecine, INSERM, CNRS, Institut Cochin (U567-UMR 8104) Paris, France 2 Service d’Hématologie biologique, AP-HP, Hôpital Cochin, Paris, France

Introduction Patients undergoing chemotherapy for cancer are at risk of developing anemia, and recombinant human EPO is an interesting alternative to replace transfusions of allogenic red blood cells in this setting. The role of EPO, a 34 kDa glycoprotein hormone, is to control red blood cell production through the promotion of survival and proliferation of the erythroid progenitors in the bone marrow. EPO is the hematopoietic growth factor which is acting specifically on the late erythroid progenitors, so-called CFU-E (colony-forming unit-erythroid). These cells correspond to the last amplification compartment of the erythroid lineage and give rise to the erythroblasts in the bone marrow. Because the main function of red cells is to transport oxygen from the lungs to the peripheral tissues, the regulation of EPO production is an important feature of the control of tissue oxygenation. Accordingly, EPO is the only hematopoietic growth factor the production of which is regulated by hypoxia. EPO acts through a specific receptor (EPO-R) belonging to the family of the hematopoietic growth factor receptors. Activation of the EPO-R by its ligand leads to the tyrosine phosphorylation of numerous proteins into the target cell; among these proteins, some migrate to the nucleus, where they stimulate the transcription of specific target genes. This article will review the regulation of EPO production, the structure of the EPO-R and the EPO-induced intracellular signaling events. We will also describe the mechanisms of EPO and EPO-R internalization and degradation.

Role of EPO in erythropoiesis Cultures of hematopoietic progenitors in semi-solid media have shown that the main targets of EPO are the late erythroid progenitors and especially the

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cells which are called colony-forming unit-erythroid (CFU-E) (Gregory and Eaves 1978). EPO and EPO-R gene disruptions in mice confirmed that EPO stimulation was absolutely required for survival and proliferation of CFU-E (Wu et al. 1995; Lin et al. 1996). Moreover, these experiments showed that EPO stimulation was not necessary for the commitment of the progenitors in the erythroid lineage. Indeed, both early erythroid progenitors, so-called BFU-E (burst-forming unit-erythroid), and CFU-E were produced to normal levels in EPO or EPO-R null mice (Wu et al. 1995; Lin et al. 1996), demonstrating that EPO is not involved in the determination of the erythroid lineage. EPO is able to sustain the proliferation of several hematopoietic cell lines either naturally expressing the EPO-R, such as HCD57 (Spivak et al. 1991) or UT7 (Komatsu et al. 1991) or after ectopic expression of the EPO-R (D’Andrea et al. 1991; Quelle and Wojchowski 1991). These cells, as well as primary erythroid cells, undergo apoptosis after EPO deprivation, thereby showing that EPO requirement is mandatory for the erythroid lineage. However, the specificity of action of EPO on erythroid progenitors is mainly due to the fact that, during erythroid differentiation, only EPO-R are present at the cell surface. Indeed, it has been shown that ectopic expression and stimulation of other cytokine receptors by their cognate ligands allowed the proliferation and maturation of erythroid progenitors in the absence of EPO (Socolovsky et al. 1998; Fichelson et al. 1999; Ghaffari et al. 1999). Similarly, the expression of anti-apoptotic proteins such as Bcl2 delayed apoptosis, allowed partial erythroid differentiation, although it did not sustain cell proliferation (Lacronique et al. 1997; Lesault et al. 2002). Thus, the overall action of EPO is to protect from apoptosis and to induce the proliferation of CFUE progenitors rather than to play a specific role in erythroid differentiation.

Regulation of EPO production EPO production is regulated by hypoxia that leads to an increase of the level of gene transcription (Schuster et al. 1989), there are no preformed stores of EPO. The identification of the transcription factor hypoxia-inducible factor 1 (HIF-1) as a DNA transcriptional complex has been a critical step to understand the regulation of EPO production. Affinity purification showed that HIF-1 was composed of two subunits (Semenza and Wang 1992; Wang et al. 1995; Wang and Semenza 1995). Molecular cloning of HIF-1 by Semenza and colleagues (Wang et al. 1995) showed that the DNA binding complex was made of two basic-loop-helix PAS proteins called HIF-1α and HIF-1β. HIF1β had previously been identified as the aryl hydrocarbon nuclear receptor translocator (ARNT), a molecule involved in the xenobiotic response. In contrast, HIF-1α was a new member of this family of PAS (Per-Arnt-Sim) pro-

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teins. The mechanism of regulation by hypoxia was first studied in hepatoma cells which produced EPO. It was further shown that identical responses could be obtained in a large array of non-EPO-producing cells and that the system of gene regulation by oxygen was widespread from mammalian to insect cells (Maxwell et al. 1993; Wang and Semenza 1993). Many genes have been identified as targets of HIF-1 function; these include, in addition to EPO, vascular endothelial growth factor (VEGF), several glycolytic enzymes, glucose-transporter 1, inducible nitric oxyde synthase, heme oxygenase and transferrin (Maxwell et al. 1993; Wang and Semenza 1993). These data show that cellular response to hypoxia is an important physiological process and that a similar mechanism for oxygen sensing and signal transduction must be shared by many tissues and cells (Bunn and Poyton 1996). The major mechanism of regulation of HIF-1α involves the ubiquitinproteasome system: HIF-1α is constitutively degraded in normoxia, while it accumulates rapidly following exposure to hypoxia (Salceda and Caro 1997). The von Hippel-Lindau (VHL) tumor-suppressor protein (pVHL) has been linked to the regulation of the transcription factor HIF-1 (Maxwell et al. 1999). Wild-type pVHL is a component of an E3 ubiquitin-ligase complex that transfers ubiquitin onto substrates to be degraded, and the α-subunit of HIF-1 is the ubiquitination target for VHL (Ohh et al. 2000). Degradation of HIF-1α under normoxic conditions is triggered by hydroxylation of a proline residue 564 located within the oxygen-dependent degradation domain (ODD) of the protein (Hon et al. 2002). A family of oxygendependent prolyl hydroxylases is responsible for the modulation of HIF stability (Epstein et al. 2001; Semenza 2001). In addition, the carboxyl-terminal transactivation domain (C-TAD) of HIF-1α is able to recruit coactivator complexes such as p300/CBP only under hypoxic conditions. This regulation also involves an oxygen-dependent hydroxylation event targeted to a conserved asparagine residue (Lando et al. 2002). The VHL gene is inactivated in 80% of sporadic clear-cell renal carcinoma; these tumors lacking functional pVHL fail to degrade HIF-1α, which stimulates the transcription of a series of hypoxia-responsive genes, among which VEGF plays an important role in tumor angiogenesis (Wiesener and Eckardt 2002). More recently, Chuvash polycythemia has been described as a congenital defect of oxygen homeostasis due to an homozygous mutation in VHL gene. An Arg200Trp substitution impairs the interaction of VHL with HIF-1α, thus reducing the rate of degradation of HIF-1α and resulting in increased expression of downstream target genes including EPO (Ang et al. 2002).

