Advances in Cancer Research Volume 93

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Michael Campoli, Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 (189) Chien-Chung Chang, Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 (189) Lisa M. Coussens, Cancer Research Institute, Department of Pathology, and Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94143 (159) Hanna Mellin Dahlstrand, Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, 171 76, Stockholm, Sweden (59) Tina Dalianis, Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, 171 76, Stockholm, Sweden (59) Armin Ensser, Institut fu¨r Klinische und Molekulare Virologie, FriedrichAlexander-Universita¨t Erlangen-Nu¨rnberg, 91054 Erlangen, Germany (91) Soldano Ferrone, Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 (189) Bernhard Fleckenstein, Institut fu¨r Klinische und Molekulare Virologie, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, 91054 Erlangen, Germany (91) Alexander Griekspoor, Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (129) Ingrid Jordens, Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (129) Marije Marsman, Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (129) Jacques Neefjes, Division of Tumor Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (129) Stephen C. Robinson, Cancer Research Institute, University of California San Francisco, San Francisco, California 94143 (159) Harry Rubin, Department of Molecular and Cell Biology, Life Sciences Addition, University of California Berkeley, Berkeley, California 94720-3200 (1)

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Central Roles of Mg2þ and MgATP2 in the Regulation of Protein Synthesis and Cell Proliferation: Significance for Neoplastic Transformation Harry Rubin Department of Molecular and Cell Biology, Life Sciences Addition, University of California Berkeley, Berkeley, California 94720-3200

I. Specific and Nonspecific Stimulators of Cell Proliferation II. Necessity for Prolonged Stimulation and Increased Protein Synthesis to Induce DNA Synthesis III. Requirement of RNA and Protein Synthesis for the Initiation of DNA Synthesis IV. Effects of Inhibitors of Protein Synthesis on Initiation of DNA Synthesis V. Cyclins and CDKs, Specific Proteins Required for the Transition from the G1 to the S Stages of the Cell Cycle VI. Role of Mg2þ in Growth Regulation VII. Protein and DNA Synthesis in Very Low Ca2þ with Variations in Mg2þ Concentrations VIII. Kinetics of Cellular Responsiveness to Mg2þ Limitation in Physiological Ca2þ IX. Mitogen-Induced Increases in Cytosolic Free Mg2þ X. Mg2þ Effects on Diverse Cellular Responses to Growth Factors XI. Possible Roles of Kþ, Ca2þ, pH, and Naþ in Growth Regulation A. Potassium B. Calcium C. pH and Sodium XII. Regulation of Protein Synthesis by the PI 3-K and mTOR Pathways XIII. Role of Cations in Neoplastic Transformation XIV. Conclusions References

Growth factors are polypeptides that combine with specific membrane receptors on animal cells to stimulate proliferation, but they also stimulate glucose transport, uridine phosphorylation, intermediary metabolism, protein synthesis, and other processes of the coordinate response. There are a variety of nonspecific surface action treatments which stimulate the same set of reactions as the growth factors do, of which protein synthesis is most directly related to the onset of DNA synthesis. Mg2þ is required for a very wide range of cellular reactions, including all phosphoryl transfers, and its deprivation inhibits all components of the coordinate response that have so far been tested. Growth factors raise the level of free Mg2þ closer to the optimum for the initiation of protein synthesis. Advances in CANCER RESEARCH 0065-230X/05 $35.00

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Copyright 2005, Elsevier Inc. All rights reserved

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Harry Rubin The resulting increase in protein synthesis accelerates progression through G1 to the onset of DNA synthesis and mitosis. None of the other 3 major cellular cations are similarly involved in growth regulation, although internal pH may play an auxiliary role. Almost 105 externally bound divalent cations are displaced from membranes for every attached insulin molecule, implying a conformational membrane change that releases enough Mg2þ from the internal surface of the plasma membrane to account for the increase in free cytosolic Mg2þ. It is proposed that mTOR, the central control point for protein synthesis of the PI 3-K kinase cascade stimulated by insulin, is regulated by MgATP2 which varies directly with cytosolic Mg2þ. Other elements of the coordinate response to growth factors such as the increased transport of glucose and phosphorylation of uridine are also dependent upon an increase of Mg2þ. Deprivation of Mg2þ in neoplastically transformed cultures normalizes their appearance and growth behavior and raises their abnormally low Ca2þ concentration. Tight packing of the transformed cells at very high saturation density confers the same normalizing effects, which are retained for a few days after subculture at low density. The results suggest that the activity of Mg2þ within the cell is a central regulator of normal cell growth, and the loss of its membrane-mediated control can account for the neoplastic phenotype. ß 2005 Elsevier Inc.

I. SPECIFIC AND NONSPECIFIC STIMULATORS OF CELL PROLIFERATION The regulation of cell proliferation almost inevitably became the subject of research with the development of monolayer culture in which all the cells could be observed microscopically and counted by simple techniques. The field is most conveniently called growth regulation, although its major concern is the rate of increase in cell number, but there is an obvious relationship between growth in mass in the form of protein and the division of cells if they are to retain proliferative capacity. When synthetic media consisting of all the known required micronutrients were developed in the middle of the last century, it was realized that cells in culture needed serum proteins to multiply and these were interacting like polypeptide hormones with receptors on the cell surface. Early passage normal fibroblasts derived from chicken or mouse embryos were initially the most commonly used cells for studying growth regulation, partly because they outgrew the other cell types in culture, but they were later joined by mutant derivative cell lines that could multiply indefinitely in culture while retaining normal, growth-regulating behavior. A prominent feature of that behavior was contact inhibition, a term first applied to the inhibitory effect of contact on cell migration but adopted for the limitation on increase in cell number when cells form a confluent sheet covering the entire surface of the culture dish. That limit, called the saturation density, is proportional to the concentration of serum up to a maximum percentage of the medium that varies with different cells. A prominent feature of growth regulation is the failure of normal cells to sustain multiplication in culture unless they attach to and spread on a solid surface. If they are placed in suspension in a semisolid medium, such as soft

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agar or a viscous liquid medium, normal cells will not undergo more than one or two divisions before they become quiescent. Neoplastic cells violate these features of normal growth regulation to a greater or lesser extent. They require less serum to multiply; they have a higher saturation density, sometimes limited only by the supply of micronutrients rather than serum; and they have little or no requirement for attachment to a solid surface. As a result, neoplastic cells can usually be identified by their ability to continue multiplying when surrounded by a confluent sheet of normal cells that has undergone contact inhibition. The neoplastic cells can therefore be quantified by the formation of discrete dense or multilayered foci arising from single cells against a monolayered confluent sheet of normal cells. A major problem of cell culture is to identify the structures, molecules, and pathways that contribute to growth regulation of normal cells and how that regulation is transcended in neoplastic cells. One method for approaching this aim is to use normal fibroblasts that have been contact-inhibited at confluence in the G1 stage of the cell cycle, sometimes further inhibited by withdrawal of serum, then stimulated with a relatively high concentration of serum. After a period of hours that varies with cell type, the cells progress through the G1 stage into the S period of DNA synthesis. The initiation of DNA synthesis and progression through the S period can be monitored through the incorporation of radioactive thymidine into DNA in confluent cultures. This short-circuits the need to measure cell proliferation over several days at low densities and provides ample material for chemically measuring other quantities. Various hormones, such as insulin in the case of chicken embryo fibroblasts, or combinations of hormones act as growth factors sufficient to induce a round of DNA synthesis, which is generally correlated with an increase in the protein content of the cell population. There are other quantities that are stimulated by growth factors within minutes or even seconds, such as the uptake of glucose measured with radioactive analogs, or of uridine which is quickly phosphorylated and destined for incorporation into RNA. These transport and phosphorylation changes are, however, not coupled to growth or DNA synthesis since the external glucose concentration can be lowered so the amount taken up by the stimulated cells is less than that taken up by the quiescent cells with no effect on the onset of DNA synthesis, and uridine is not required at all for growth. The transport of Kþ, Naþ, and Ca2þ into cells increases quickly after stimulation (Rozengurt, 1986) and their putative role in regulating growth will be discussed in the following text. There are other early responses, such as the stimulation of protein and RNA synthesis and a decrease in the rate of protein degradation, which are connected to the onset of DNA synthesis. The sum of such early reactions was initially called the ‘‘pleiotypic response’’ and related to the stringent response of bacteria to amino acid starvation (Hershko et al., 1971). However, no further evidence