Structure of the EPO receptor EPO acts on its target cells through specific membrane receptors. They are mainly expressed at the CFU-E stage, receptor expression then decreases

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with erythroid maturation (Mayeux et al. 1987). The number of EPO-R at the cell surface of normal or transformed erythroid cells is low: around one thousand per cell (reviewed in D’Andrea and Zon 1990). EPO-R are present on the surface of erythroid cells (Broudy et al. 1991), on megakaryocytes (Fraser et al. 1989), on endothelial cells (Anagnostou et al. 1994), neuronal cells (Masuda et al. 1993) and probably many other cell types according to the wide expression of its mRNA (Suzuki et al. 2002). The EPO-R cDNA has been cloned by A. D’Andrea (D’Andrea et al. 1989) and was shown to encode a single membrane-spanning protein of 507 amino-acids which does not possess catalytic activity in its intracellular region. This receptor belongs to the cytokine receptor family. Most of these receptors form multimeric complexes; several chains have been cloned for the receptors for IL-2 to IL7, for the GM-CSF and the leukemia inhibitory factor (LIF) receptors. The 66 kDa chain cloned in the EPO-R is responsible for intracellular signalling, since the transfection of this protein in hematopoietic cells such as Ba/F3, 32D or DA3, which do not possess EPO-R at their cell surface, allows their proliferation in response to EPO alone (Gobert et al. 1995b). However, chemical cross-linking experiments with 125 iodine-labelled EPO have detected at the surface of erythroid progenitors two additional proteins of 85 and 100 kDa, respectively, which are not recognized by anti p66 antibodies (Mayeux et al. 1991). These proteins probably belong to the receptor complex but are not able to bind to the ligand. Their cloning will be required to better understand their specific role. The fixation of EPO on its cognate receptor leads to dimerization of the p66 EPO-R as shown by cristallization of the complex EPO/EPO-R (Syed et al. 1998). One EPO-R molecule binds to the ligand with a rather high affinity (Kd = 1 nM), the second receptor molecule binds to the complex with a lower affinity (Kd = 1 μM) and both associations give a high-affinity binding (Kd = 160 pM). Thus, EPO is a bivalent molecule for the fixation of the EPO-R. Recently, the presence of EPO-R has been reported on the surface of several cells not belonging to the erythroid lineage. Therefore, it appears that the anti-apoptotic role of EPO is not restricted to erythropoiesis but is enlarged to many other tissues of normal origin. The role of EPO-R expression on tumor cells is the subject of a separate chapter in this book.

EPO-induced intracellular signalling As mentioned above, the dimerization of the EPO-R after binding to one molecule of EPO leads to its activation and subsequent downstream intracellular signalling. Many groups showed that the EPO-induced activation led to the rapid tyrosine phosphorylation of a number of proteins, even though the EPO-R does not possess endogenous tyrosine kinase activity. The two Jak2 tyrosine kinase molecules are pre-associated to the EPO-R. This asso-

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ciation between Jak2 and EPO-R occurs in the endoplasmic reticulum and seems to be required for the maturation and expression of the receptor at the cell surface (Huang et al. 2001). The fixation of EPO on its receptor leads to a change of conformation of the complex; the two Jak2 molecules become positioned in sufficient proximity for their reciprocal transphosphorylation and activation (Remy et al. 1999). Activated Jak2 proteins in turn phosphorylate the EPO-R tyrosine residues (Dusanter-Fourt et al. 1992, 1994). These phosphorylated tyrosines become secondary binding sites for signalling proteins containing SH2 (SRC homology 2) domains. Thus, a complex of signalling proteins is generated around the dimerized and activated receptor.

Signalling pathways activated by EPO The PI 3-kinase/Akt pathway PI 3-kinase is associated to EPO-R in response to EPO stimulation (Mayeux et al. 1993). It was first reported that one SH2 motif of the PI 3-kinase p85 subunit was bound to the last tyrosine residue of the EPO-R (Damen et al. 1995). Other adaptor mechanisms of PI 3-kinase activation have since been described: two adaptor proteins, IRS2 (Verdier et al. 1997) and GAB1 (Lecoq-Lafon et al. 1999) are phosphorylated following EPO stimulation and associate with PI 3-kinase. We demonstrated that these three alternative pathways independently led to EPO-induced activation of PI 3-kinase (Fig. 1). Ly294002, a specific inhibitor of PI 3-kinase activation inhibits EPOinduced cell proliferation, thereby suggesting that the PI 3-kinase pathway plays an important role in the mode of action of EPO. PI 3, 4, 5 trisphosphate, a metabolite of the PI 3-kinase pathway, activates the serine/threonine kinase AKT which is known to play a major role in the inhibition of cellular apoptosis (Franke et al. 1997). We showed that PI3K signalling occurred through modulation of the E3 ligase SCFSKP2 which downregulated p27Kip1 inhibitor via proteasome degradation. Thus, the activation of PI 3-kinase in response to EPO stimulation is an important event, contributing to the inhibition of apoptosis of erythroid progenitors and required for their proliferation (Bouscary et al. 2003).

The Ras/MAP kinase pathway Ras, Raf and MAP kinase proteins are all activated by EPO (Gobert et al. 1995a). The adaptor proteins SHC and Grb2 are associated to the EPO-R, together with the tyrosine phosphatase SHP-2, which is also able to bind Grb2 (Tauchi et al. 1995). The Ras/MAP kinase pathway could be activated by EPO via several different mechanisms; however, these mechanisms have

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Epo

Jak-2

IRS-2

Y1 Y2

GAB1

PI 3-kinase

Y8

Fig. 1. Mechanisms of PI 3-kinase activation by EPO. The IRS2 adaptor is constitutively associated to the proximal part of the intracellular EPO-R domain. After EPO stimulation, its phosphorylation leads to fixation of PI 3-kinase. Phosphorylation of Y1 and Y2 tyrosine residues of the EPO-R allows GAB1 fixation and its phosphorylation. After tyrosine phosphorylation, GAB1 associates with the PI 3-kinase. The third possibility of PI 3-kinase activation is its direct fixation to the last phosphorylated tyrosine of the EPO-R

not been definitively identified. This pathway is also involved in EPO-induced cell proliferation (Damen and Krystal 1996).

The STAT pathway The STAT (Signal Transducer and Activator of Transcription) proteins are transcription factors activated in response to several cytokines (Ihle 1995). EPO activates the two isoforms of STAT5, STAT5a and STAT5b (Gouilleux et al. 1995; Pallard et al. 1995). The STAT proteins bind to the Tyr 343 and 401 of the EPO-R, they become phosphorylated and activated and translocate into the nucleus (Gobert et al. 1996). The role of STAT transcription factors during EPO stimulation has been a matter of debate: some reports established a correlation between STAT activation and cell proliferation (Chrétien et al. 1996), whereas others attributed a role for STAT in erythroid differentiation (Wakao et al. 1997). A double knock out for STAT5a and STAT5b genes did not lead to any major defect of erythropoiesis (Teglund et al. 1998). It was further shown that STAT5 was essential for the high erythropoietic rate during fetal development, because it bound to the promoter of the Bcl-X gene and played a crucial role in EPO-R antiapoptotic signaling (Socolovsky et al. 1999).

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Pathways leading to signalling arrest The signal of activation which results from stimulation by a cytokine needs to be terminated by additional pathways leading to signal interruption. Several proteins have been recently isolated that play a negative role in EPOinduced signal transduction.