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developed for this relationship, and the stereotyped set of animal cell reactions to growth factors and other external conditions will be referred to as the ‘‘coordinate response’’ or ‘‘coordinate control’’ (Rubin, 1975a). Individual hormonelike protein growth factors undoubtedly interact with specific receptors on the external surface of the cell and some schemes of growth regulation call for the activation of different pathways for stimulation of growth (Rozengurt, 1986; Zetterberg and Engstro¨ m, 1983). However, this does not explain the need for attachment and spreading on a solid surface in order for freshly explanted normal cells to grow (O’Neill et al., 1986; Stoker et al., 1968) (Table I) nor the inhibition of growth at confluence. The latter is most clearly shown by the so-called wound-healing experiment in which a strip of cells is removed from a quiescent confluent layer and the cells on the edge of the strip move into the denuded area and multiply at a high rate until confluence is restored (Gurney, 1969) (Table I). It should be noted, however, that cell migration is correlated with proliferation (Barrandon and Green, 1987; Fischer, 1946) and migration stops when a culture becomes confluent. Migration on a solid surface involves continuous perturbation of the plasma membrane and possibly internal membranes such as endoplasmic reticulum as well, which decreases binding capacity for divalent cations, replacing them with monovalent cations (Dawson and Hauser, 1970). Actually, DNA synthesis in the established line of Swiss mouse 3T3 fibroblasts was stimulated sixfold over that of completely suspended cells by attachment of such a small area of their plasma membrane that there was no increase in surface area (O’Neill et al., 1986), indicating a limited perturbation of the membrane is sufficient to activate many cells. In another example of membrane changes, serum-starved quiescent cells exhibit few microvilli at their surface whereas logarithmically growing cells in serum-containing medium reveal an abundance of such microvilli (Evans et al., 1974). Within 1 hr of insulin treatment, microvilli appear at the surface in numbers and subcellular organization characteristic of exponential growth. Since the microvilli are bounded by plasma membrane, their Table I

Nonspecific Mitogenic Stimulation of Nontransformed Animal Cells Treatment

Reference

Attachment and spreading on a solid surface Migration (wound healing)

O’Neill et al., 1986; Stoker et al., 1968 Barrandon and Green, 1987; Fischer, 1946; Gurney, 1969 Carney et al., 1978; Sefton and Rubin, 1970 Rubin and Koide, 1973; Sanui and Rubin, 1984 Bowen-Pope and Rubin, 1983; Rubin and Sanui, 1977

Trypsin and selected proteases Subtoxic concentrations of heavy metals Ca pyrophosphate and Ca phosphate precipitates

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large numbers after insulin treatment represent a significant increase in surface area and movement, which is also likely to decrease the binding capacity of divalent cations. The nonspecificity of stimulation is further brought out by the addition of nonphysiological stimulants to cells. This first drew my attention with the finding that trypsin and other proteolytic enzymes in amounts too small to even retract, much less detach, cells stimulated growth of contact-inhibited cultures of chicken embryo fibroblasts (CEF) (Sefton and Rubin, 1970), and thrombin did the same for mouse fibroblasts (Carney et al., 1978) (Table I). The proposed nonspecificity of stimulation was bolstered by discovering the stimulation of DNA synthesis and hexose uptake in CEF by subtoxic concentrations of Zn2þ, Cd2þ, Mn2þ, Hg2þ, and Pb2þ (Rubin, 1975b; Rubin and Koide, 1973; Sanui and Rubin, 1984). Further support for nonspecificity came from the stimulation of DNA synthesis in Balb/c 3T3 mouse fibroblasts by sodium pyrophosphate at concentrations just sufficient to form flocculent precipitates with calcium (Rubin and Sanui, 1977). (It should be noted that higher concentrations sufficient to bind the Mg2þ levels present in the medium sharply inhibit DNA synthesis.) The stimulatory concentrations also enhance hexose uptake, and exert all their effect at the cell surface (Bowen-Pope and Rubin, 1983). The variety of nonspecific treatments that elicit the same set of early responses and are followed by DNA synthesis and mitosis indicated that they had in common a relatively simple effect on the cell membrane which initially entrained a shared intracellular response. When that stimulus was maintained over the length of the G1 period, it led to the initiation of DNA synthesis followed by mitosis. The same set of responses could, of course, be evoked by polypeptide hormones binding with their specific receptors at the cell surface. The diversity of intracellular effects induced by perturbing the cell membrane suggested that alterations in cation content of the cytosol might serve as a second message to stimulate the pleiotypic response and coordinate with growth and proliferation.