The tyrosine phosphatase SHP-1 SHP-1 protein is involved in EPO signalling, this protein binds both to the third Tyr of the EPO-R and to Jak2 tyrosine kinase. This association leads to Jak2 dephosphorylation and thus to the signal arrest (Klingmuller et al. 1995). Several cases of familial erythrocytosis have been reported, due to a truncation of the cytoplasmic domain of the EPO-R, which lacked the binding site for SHP-1 protein and thereby became hypersensitive to EPO stimulation in vivo (De La Chapelle et al. 1993; Kralovics et al. 1997). The erythroid progenitors derived from mice knocked out for the Shp-1 gene are also hypersensitive to EPO (Van Zant and Shultz 1989).

The Cis and SOCS3 proteins The Cis protein (for cytokine-inducible SRC homology 2-containing protein) is one of the known targets of STAT5 factor (Yoshimura et al. 1995). Cis is an inhibitor of EPO-induced cell proliferation. We showed that Cis was associated to the Tyr 401 of the EPO-R and was ubiquitinated. This ubiquitination of Cis suggests that this protein could play an active role in the sequestration of the EPO/EPO-R complex by the proteasome (Verdier et al. 1998). Indeed, we showed that the proteasome controls the down-regulation of EPO-R in EPO-stimulated cells by inhibiting the cell surface replacement of internalized EPO-R (Verdier et al. 2000). A second member of the Cis family called SOCS3 (for Suppressor Of Cytokine Signaling) has been described as essential in the regulation of erythropoiesis. However, contradictory results have been published and the precise role of SOCS3 in erythropoiesis remains to be understood (Marine et al. 1999; Roberts et al. 2001).

Role of the proteasome system The duration of activation of the EPO-R is rather short when compared to that of other cytokine receptors such as the thrombopoietin (TPO) receptor. Indeed, after EPO stimulation, the different intracellular pathways are acti-

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vated during a very short period (up to 10 min), whereas these pathways are still activated one hour after TPO stimulation. For the EPO-R, two mechanisms involving the ubiquitin/proteasome system are responsible for the control of duration of receptor activation: first, the stimulated receptors are internalized and degraded, second, new synthetized EPO-R are hampered to reach the plasmic membrane after EPO stimulation. We showed that, following EPO-R activation, the EPO-R is ubiquitinated at the cell surface by a Jak-2 dependent mechanism. As a consequence, there is a cleavage by the proteasome of the intracellular part of the EPO-R which probably leads to a rapid arrest of the signalling process. The remaining of the EPO/EPO-R complex is internalized and degraded by the lysosomes (Verdier et al. 2000; Walrafen et al. 2005) (Fig. 2). The E3 ligase responsible for the EPO-R ubiquitination is not yet identified.

Fig. 2. Mechanisms of EPO-R down-regulation during EPO stimulation. Ia: Upon EPO binding, the EPO-R is tyrosine-phosphorylated, triggering intracellular signaling. II: The EPO-R is ubiquitinated at the cell surface. III: The proteasome degrades the EPO-R cytoplasmic tail, removing all the phosphorylated tyrosine residues and preventing further signal transduction. IV: The cleaved EPO-R is internalized and degraded in the lysosomes. Ib: If Jak2 activation is prevented by the inhibitor AG490, neither phosphorylation nor ubiquitination of the EPO-R occur. EPO-EPO-R complexes are still internalized but the complexes are not degraded and recycle to the cell surface. This research was originally published in Blood. Walrafen et al. Both proteasomes and lysosomes degrade the activated erythropoietin receptor. Blood. 2005. 105:600–8.© the American Society of Hematology

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The efficient mechanisms of downregulation explain why the EPO-R very rapidly disappears from the cell surface of erythroid cells in contrast to the TPO receptor that is still present on the platelet surface. These mechanisms of EPO-R downregulation could also improve erythroid terminal differentiation. Indeed, erythroid terminal differentiation requires caspase and FOXO 3A activation (Zermati et al. 2001; Bakker et al. 2004). Both activations are blocked by pathways activated after EPO-R stimulation like the PI 3-kinase/Akt pathway. Therefore, the disappearance of the EPO-R favors a balance towards caspase and FOXO 3A activation, thereby leading to terminal differentiation of these erythroid cells.

Acknowledgements This work was supported by a grant from Association pour la Recherche contre le Cancer (ARC) and a grant from Ligue Nationale Contre le Cancer (LNCC).

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Correspondence: Catherine Lacombe, MD, PhD, Hematology Department, Hôpital Cochin, 27 rue du Faubourg St Jacques, 75014 Paris, France, E-mail: [email protected]

Chapter 3

The role of erythropoietin receptor expression on tumor cells J. Fandrey Institut für Physiologie der Universität Duisburg-Essen, Essen, Germany

Introduction Based on the successful treatment of renal anemia with recombinant erythropoietin (rhEPO), EPO was soon considered for the use in other forms of anemia, such as the cancer associated anemia. Anemia is common in patients with malignant disease (Jumbe 2002; Weiss 2003). It is either due to the myelosuppressive effects of chemotherapy and/or radiotherapy or sometimes infiltration of the bone marrow. In many cases, cancer patients suffer from the anemia of chronic disease which is caused mainly by functionally impaired iron availability, reduced EPO levels and direct suppressive effects of cytokines on erythroid progenitor cells (Jelkmann 1998). Experimental evidence suggested that proinflammatory cytokines that may be increased during the course of malignant disease, effectively inhibit endogenous EPO synthesis (Jelkmann et al. 1992). Thus, the resulting anemia impairs the survival of cancer patients by 1. making chemotherapy or radiotherapy less effective due to the reduced oxygen supply, 2. limiting the intensity of treatment due to fatigue and overall exhaustion, 3. decreasing the patients’ quality of life and thus indirectly influencing therapy and 4. impairing tumor oxygenation which causes areas of hypoxia within the tumors where angiogenesis may be stimulated or the potential for tumor growth and metastases is enhanced. The introduction of EPO as a supportive measure in the treatment of cancer patients can correct the anemia and has improved the quality of life of the patients (Vaupel et al. 2005). In addition, at least in a meta-analysis in which 200 publications were critically reviewed anemia has been identified as an independent prognostic factor for survival (Caro et al. 2001). Whether the correction of anemia has an impact on survival will be subject of other chapters in this book.

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However, from the very beginning when rhEPO became available for therapy there has been the serious concern that a growth factor such as EPO might also promote the growth of tumor cells. Although these safety considerations were not followed by any unfavorable observations in millions of renal patients two recent publications have fueled the discussion whether EPO treatment may actually be dangerous for patients with cancer. Both studies (Henke et al. 2003; Leyland-Jones 2003) diverge from the generally positive results obtained with EPO treatment in cancer patients and have initiated a hearing at the FDA in May 2004 (FDA 2004). In addition, both studies have been extensively discussed in letters to The Lancet and some of the potential pitfalls with these studies or differences in interpreting their data have been documented in a recent publication to which the reader is referred to here (Vaupel et al. 2005). Interestingly, at the above mentioned hearing the FDA panel considered that determination of the EPO receptor status of tumors is “difficult and (may) not (be) totally relevant” (FDA ODAC on EPO 2004). Still, the clinical studies by Henke et al. (2003) and Leyland-Jones et al. (2003) most probably received so much attention because a number of in vitro studies was published in which established tumor cell lines were claimed to respond to recombinant EPO with enhanced proliferation. Although the safety issue in patients can only be answered in large clinical studies that are underway (FDA ODAC on EPO 2004) it also appears necessary to selectively review some of the in vitro studies particularly with respect to the EPO receptor and its intracellular signaling pathways.

Does EPO cause proliferation of tumor cells in vitro? Westenfelder and Baranowski (2000) studied cell lines from patients with renal cell carcinoma (RCC) and also established renal tumor cell lines of human origin (Caki-2, 786-0) as well as a mouse cell line of a renal adenocarcinoma (RAG). EPO receptor mRNA and protein were detected and 125 Iodine-labelled EPO binding was found but only to a single class of receptors (erythroid progenitors have a high and a low affinity receptor binding site (Philo et al. 1996). Proliferative effects of EPO, however, were weak and only observed in serum deprived cultures. In one cell line the addition of 10% serum had a more prominent effect than EPO. Whatever component(s) of the serum may have caused this effect it is conceivable that they may be present in a tenfold higher concentration in vivo and may thus be much more relevant as tumor proliferation supporting factors than EPO. Interestingly, in samples from the tumors low level expression of the EPO gene was detected while the permanent cell lines had no EPO mRNA. Thus, a potential autocrine role of EPO in renal tumors might be suggested although only a minor fraction of renal cell carcinomas (RCC) express EPO mRNA (Gross

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et al. 1994). However, a study by Shouval et al. (1988) which was also quoted in the work by Westenfelder and Baranowski (2000) demonstrated that EPO secreting RCC cells were less tumorigenic when transplanted into nude mice than non-EPO-secreting cells. Acs et al. (2001) suggested that either endogenous EPO or exogenous rhEPO could support the growth and survival of EPO receptor expressing breast carcinoma cells. In their study they used five breast carcinoma cell lines that are well-established but, on the other hand, have gone through many passages in culture. It is therefore questionable whether they actually reflect the in vivo system of a breast carcinoma. When studying these cells, the authors deprived the cells of serum which may synchronize the cells and thus make them even less suitable as a model. Extremely high concentrations of up to 10,000 mU EPO/ml were used to show increased DNA synthesis and even a 25-fold higher concentration of EPO was claimed to increase tyrosine phosphorylation of the EPO receptor protein. Interestingly, as a positive control for the EPO receptor detection Acs et al. (2001) used Hela cells which served as a negative control in the above mentioned study by Westenfelder and Baranowski (2000). In a more recent study Acs et al. (2002) proposed an autocrine or paracrine effect of EPO produced by tumor cells to promote breast carcinogenesis (Acs et al. 2002). Immunohistochemical staining of tumor cells showed cytoplasmic localization of the EPO receptor which was not refined to malignant cells but also to normally appearing ductal cells. Acs et al. (2002) and in an additional study Arcasoy et al. (2002), who detected EPO receptor protein by immunohistochemical staining in breast cancer cells, both used an antibody that was directed against the C-terminus, i.e. the intracellular part, of the EPO receptor. By this procedure of immunohistochemical staining splice variants or soluble receptors will be missed (see below). Moreover, the specificity of the antibody used (Santa Cruz C20) has recently been questioned since it crossreacted with multiple proteins of sizes different from that predicted for the EPO receptor in Western blot analysis (Busse et al. 2005). More recently, Pajonk et al. (2004) used HeLa cells which were stably transfected with the EPO receptor cDNA to express the EPO receptor. They reported tyrosine phosphorylation of STAT5 which is of central importance for EPO receptor signaling in erythroid progenitors (see below; (Klingmuller 1997)) and strong activation of NFκB by EPO receptor signaling. However, NFκB activation was unusual in the way that it did not require degradation of IκBα and activation was not prevented by inhibition of proteasomal function. This mode of NFκB activation is of considerable interest since it may reveal a tumor cell specific process but it requires further studies. Although treatment with EPO of EPO receptor expressing cancer cells did not change intrinsic radiosensitivity or sensitivity to chemotherapeutics, the authors “advocate a restricted use of erythropoietin to patients suffering from erythropoietin-receptor-expressing cancers” (Pajonk et al. 2004).

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In contrast to the above mentioned work many other authors do not find any influence of EPO on tumor cell proliferation in vitro. Rosti et al. (1993), tested the effect of recombinant human EPO on clonogenic growth and DNA synthesis in 10 different cell lines derived from hematologic malignancies or solid tumors. They included cell lines like K-562 and HEL which both express EPO receptors. Despite significant EPO receptor expression, concentrations up to 10,000 mU/ml of EPO did not affect the clonogenic growth of any of these human tumor cell lines. Likewise, a study by Selzer et al. (2000) confirmed that normal melanocytes and human melanoma cell lines which express the EPO receptor, were not affected in their cell growth by treatment with recombinant EPO. Interestingly, the authors of this study suggested that the expression of EPO receptor may be a progression marker of the more malignant phenotype, but nevertheless did not have functional influence on tumor growth. A study with a large number of 25 different benign and malignant cell lines by Westphal et al. (2002b) revealed that most of the cell lines expressed EPO receptor mRNA and proteins, but did not respond with an increase in proliferation to treatment with EPO. In this study several cellular response markers like 3H-thymidine uptake as a measure of DNA synthesis, Northern blot for c-fos expression (as an early growth response gene) and tyrosine kinase activity were measured. Since tumor cells that were EPO receptor positive did not respond with an increase in c-fos mRNA or stimulated tyrosine kinase activity, the authors suggested that the EPO signal was probably not transduced in these cells despite the detection of EPO receptor protein in whole cell lysates. The authors thus conclude that a deleterious effect on cancer patients may not be expected. This notion is supported by the finding that several tumor cell lines that expressed high levels of EPO receptor protein as detected by Western blot showed no detectable binding of 125 Iodine-labelled EPO (Sinclair et al. 2005). In another study Westphal et al. (2002a) measured soluble EPO receptors that were produced by tumor cells in culture from well established cell lines that were also used for most of the so far mentioned in vitro studies. Many of these cells secreted soluble EPO receptors into the culture supernatant. The meaning of soluble receptors will be discussed in further detail below. Finally, a very recent study by Liu et al. (2004) aimed at investigating the potential effects of GM-CSF and EPO on tumor cells and speculated whether growth factor treatment during chemotherapy may only be detrimental in those cancers in which the tumor cells express high levels of the specific receptor. For their study the authors selected seven cell lines including two RCC cell lines of which one was also used in the previously mentioned study (Westenfelder and Baranowski 2000). Neither EPO nor GM-CSF increased the rate of proliferation which was both measured by cell counting and bromo-2′-deoxyuridine (BrdU) incorporation. Interestingly, EPO caused an increase in mitogen activated protein (MAP) kinase activity in several cell

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lines but did not change the status of Bcl-2, which is part of the antiapoptotic pathway. Indeed, although MAP kinase activation is part of the intracellular signaling of the EPO receptor (Klingmuller 1997), Kolonics et al. (2001) have suggested that the MAP kinase pathway may not be the principal mechanism of EPO receptor activity. Consequently, any adverse effects associated with increased MAP kinase activity which result in reduced chemotherapeutic drug sensitivity may not be an issue of EPO treated cells (Liu et al. 2004). Finally, the authors conclude that EPO therapy may be even safe in renal cancers and other tumors that show high EPO receptor levels.