II. NECESSITY FOR PROLONGED STIMULATION AND INCREASED PROTEIN SYNTHESIS TO INDUCE DNA SYNTHESIS In any analysis of growth regulation, it is necessary to establish the essential parameters of the problem. One such parameter is how long a stimulatory agent has to be applied to quiescent (G1 or G0) cells in order to initiate and sustain DNA synthesis in a population. It is generally understood that serum or hormones have to be applied for several hours, well into

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the G1 period, before DNA synthesis begins in a large fraction of animal cells. Early studies on CEF cultures indicated that removal of serum growth factors in the first few hours of the G1 period of the cell cycle kept most cells from entering the S period of DNA synthesis, but the cells were committed to DNA synthesis about 4 hr before the actual start of DNA synthesis, i.e., removal of serum during this late period, about halfway through G1, did not delay the S period (Temin, 1971). Removal of serum from the medium, however, is not equivalent to its removal from the cell. Serum proteins adsorb to the cell surface and are not entirely removed by repeated washing or even by trypsinization (Hamburger et al., 1963). Lowering the pH of the medium from 7.4–7.5 to 6.8 inhibits the proliferation of CEF at high population densities but not at very low population densities (Rubin, 1971b). A combination of removing serum and lowering pH overnight proved to be a particularly effective method for inducing a reversible quiescence in high population densities of CEF (Rubin and Steiner, 1975). When serum and higher pH were restored, a small fraction of the CEF began synthesizing DNA between 2 and 4 hr, and the fraction then increased steeply up to 7 hr (Fig. 1). If either serum or pH or both were restored to inhibitory levels at 2 hr, there was no increase in DNA synthesis in the cultures. If these inhibitory operations were begun at 4 hr when DNA synthesis had already been initiated in a few cells, the removal of serum alone slowed the ensuing steep entry into S by about two-thirds, but the reduction in pH, and particularly the combined treatment, effectively stopped the progression. When the same operations were instituted at 6 hr in the midst of the steep ascent of cells entering S, the removal of serum alone or reduction of pH decreased the rate of entry into S by about one-half or three-fourths, respectively. The combination treatment stopped further progression into S but allowed continuing DNA synthesis in those cells already in the S phase. In effect, then, the passage from G1 into the S phase required the presence of the full stimulatory conditions throughout the entire G1 period of individual cells, though partial escape into S was permitted by maintaining a lower degree of stimulation (Rubin and Steiner, 1975). A similar conclusion could be derived from the use of a purified growth factor MSA (multiplication stimulating activity), which had weaker activity than serum as the stimulant of quiescent CEF (Bolen and Smith, 1977). Unlike removal of serum at different times after the beginning of the S phase in the CEF population, which gave similar partial inhibitions to those already described for the removal of serum, the removal of MSA stopped further progress just as the combination treatment did. The authors concluded that irreversible commitment to DNA synthesis occurred at or near the G1–S boundary. In light of these observations and similar ones in other systems (Prescott, 1968), it is apparent that an early triggering event that irreversibly sets off other processes leading to DNA synthesis cannot account

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Fig. 1 DNA synthesis after growth stimulation of high-density quiescent CEF cultures, and reversal of the stimulation at later intervals. DNA synthesis was turned off by overnight removal of serum and lowering of pH to 6.8. Serum was then restored and pH raised to 7.5 and the cells labeled with 3H-thymidine at 0, 2, 4, 6, and 7 hr. — . At 2, 4, and 6 hr, several cultures were changed to no serum at pH 7.5, – –&; serum at pH 6.8, – –!; no serum at pH 6.8, – –~; and were labeled with 3H-thymidine at 7 hr (Rubin and Steiner, 1975).





 



for the onset of the S phase. Evidence to be considered in the following text indicates that the rate of protein synthesis is the key process that determines the rate of progress through G1, and that the transition to S is brought about by synthesis of a particular protein (cyclin?) just before the transition.

III. REQUIREMENT OF RNA AND PROTEIN SYNTHESIS FOR THE INITIATION OF DNA SYNTHESIS Reproduction of the cell requires a doubling in all its constituents. Protein makes up the largest part of the structure of the cell and RNA, particularly that associated with ribosomal structure, makes a significant contribution to the dry mass of the cell. The rate of protein synthesis in a cycling cell

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increases throughout the entire interphase, reaching a level at mitosis twice that at the beginning of G1 (Zetterberg and Killander, 1965). Analysis of growth in single cells suggests that the initiation of DNA synthesis depends on cell mass (Killander and Zetterberg, 1965). A sharp reduction in serum concentration of perfusion-grown normal human fibroblasts decreases the rate of protein synthesis within 2 hr and increases the rate of protein degradation, resulting in cessation of protein accumulation (Castor, 1977). DNA synthesis only begins to decline at 6 hr. Contact inhibition between normal human fibroblasts also reduces the rate of protein, RNA, and DNA synthesis, and these changes are associated with a large reduction in free cytoplasmic polysomes (Levine et al., 1965). A 10-fold reduction in the rate of protein accumulation is accompanied by only a 37% decrease in the rate of protein synthesis, indicating a role for degradation. Contact inhibition in hamster cells is associated with about a two-thirds decrease in the rate of protein synthesis (Stanners and Becker, 1971). The reduction can be accounted for by the observations that (a) the average cell in stationary phase contains about half the total number of ribosomes per cell as the average cell in exponential growth and (b) only two-thirds of the ribosomes are bound in polysomes in stationary phase while virtually all of them are bound in polysomes in the exponential phase. The polysomebound ribosomes of the stationary phase function with the same efficiency as those in the exponential phase, and produce proteins of about the same average length. The results suggest that the higher proportion of free ribosomes in stationary phase is not due to a limitation of messenger RNA but to a decreased probability of attachment of ribosomes to messenger RNA (Stanners and Becker, 1971). Related results were found after treatment of chick embryo epidermis with epidermal growth factor (EGF) (Cohen and Stastny, 1968). Within an hour after addition of EGF to the epidermal cells, there is a conversion of preexisting ribosomal monomers into polysomal structures with an increase in protein synthesis accompanied by synthesis of all classes of cytoplasmic RNA. The conversion of ribosome monomers to polysomes and the stimulation of RNA synthesis do not require synthesis of new protein, nor do they require the increased transport of glucose and amino acids induced by EGF. The ribosome monomers decrease to 70% of their unstimulated value within half an hour and to a stabilized level 30 to 50% of their unstimulated value at 1.5 hr. None of the articles describing the changes in polysome formation with growth state has proposed a mechanism based on observations of the in vitro formation of polysomes. It should be noted, however, that the rate of protein synthesis in vitro, which is based on the formation of polysomes, is acutely dependent on free Mg2þ concentration, estimated in some mammalian cells to be about 1 mM (Rink et al., 1982). However, a wide variety of free Mg2þ concentrations have been reported depending on the method

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used for measurement, the tissue involved, and its metabolic state (Garfinkel and Garfinkel, 1988). Most of the estimates fall below 1 mM, some as low as 0.1 to 0.2 mM, with many in the range of 0.4 to 0.6 mM. The results as a whole show that only a small fraction of the total cell Mg2þ of 10 to 20 mM is free, and that varies with metabolic conditions. Newer estimates of cytosolic Mg2þ fall in the range of 0.25 to 1.0 mM (Grubbs, 2002), where it can exert substantial control on metabolism (Garfinkel and Garfinkel, 1988). Protein synthesis on mammalian polysomes in vitro increases sharply between 0.6 and 2.6 mM Mg2þ and decreases almost as sharply between 2.6 and 5.6 mM Mg2þ in a bell-shaped curve when corrected for the chelating action of ATP and GTP (Schreier and Staehelin, 1973) (Fig. 2). The optimal, uncorrected Mg2þ concentration varies with the source of messenger RNA from 2 to 3 mM Mg2þ concentration (Brendler et al., 1981; Ilan and Ilan, 1978). Protein synthesis in vitro also varies with Kþ concentration over a much broader range of about 40 to 120 mM Kþ. There is not an absolute requirement for Kþ, however, since it can be replaced by NH4þ

Fig. 2 Mg2þ and Kþ concentration dependencies of globin synthesis in vitro by mouse liver ribosome subunits labeled with 14C-leucine. (A) Mg2þ-dependence in 70 mM Kþ. After correction for chelation of Mg2þ by ATP and GTP, the effective Mg2þ concentration is 1.4 mM lower than that indicated. (B) Kþ-dependence in 3 mM Mg2þ – – ; in 4 mM Mg2þ — (Schreier and Staehelin, 1973).

 

 

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in bacterial polysome formation in vitro (Meselson et al., 1964). The initiation of protein synthesis requires a higher Mg2þ concentration than elongation of the peptide chain (Revel and Hiatt, 1965; Schreier and Noll, 1971). Contrary to the general view, the Mg2þ-sensitive step is not the binding of messenger RNA to the small ribosomal subunit but is thought to be the first translocation (Schreier and Noll, 1971). The significance of these findings for the role of Mg2þ in control of protein synthesis in cultured animal cells will be discussed later in the appropriate places.