EPO receptors are found on many cells It appears to be generally accepted now that EPO receptor expression is not only found in erythroid progenitor cells, but also in many nonmalignant nonerythroid cells like endothelial cells, neuronal cells, myoblasts and megakaryocytes (Farrell and Lee 2004). Expression of erythropoietin in neural tissue appears to be of considerable importance and is also of interest with respect to EPO receptor activation. More and more evidence accumulates that EPO in the brain is neuroprotective and even neuroregenerative. These effects have recently been extensively reviewed and the reader is referred to this work (Jelkmann 2005). Physiologically EPO and EPO receptors are expressed in the human brain, even very early during embryonic development (Marti et al. 1996; Juul et al. 1998; Dame et al. 2000). Recombinant EPO has been found to actively cross the blood brain barrier in animals, although much higher doses than conventionally used for the treatment of anemia were required (Brines et al. 2000). This has been confirmed for humans, where doses of 5,000 units/kg were required to increase erythropoietin concentration in the cerebral spinal fluid after intravenous administration (Jumbe 2002). Still systemic erythropoietin administration has been found to reduce injury from brain ischemia both in an experimental animal model (Brines et al. 2000; Siren et al. 2001) and in patients at doses similar to those used in clinical practice (Ehrenreich et al. 2002). Other beneficial effects of erythropoietin on neuronal cells include an attenuated brain injury after different forms of trauma and/or toxic effects of glutamate, a representative of the excitotoxicity found in many forms of brain injury (Morishita et al. 1997; Jelkmann 2005). The role for erythropoietin in the brain is underlined by observations of mouse embryos with targeted disruption of the EPO and the EPO receptor gene. Although these animals die from severe anemia in mid gestation, an increased rate of apoptosis in the brain of these mice has been reported (Wu et al. 1995; Yu et al. 2002). In contrast, however, mice that exclusively express the EPO receptor in hematopoietic tissues using GATA-1 directed erythroidspecific expression show no apparent neurological deficits (Suzuki et al.

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2002). Thus, it is conceivable that EPO receptor signaling plays an important role in limiting the damage incurred by various neurological stresses. These protective effects could depend on different classes of EPO receptors and on intracellular signaling pathways that are different from those known in erythroid progenitor cells (Masuda et al. 1993). In this respect the work of Digicaylioglu and Lipton (2001) is of great interest. The authors reported the activation of NFκB following EPO treatment which caused a delay in apoptosis of neuronal cells induced by N-methyl-D-aspartate or nitric oxide. Very recently, it was shown that carbamylated EPO does not stimulate erythropoiesis but is tissue-protective (Leist et al. 2004). Detailed analysis based on the assumption that EPO receptor signalling is different from the known pathways revealed the association of the EPO receptor with the common β receptor (βcR) in nonhematopoietic cells (Brines et al. 2004). βcR acts as the signal transducing subunit for several other cytokine receptors like GM-CSF, IL-3 and IL-5. It remains open whether this “new” signaling from the EPO receptor plays any role in tumor cells. Whereas the effect on neuronal cells is very welcome since EPO may indeed be neuroprotective and potentially have a role in neuronal differentiation, the detection of EPO receptors and potential anti-apoptotic effects of EPO on tumor cells have raised serious concerns. Although tumor cell lines derived from breast, liver or cervical carcinoma, neuroblastoma, glioblastoma and glioma were used for these studies they had been propagated through many generations in culture (Acs et al. 2001). Tissue biopsies from RCC (Westenfelder and Baranowski 2000), breast carcinoma (Acs et al. 2001) and melanoma (Selzer et al. 2000) have also been studied. Although results from experiments with these tissue samples in general did not reflect the findings from cells in culture, e.g. (Westenfelder and Baranowski 2000), it was inferred that administration of rhEPO to treat the anemia in cancer patients may positively influence the growth and/or survival of cancer cells or stimulate angiogenesis to promote tumor growth. It remains doubtful whether the receptors found on tumor cells in culture or in the tissue have any functional activity when exposed to clinically relevant concentrations of EPO. Nevertheless the theoretical consideration of such an adverse effect fell on fertile soil when two clinical trials with negative outcome in the EPOtreated group of patients were reported (Henke et al. 2003; Leyland-Jones 2003). Both studies have recently been critically evaluated and it was concluded that the negative outcome cannot be unambiguously attributed to the anemia-correcting treatment by rhEPO (Vaupel et al. 2005). Even a considerable number of in vivo – mostly animal – studies does not help to clarify the role of EPO on tumors: EPO was found to induce tumor regression (Rubins 1995; Gagic et al. 1997; Mittelman et al. 2001; Mittelman et al. 2004) or improve tumor sensitivity to radiation or chemotherapy by increasing the oxygenation of tumors (Silver and Piver 1999; Thews et al. 2001). In fact, the improvement of tumor oxygenation has been one

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reason to consider treatment with rhEPO not only to increase the quality of life by correcting the anemia, but also to increase the oxygen capacity of the blood and thus the oxygen supply of the tumor tissue. Human tumors and metastases commonly have an inadequate vessel supply and are therefore hypoxic (Vaupel et al. 1989). Tumor tissue hypoxia can lead to poorer outcomes and is generally considered as an indicator of a negative clinical prognosis (Hockel et al. 1993). In addition, hypoxia causes resistance to radiation treatment, and a higher hematocrit would thus be predicted to be beneficial. Indeed, Stüben et al. (2001) conducted a study with six groups of nude mice which had glioblastoma cell line tumors implanted. Four groups of mice were anemic induced by total body irradiation 6 h before implantation of the tumor cells. Two of these groups were pretreated with EPO to prevent anemia. Subsequently, one half of all animals was irradiated with a single dose and tumor volume was assessed. None of the EPO treated animals showed any increase in tumor growth. On the contrary, anemic mice were increased in their sensitivity to radiotherapy when their anemia was corrected by EPO treatment. In another animal study Kelleher et al. (1996) implanted DS sarcomas onto the foot of rats and a tumor associated anemia was induced. Again, treatment with EPO improved the anemia and also increased the pO2 in the tumor tissue as measured by polarographic oxygen electrodes. However, EPO administration did not influence tumor growth or showed any other adverse effects. Obviously these animal experiments are closer to the clinical situation. Still, the in vitro data together with the above-mentioned two clinical studies with potential adverse effects have fueled the discussion on the potential tumor-proliferative effects of EPO. Only in vitro studies will allow to completely elucidate the signaling from the EPO receptors inside the cell. Methodological differences and, in part, over-interpretation of in vitro data are not helpful to define the potential risk of EPO treatment in cancer patients. Therefore, in vitro studies need to very carefully address at least the five following points to help clarifying the meaning of EPO receptors on tumor cells.