IV. EFFECTS OF INHIBITORS OF PROTEIN SYNTHESIS ON INITIATION OF DNA SYNTHESIS The foregoing results indicate essential roles for protein and RNA synthesis in the initiation of DNA synthesis. Specific inhibitors of these syntheses can provide a more precise indication of the roles of each of these macromolecular species in growth regulation. Cycloheximide or actinomycin were added to synchronized HeLa cells to inhibit protein synthesis or RNA synthesis, respectively (Kim et al., 1968). Exposure to either drug in the G1 period completely prevented the onset of DNA synthesis. Exposure to a relatively large dose of cycloheximide in the DNA synthetic phase that immediately abolishes protein synthesis also shuts down DNA synthesis, whereas actinomycin D allowed the cells to continue DNA synthesis for 2 hr before steeply reducing its synthesis. The results indicate that DNA synthesis is directly dependent on concurrent synthesis of protein and only after a 2-hr delay on the synthesis of RNA. That delay may be due to the need for exhaustion of messenger RNA molecules before its effect on protein synthesis is felt. The initiation of DNA synthesis is more sensitive to inhibitors than is the continuation of DNA synthesis. The activities of several enzymes necessary for the formation of DNA were not reduced by several hours of treatment during the S period with either drug while the rate of DNA synthesis was markedly reduced. It was proposed that there is a protein with a very short half-life that is necessary for continuing DNA synthesis (Kim et al., 1968). The addition of low concentrations of cycloheximide at the end of the G1 period to synchronized 3T3 cells rapidly reduced the rate at which the cells enter the S phase by an amount proportional to the inhibition of protein synthesis (Brooks, 1977) (Fig. 3). This suggests that the initiation of DNA synthesis depends on the continuous synthesis of a protein with a short halflife. The rate-limiting transition occurs within 2 hr of the start of DNA synthesis. The addition of a very low dose of cycloheximide (33 ng/ml) at the beginning of the G1 period completely suppressed DNA synthesis over a

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Fig. 3 Inhibition of protein synthesis by cycloheximide and its effect on the initiation of DNA synthesis in Swiss 3T3 cells. Quiescent cells were stimulated with serum in the presence of the indicated concentrations of cycloheximide. Cultures were pulse-labeled for protein synthesis with 3H-leucine at 2 hr (d), and continuously labeled for 24 hr with 3H-thymidine ( ). Ordinate: radioactivity incorporated as percentage of control (no cycloheximide) (Brooks, 1977).



24-hr period although protein synthesis itself was reduced by less than 30%, again indicating the acute sensitivity of the initiation of DNA synthesis to the rate of protein synthesis. These results suggested that the transition from G1 to S periods depends on the synthesis of a protein with a short half-life (Brooks, 1977). Additional evidence for a labile protein needed for the initiation of DNA synthesis came from a variety of experimental manipulations of protein synthesis during the G1 period (Rossow et al., 1979; Schneiderman et al., 1971).

V. CYCLINS AND CDKs, SPECIFIC PROTEINS REQUIRED FOR THE TRANSITION FROM THE G1 TO THE S STAGES OF THE CELL CYCLE When cells are maintained in culture under conditions that increase their generation time, they do so by expanding the G1 period (Prescott, 1968). For example, CEF maintained in the absence of serum and/or at low pH can be maintained in healthy condition for 2 days during which very few cells

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enter the S period, but the addition of serum and/or raising pH allows most of the cells to initiate DNA synthesis within 4 to 6 hr (Rubin and Steiner, 1975). Different epithelia of the living mouse have average generation times varying from 16.7 hr in the lower ileum to 181 hr in the esophagus, but the S period remained about 7 hr in all the tissues (Cameron and Greulich, 1963). Although there may be some variation in the length of the G2 period, the greatest flexibility undoubtedly lies in the G1 portion of the interphase. G1 is primarily a period of growth during which the cell has to reach a minimal mass before initiation of DNA synthesis, and the rate of its accomplishment apparently depends on the amount of general metabolic machinery. The regulation of cell proliferation therefore depends on the rate of protein synthesis during G1 (Liskay et al., 1980; Rossow et al., 1979). As has been indicated, however, the requirement of protein synthesis up to the time of DNA synthesis suggested that synthesis of a particular, unstable protein (or proteins) is needed to bring about the transition to the S period (Brooks, 1977; Kim et al., 1968; Liskay et al., 1980; Rossow et al., 1979; Schneiderman et al., 1971). Although existence of an unstable protein that is required for the initiation of DNA synthesis in animal cells has been posited for some time (see Prescott, 1968), one of the first, if not the first, isolation of a protein that apparently had the requisite properties was reported only in 1983 (Croy and Pardee, 1983). At this time, the concept was developing that there were cell division cycle (CDC) mutants in yeast that arrested division at unique stages of the cell cycle regardless of the time they were shifted from permissive to restrictive temperature (Hartwell, 1991). It should be noted, however, that ribonucleotide reductase, the enzyme that converts cytosine monophosphate to dCMP, exhibits a strict parallelism with DNA synthesis (Turner et al., 1968). It was estimated that there were as many as 500 genes with stage-specific functions, or 10% of the yeast genome. Attention turned to mammalian cells and proteins that control the transition from G1 to S. Assuming that vertebrates have the same genomic proportion of stagespecific functions as does yeast, there would be thousands of genes controlling such functions in animal cells. Candidate regulators of the G1/S transition included cyclins and their associated protein kinases (Matsushime et al., 1991). Cyclins were first identified in marine invertebrates as proteins that undergo periodic fluctuations during each cell cycle. Genes required for G1 progression in yeast include cyclins 1, 2, and 3. Because the mutants of the 3 genes are functionally redundant, their combined inactivation is required to induce cycle arrest in G1. Observations that the transcripts of cyclin 1 and 2 increase during G1 and decrease as the cells enter the S phase, and noting the association of cyclin 2 protein with a CDC kinase, reinforce the view that these genes form part of the regulatory apparatus that governs the G1/S transition in yeast. The first cyclinlike (CYL) protein was

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reported in mouse macrophages (Matsushime et al., 1991). Deprivation of a growth factor during G1 leads to degradation of a CYL protein and correlates with failure to initiate DNA synthesis. The timing of this CYL expression, its rapid turnover in absence of the growth factor, and its transient binding to a CDC-related polypeptide suggested that this CYL gene functions during S phase commitment. It was later called cyclin D1 and was recognized as a member of a family that includes two other related genes, cyclins D2 and D3 (Sherr, 1993). The cyclins bind to and activate cyclin-dependent kinases (CDKs) which phosphorylate proteins that control the transition from the G1 to the S phase of the cell cycle. The levels of mRNAs for cyclins D1 and D3 decrease in human diploid fibroblasts upon serum depletion or at high cell densities (Won et al., 1992). Following stimulation of quiescent fibroblasts with serum, the mRNA levels increase gradually to a peak at 12 hr prior to the onset of the S phase and then decline, which suggests a correlation between their gene products and the induction of DNA synthesis. However, induction of these genes is not sufficient for the transition from quiescence into S phase (Won et al., 1992). Cyclin E was maximal in synchronized Hela cells near the G1–S phase boundary as was the cyclin E-associated protein kinase activity (Dulic et al., 1992). The kinase activity declined when cells entered G2. Cyclin E associated with Cdk2 and induced maximal levels of cyclin E-dependent kinase activity at the G1–S transition (Dulic et al., 1992; Koff et al., 1992). It is thought that cyclins D and E might fulfill different cyclinlike functions required for controlling late G1 restriction point control, with cyclin A triggering S phase; alternatively, cyclin E might be necessary for the actual onset of DNA synthesis (Sherr, 1994). Many other elements of cell cycle control have been identified, including ubiquitin ligases for degrading cyclins and proteins that inhibit Cdk activity by dephosphorylation (Alberts et al., 2002). Since there are likely to be thousands of stage-specific proteins in vertebrate cells based on the estimates of the number in yeast (Hartwell, 1991), the level of complexity of checkpoints controlling the progress of cells through G1 and the transition to the S phase will continue to grow as the number of identified proteins increases. The cyclin-controlled checkpoints, however, are sequential obligatory gatekeepers that are dependent on the orderly progression of cells through the cycle. They are not determinants of the rate of progression. They occur in proper order whether cells are growing slowly or rapidly. Our focus here is on the regulation of the rate at which cells cycle, and particularly what moves them from a relatively stationary state with an expanded G1 phase to a rapidly multiplying state with a much shortened G1 phase. That change in state depends on events that begin with a persistent perturbation of the cell membrane by specific growth factors or nonspecific agencies of various kinds and is maintained at least through the G1 period for a single cycle of initiation of DNA synthesis, or throughout the

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cell cycle for a doubling of an entire cell population. Basically, we are interested here in what changes are entrained within the cell by the membrane perturbations that drive the cell through the G1 period at an accelerated rate. The molecular biology of such changes will be considered in a later section.