What does the detection of EPO receptors on tumor cell mean? Five points to consider Signal transduction from the EPO receptor to the nucleus The EPO receptor is a member of the type I cytokine receptor super family (Klingmuller 1997). Other members of this family are receptors for growth hormone, prolactin, G-CSF, GM-CSF thrombopoietin and several interleukins. Common features shared by these receptors in the extracellular

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domain are four spaced cysteins near the N-terminus and a Trp-Ser-X-TrpSer (WSXWS) motive located proximal to the cell membrane (Klingmuller 1997). For the EPO receptor in particular, crystallographic analyses have shown that two EPO receptor monomers are bridged by EPO as a symmetrical T-shaped dimer (Livnah et al. 1996). These analyses and data from Syed et al. 1998 clearly indicate that EPO receptor activation depends upon ligand binding to the receptor dimer. This is in line with previous observations that certain receptor dimerizing antibodies can activate the EPO receptor in EPO responsive cells or that disulfide bridging of β-chains due to a mutation at position 129 of the EPO receptor (R129C mutation) leads to constitutive proliferation in BAF/3 cells (Yoshimura et al. 1990; Elliott et al. 1996). However, when EPO receptors were successfully crystallized, it turned out that the receptors are found as preformed dimers caused by interaction between two fibrinonectin III-like subdomains in the extracellular domain of each monomer in the absence of their ligand EPO. However, unliganded dimers have a distance of 97Å whereas binding of the ligand reduced the spacing between the 2 receptor monomers to 39Å. Thus it has been concluded that receptor activation is driven by a ligand induced conformational change which then causes Janus kinase 2 (Jak2) activation (see below). This model was nicely confirmed by fluorescence complementation measurements which clearly indicated that unliganded EPO receptor is preformed in the membrane and that these dimers are brought into functional proximity by EPO or EPO mimetic peptides (Remy et al. 1999). Importantly, the cytoplasmic domain of the EPO receptor lacks intrinsic signaling activity. This requires the recruitment of cytoplasmic kinases, in particular Jak2 to promote signal transduction. Upon EPO binding the conformational switch facilitates the binding of Jak2. A continuous stretch of residues in the membrane proximal region of the EPO receptor is required to activate intracellular signal transduction by recruitment of Jak2. Interestingly, the same juxta-membrane motive is critical for the transport of the EPO receptor from the endoplasmic reticulum to the cell surface (Huang et al. 2001). Activated Jak2 is then involved in activation of signal transducer and activator of transcription protein-5 (STAT5) (Ketteler et al. 2002). Activation of STATs promotes signal transmission from the cell surface to the nucleus to induce changes in the expression pattern of EPO responsive genes. One of the key targets are the antiapoptotic proteins Bcl-2 and Bcl-XL. This clearly illustrates one primary action of EPO in erythroid cells which is to inhibit programmed cell death (Koury and Bondurant 1990). In contrast, the signaling of EPO to act as a mitogen has been more difficult to define. The addition of EPO to early erythroid progenitor cells supported their proliferation and was shown to activate several known effectors of mitogenesis like c-myc, mitogen activated protein (MAP) kinases and phosphoinositol-3 kinase (PI-3K). In addition to these receptor associated effectors at least 19 additional signaling factors have been described to bind to the activated EPO

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receptor complex (for review Wojchowski et al. 1999). Among them are further activating but also negative inhibitory molecules including suppressors of cytokine signaling (SOCS) and tyrosine phosphatases that terminate EPO signaling. Proliferation of immature red blood cells and their differentiation into mature erythrocytes are mediated by binding of erythropoietin to its cell surface receptors (D’Andrea et al. 1990). Ligand binding to the EPO receptor on the cell surface was found to accelerate the rate of receptor internalization from a half-life of 3 hours in the absence of EPO to 15 minutes in its presence (Sawyer and Hankins 1993). Now, obviously the binding of the hormone EPO takes place at the surface but EPO receptor internalization is required for EPO-mediated proliferation of erythroid progenitor cells. Truncated receptors lacking amino acid 268 to 276 were defective in their capacity for endocytosis of the EPO/EPO receptor complex and did not promote cellular growth. Thus, surface localization of the EPO receptor and the 9 amino acid stretch, the internalization motif, of the EPO receptor responsible for internalization upon binding of EPO are required for proper signaling (Flint-Ashtamker et al. 2002). Further intensive studies to determine the role of the internalization motif within the cytosolic part of the EPO receptor revealed that it is not only important for internalization but also for association of Jak2 and thus intracellular signal transduction since tyrosine phosphorylation mediated by Jak2 was abolished in mutants lacking the nine amino acids (FlintAshtamker et al. 2002). Importantly endocytosis of EPO via the EPO receptor is essentially similar in different cellular systems (Levin et al. 1998; Beckman et al. 1999). This supports the notion that the process of internalization of EPO via the receptor relies mainly on intrinsic properties of the receptor molecule and would not depend on the cell type in which the receptor is expressed. If de novo synthesis of EPO receptor is blocked by the translational inhibitor cycloheximide, EPO receptors decline by 50–60% after 4 hours of cycloheximide treatment. The absence of the internalization motif abrogated the decline. All these data were obtained in the absence of ligand indicating the considerable amount of ligand-independent EPO receptor turnover via internalizations is present at least in hematopoietic cells. In addition, particular events in the activation of proliferative signals from the EPO receptor were defined. The above mentioned anti-apoptotic effects of EPO on neuronal cells appear to involve activation of NFκB through cross-talk between the Jak2 and the NFκB pathways (Digicaylioglu and Lipton 2001). This appears different from the regular signaling in erythroid cells but the signaling chain from the receptor to the target genes was successfully followed. This has not been the case for several in vitro studies with tumor cells in which activation of the EPO receptor was claimed. In most studies only parts of the signaling were studied, i.e. sometimes only tyrosine phosphorylation of the EPO recep-

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tor protein. Interestingly, in a recent study using HeLa cells stably transfected with the EPO receptor activation of NFκB was suggested without phosphorylation and degradation of IκBα, i.e. an untypical activation mechanism of NFκB. If such “new” signaling pathway would exist in tumor cells a more complete dissection of the signaling cascade is required to draw any firm conclusions as to the potential impact of EPO on tumor cells in vivo. Moreover, the above-mentioned known components of the signaling cascade from hematopoietic progenitors should be scrutinized in tumor cells. It appears mandatory to run hematopoietic progenitors as positive controls in parallel to either confirm activation of known pathways or to compare new intracellular signals with respect to receptor affinity, signaling kinetics and the response of target genes. Of note, in view of the recent work on signaling in neuronal cells (Brines et al. 2004) one should be prepared to find new modes of EPO receptor activation in tumor cells.

Soluble receptors and EPO receptor splice variance In addition to the classical membrane spanning EPO receptor with its intracytoplasmic region for recruitment of Jak2, splice variants resulting in soluble forms of the EPO receptor have been described (Yet and Jones 1993; Ku et al. 1996). Soluble forms of cytokine receptors or growth factor receptors are often expressed in cancer cells and have been implicated in various biological functions. On the one hand it is known that soluble receptors can antagonize the effect of membrane spanning isoforms, whereas in other cases they prolong the half-life of the respective ligand (Rose-John and Heinrich 1994; Heaney and Golde 1996). With respect to EPO it has been shown that ligand binding to soluble EPO receptors decreases receptor-mediated signal transduction (Shimizu et al. 1996). Therefore, a report on different receptor splice variants in human cancer cells potentially has a great impact (Arcasoy et al. 2003). The authors examined a considerable number of tumor cells and were able to detect full-length EPO receptor mRNA in lung, colon, breast, prostrate and ovary cancer cells. In addition to the full-length receptor which was also detected on the protein level, at least 5 isoforms resulting from different splicing of the seven introns of the EPO receptor gene were isolated. Interestingly, three of these isoforms resulted in truncated EPO receptor that still contained the trans-membrane domain and can be expected to be found on the cellular surface. The structures of the deduced EPO receptor proteins for the first three isoforms had severe truncations deleting the membrane distal intracellular part of the receptor including the Jak2 docking site. Thus, these truncated forms are expected to lack functional activity. Isoform 3 of these truncated EPO receptors is identical to previously described isoform from erythroid progenitor cells that showed a dominant negative effect on EPO-induced differentiation and inhibition of apoptosis (Shimizu et al.

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1996). Thus, although only isoform 3 truncation of the cytoplasmic domain has been clearly associated with an inhibitory function, truncated EPO receptors isoforms 1 and 2 may likewise negatively modulate EPO-mediated signaling in cancer cells. It is noteworthy that in addition to the detection in permanent cancer cell lines these isoforms were also isolated from primary cancer tissue of breast, colon, lung and ovary carcinomas. Two forms, isoforms 4 and 5, that lack the transmembrane domain are most likely soluble forms and the authors succeeded in detecting a 26 kD immunoreactive protein in the supernatant of prostate and breast cancer cells by Western blot using an antibody that recognizes the extracellular domain (Arcasoy et al. 2003). These proteins were not detected in the supernatants of Chinese hamster ovary cells or in non-conditioned medium but corresponds to data from Westphal et al. (2002b) who found soluble EPO receptors in the supernatant of many cancer cell lines. As pointed out above, soluble receptors may be ambivalent with respect to the effect on EPO signaling, but the competition with membrane-bound receptors for EPO has been shown for hematopoietic cells (Yet and Jones 1993). Moreover, in some of the splice variants that still contain the transmembrane part of the EPO receptor, splicing also affects the immediate cytoplasmic part. This stretch of amino acids is close to the membrane spanning domain and overlaps with amino acids 268–276 that are important for internalization (see above). Therefore, some of the splice variants may be defective in internalization and thus not convey a proliferative signal. Tumor cells secrete soluble receptors and have different splice variants expressed. The issue of EPO receptor isoforms in human cancers has not been thoroughly studied. Soluble receptors in the tissue are hard to identify but may well significantly affect the effects of any EPO, endogenous or exogenous.

Cellular localization of the EPO receptor Since the EPO receptor lacks intrinsic kinase activity, recruitment of Jak2 is essential to transmit the signal from the receptor to the nucleus. The precise orientation of critical residues in the juxta membrane motif is essential for Jak2 activation and subsequent signaling upon binding of EPO to its receptor. It is of note that the membrane proximal domain of the EPO receptor that mediates binding of Jak2 is also responsible for transport of the EPO receptor from the endoplasmic reticulum to the cell surface. To visualize intracellular trafficking of the receptor Ketteler et al. (2002) generated fusion proteins of green fluorescent protein (GFP) and different parts of the EPO receptor. After transient transfection of both Ba/F3 cells (serving as a model for erythroid progenitors (D’Andrea et al. 1991)) and HEK293T-cells (as a permanent tumor cell line) the authors observed different cellular localiza-

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tion of the fusion proteins in the membrane or within the cell. Interestingly, fusion protein constructs that resided within the endoplasmic reticulum and did not reach the cell surface, were unable to trigger the activation of signaling. Signaling was assessed by detecting association of Jak2, subsequent tyrosine phosphorylation of the EPO receptor and association of STAT5, SHP-1 and p85 (as part of the PI3-kinase) with the activated EPO receptor (Ketteler et al. 2002). In addition, proliferation and differentiation of Ba/F3 cells was determined. All these clear signs of receptor activation were absent with the above-mentioned fusion proteins that stayed in the endoplasmic reticulum. Other fusion proteins showed punctuated cellular fluorescence, but were also localized in the membrane and able to bind EPO as shown by 125 iodine-labelled EPO bound to the surface. Flow cytometry using EPO receptor antibodies that detect extracellular epitopes of the EPO receptor supported the notion that – depending on the construct – inactive receptors were not transported to the membrane and thus not detected by flow cytometry. Collectively, the authors concluded that the extracellular, transmembrane and membrane-proximal segments of the cytoplasmic domain form an entity of which the precise orientation is essential for initiation of signaling of the EPO receptor. Although the cytoplasmic domain had some flexibility in adopting an activated confirmation of the receptor, fusion proteins that were retained in the endoplasmic reticulum were signaling-incompetent. On the other hand, some cell surface expressed receptors could bind EPO, but were signaling-incompetent due to disruption of the intracellular parts of the receptor. Finally, as a positive control, however, and to exclude any unspecific effects through the presence of the GFP within the fusion proteins some of the constructs were detected on the cell surface, bound EPO and initiated signaling with subsequent proliferation, although the receptor had been fused to GFP. These data are of critical importance with respect to the immunohistochemistry of EPO receptor protein that has been reported in tumor cells so far. Several studies (Acs et al. 2002; Arcasoy et al. 2002) have included immunohistochemical staining for the EPO receptor in which the protein was clearly found in the cytoplasm, partly surrounding the nucleus and most likely in the endoplasmic reticulum. From the study of Ketteler et al. (2002) and also earlier work from Yoshimura et al. (1990) it is evident that EPO receptors retained in the endoplasmic reticulum are unable to interact with the ligand and thus transmit no signal to the nucleus. In view of the recent reports of the questionable specificity of the commercially available antibodies against the EPO receptor (Busse et al. 2005) and the fact that despite the detection of EPO receptor protein by Western blot no binding of 125Iodinelabelled EPO was observed (Sinclair et al. 2005), it is of utmost importance to show functionality of EPO receptor signaling. The sole detection of EPO receptor mRNA by PCR and immunohistochemical staining for the EPO receptor protein by the currently available antibodies do not justify the conclusion of potentially proliferative effects of EPO on tumor cells.

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Promiscuous activation of cytokine type 1 family receptors by high concentrations of EPO Extremely high concentrations of recombinant EPO have been used for in vitro studies with different tumor cell lines. Some of the investigators used 10,000–250,000 mU/ml (Acs et al. 2001, 2003) to stimulate their tumor cells in vitro while comparable work on EPO receptor signaling on hematopoietic cells provides clear results with EPO concentrations between 100 and 1,000 mU/ml (Ruan et al. 2004). This is several orders of magnitude higher than would ever be achieved in patients by exogenous EPO even after intravenous injection. These high concentrations may be problematic with respect to specificity of the EPO effects. In earlier studies to investigate the putative effect of EPO on smooth-muscle-like cells with respect to the observed increase in blood pressure in patients with renal anemia, the effects of EPO on changes in intracellular calcium concentration and subsequent cell contraction were determined (Morakkabati et al. 1996). Upon treatment with EPO between 2,000 and 10,000 mU/ml the cells showed clear shape changes as a result of contraction and actomyosin activation after an increase in their cellular calcium concentration. However, despite highly sensitive PCR methods no EPO receptor mRNA was detected. Thus, despite the reproducible effects on cell calcium and contraction no expression of the receptor for EPO was found and it remained open whether EPO concentrations in the range of several thousand mU/ml as used in this study caused promiscuous activation of other cellular receptors of the cytokine receptor I family (Morakkabati et al. 1996).

Local concentrations of EPO in tumors In addition to the just mentioned high concentrations needed for some in vitro studies it appears mandatory to consider which concentrations are likely to be achieved in the tumor when EPO is given to cancer patients. Often large parts of solid tumors are mal-perfused and thus hypoxic as pointed out above (Vaupel et al. 1989). Consequently, EPO concentrations that can be reached in the tissue after injection, will be extremely low, definitely several orders of magnitude lower than those used in all in vitro studies. Berdel et al. (1991) used serum concentrations considered to be in the therapeutic range when patients receive EPO. None of these concentrations stimulated growth of tumor cells. Subcutaneous injection of EPO which has been recently favored in renal patients will even show more sustained plasma levels of EPO but much lower peak concentrations. Figure 1 summarizes these five issues as they were raised. The situation regarding local EPO concentration within the (tumor) tissue may be different when EPO is produced by and/or within the tumor.

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2

soluble EPO-R

4

EPO

5

local EPO concentration

EPO-R P-

non EPO-R

-P Jak2 1

Ca2+

?

?

STAT5

NFkB

3 ER

nuclear signals

Fig. 1. The figure summarizes the open questions with respect to EPO and EPO receptor signaling in tumor cells. 1 EPO signaling depends on recruitment of several kinases and subsequent signal transduction to the nucleus. In addition, however, other signalling pathways like increase in intracellular calcium or activation of NFκB in neuronal cells have been described (see text for details). It is still unresolved whether tumor cell specific signaling pathways exist that have not been described in erythroid progenitor or neuronal cells so far. 2 Soluble EPO receptors may well antagonize membrane spanning receptors, as has already been described for hematopoietic cells (Yet and Jones 1993). On the other hand, EPO bound to soluble EPO receptors may have a longer half-life in the serum or even tissue. 3 EPO receptors retained in the ER are unable to interact with the ligand and are thus non-functional (Yoshimura et al. 1990; Ketteler et al. 2002). 4 EPO concentrations to stimulate tumor cell responses are several orders of magnitude higher than concentrations to be expected in the serum. Considering a mal-perfused tissue concentrations of EPO that can be expected in close proximity to tumor cells may be very low. Thus, the concentration of EPO within the tumor needs to be critically evaluated. 5 Non-specific effects of high concentrations of EPO in the absence of EPO receptor mRNA have been found in smooth muscle like cells (Morakkabati et al. 1996). Thus, unspecific effects, potentially promiscuous activation of other cellular receptors by extremely high EPO concentrations used in vitro have to be considered

The expression of EPO receptor and EPO mRNA in tumors of the female reproductive tract has made some authors to propose an autocrine or paracrine role of EPO supporting tumor growth (Arcasoy et al. 2002; Acs et al. 2003). Two further studies by Yasuda et al. (2002, 2003) reported that antagonists of EPO can reduce tumor growth of EPO and EPO receptor expressing tumors of the female reproductive tract. EPO alone will not cause tumors – at least not in animals – because several transgenic mice that over-

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express EPO in an oxygen independent fashion suffer from polycythemia due to the continuous stimulation of erythropoiesis. However, these animals do not develop erythroleukemia (Wagner et al. 2001; Madan et al. 2003). It will therefore be critical to determine local concentrations of EPO and also consider the potential occurrence of soluble receptors (see above). Moreover, if EPO synthesized within the tumor reaches a sufficient concentration to play an autocrine role in tumor development and progression it is questionable whether a small increase that may arise from treatment will have any effect on the tumor cell. This does not argue against a potential role for EPO expressed in the tumor which is worth to be carefully studied. But this is an issue that is completely different from the questions whether exogenous EPO would cause any negative effects with respect to tumor proliferation.

Concluding remarks The concern that the hematopoietic growth factor and hormone EPO may affect tumor cell growth has to be taken seriously. However, until today solid evidence that EPO promotes tumor cell proliferation or delays tumor cell death is missing. Even determination of EPO receptor mRNA expression on tumor cells does not ensure that signals from this receptor are transduced to the nucleus to affect cell growth and survival. Clearly in vitro studies are needed to fully define the signaling pathways – if there are any – connected to EPO receptors in tumor cells. Moreover, however, pre-clinical in vivo studies using animal models with EPO receptor positive tumors are needed in which treatment with EPO is critically evaluated with respect to changes in proliferation, apoptosis or even changes in the phenotype of the tumors. In addition, large databases are available with millions of patients treated successfully with recombinant EPO due to end stage renal disease. These databases are an excellent source for retrospective analyses to detect potential adverse effects on tumor development or treatment. Apart from the discussion whether exogenous EPO can stimulate tumor growth directly Yasuda et al. (2002) have suggested that tumor angiogenesis may be affected by EPO. They reported that the capillary endothelium within tumors showed EPO receptor immunoreactivity and that injection of a monoclonal antibody against EPO or soluble forms of the EPO receptor into tumors reduced capillaries and caused tumor destruction in a dosedependent manner. If local intra-tumor levels of EPO are sufficiently high to activate EPO receptors in endothelial cells this might in fact be a new therapeutic approach supporting the efforts of anti-angiogenic therapies. However, it should again be emphasized that these concentration will most likely not achieved from outside in a mal-perfused tumor with therapeutic doses. Intracellular signaling in non-erythroid cells obviously differs from the regular pathways that have been thoroughly studied in erythroid progenitor

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cells (Digicaylioglu and Lipton 2001; Brines et al. 2004). There is still lack of convincing evidence that classical or novel signaling of the EPO receptor is fully active in tumor cells. A very recent survey on different cellular responses towards EPO may be helpful in this respect: Korbel et al. (2005) using a proteomic approach showed a full array of signaling proteins in EPO responsive cells. Profiling the cellular response in erythroid progenitors could provide a “standard” of signaling pathways to be expected in an active state. It obviously remains to be studied whether most or even a subset of these signaling molecules are activated in tumor cells in vitro and also in vivo. Treatment of anemia in cancer patients by recombinant EPO has been for the benefit of many patients. Great care should be taken to avoid overtreatment, i.e. EPO treatment currently should be limited to alleviating the anemia as suggested in a recent review (Vaupel et al. 2005). Based on the in vitro and preclinical data, however, it does not appear to be justified to refrain from treating cancer associated anemia by recombinant EPO according to the clinical practice guidelines for the use of EPO developed by the American Society of Clinical Oncology and the American Society of Hematology (Rizzo et al. 2001).

Acknowledgements I gratefully acknowledge the excellent secretarial assistance by Ms. Gundula Endemann.

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102 J. Fandrey: The role of erythropoietin receptor expression on tumor cells 83. Yu X, Shacka JJ, Eells JB, Suarez-Quian C, Przygodzki RM, Beleslin-Cokic B, Lin CS, Nikodem VM, Hempstead B, Flanders KC, Costantini F, Noguchi CT (2002) Erythropoietin receptor signalling is required for normal brain development. Development 129: 505–516 Correspondence: Univ.-Prof. Dr. med. Joachim Fandrey, Institut für Physiologie, Universitätsklinikum Essen, Universität Duisburg-Essen, Hufelandstrarse 55, 45147 Essen, Germany, E-mail: [email protected]

Chapter 4

Problems associated with erythropoietin receptor determination on tumor cells A. Österborg Department of Oncology and Hematology, Karolinska University Hospital, Stockholm, Sweden

Erythropoietin is a glycoprotein hormone which regulates erythropoiesis by stimulating proliferation, preventing apoptosis and inducing differentiation of red blood cell precursors in the bone marrow (Spivak 2005). Not surprisingly, erythropoietin receptors (EPO-R) were first observed in these target erythroid cells (D’Andrea and Zon 1990). Various studies confirm that the number of receptors located on the cell surface is low, in the range of 100–