VI. ROLE OF Mg2þ IN GROWTH REGULATION As noted earlier, there is a direct relationship in cells between the rate of protein synthesis in G1 and the rate of passage through G1 into the S phase of DNA synthesis (Brooks, 1977; Kim et al., 1968; Stanners and Becker, 1971). Protein synthesis itself depends on polysome formation which is acutely sensitive to the concentration of free Mg2þ (Brendler et al., 1981; Schreier and Staehelin, 1973). Also, ribosomal monomers combine with mRNA and are converted into polysomes in the absence of protein synthesis within half an hour of adding growth factor to cells and without the increased glucose or amino acid transport evoked by the growth factor (Cohen and Stastny, 1968). The external stimulus has to be present through most, if not all, of G1 in order to initiate DNA synthesis (Bolen and Smith, 1977; Brooks, 1977; Rubin and Steiner, 1975). These relationships suggested that perturbation of the cell membrane by specific or nonspecific stimulants of growth releases Mg2þ from binding sites on phospholipids and proteins on the internal surface of the plasma membrane (Dawson and Hauser, 1970) or other membrane sites (Sanui, 1970), thereby increasing the free Mg2þ concentration to accelerate the rate of protein synthesis and progress through G1 to S. Changes in Mg2þ pump activity could also contribute to increased cellular Mg2þ (Lostroh and Krahl, 1974). To determine whether altering the cellular content of Mg2þ would influence the rate of DNA synthesis, the author varied the concentration of Mg2þ in the medium downward from physiological levels to almost zero and the effect on DNA synthesis was recorded in sparse and crowded CEF cultures (Rubin, 1975a). There was a sharp decrease in the rate of DNA synthesis in the sparse cultures when measured after 16 hr when the concentration of external Mg2þ was reduced below 0.2 mM. There was a less dramatic decrease of DNA synthesis in the crowded cultures, with less than 0.1 mM Mg2þ. The results obtained at the low levels of Mg2þ were erratic, so a buffering agent was sought that would complex most of the Mg2þ in the medium but maintain the free Mg2þ at a constant level. Phosphorylated compounds like ATP and inorganic pyrophosphate, which bind Mg2þ a little more firmly than Ca2þ, inhibited DNA synthesis at 16 hr when their concentration exceeded that of Mg2þ, but were largely independent of the concentration of Ca2þ. The effect of Mg2þ binding by inorganic pyrophosphate was examined on other elements of the coordinate response to growth factors.

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15

There were decreases in the rates of 2-deoxyglucose uptake, synthesis of RNA and protein, and lactic acid production. The cells remained in healthy condition, and the effects were fully reversed by adding excess Mg2þ, but not Ca2þ, to the medium after 16 hr of inorganic pyrophosphate treatment. Similar results were obtained in crowded CEF cultures by simply lowering Mg2þ in the medium below 0.1 mM (Kamine and Rubin, 1976). The intracellular concentration of Mg2þ and Kþ decreased almost twofold when external Mg2þ was lowered from 0.8 to 0.016 mM; by contrast, intracellular Naþ and Ca2þ levels increased 2.2- and 1.5-fold, respectively (Sanui and Rubin, 1977). The external Mg2þ effects were greater when the Ca2þ concentration of the medium was decreased from 1.0 mM to 0.2 mM (Fig. 4). The decreases in DNA synthesis paralleled the decrease of intracellular Mg2þ. The results also indicated that the decreased level of Ca2þ in the medium increased the permeability of the cells, thereby allowing freer exchange of Naþ, Kþ, and Mg2þ.

Fig. 4 Mg2þ dependence of DNA synthesis and cation content in CEF. Quiescent cells were stimulated with medium containing serum in 0.22 mM Ca2þ and varying concentrations of Mg2þ. At 16 hr, the cellular cation concentrations were determined in some cultures, while others were labeled with 3H-thymidine for 1 hr (Sanui and Rubin, 1977).

16 Table II

Harry Rubin Intracellular Cation Changes 16 to 17 hr After Adding Mitogen

Cation

Insulin 0.1 unit/ml on CEF (59)

Calf serum 20% on Balb/c 3T3 cells (68)

Mg2þ Kþ Ca2þ Naþ

þ22% þ14% No change Negligible

þ14% þ3% 58% 9%

The significance of these observations for physiological growth control was evaluated by measuring changes of intracellular cation content after adding growth factors to quiescent cell cultures. The addition of insulin in graded concentrations from 0 to 0.1 U/ml to serum-free medium on confluent CEF cultures for 16 hr resulted in a 17-fold increase in DNA synthesis which began to approach a maximum with as little as 0.01 U/ml of insulin (Sanui and Rubin, 1978). Concomitantly, there were graded increases in intracellular Mg2þ (22%) and Kþ (14%) with no change in intracellular Ca2þ (Table II). Naþ rose slightly with 0.01 U/ml insulin but remained at control levels with 0.1 U/ml insulin. The intracellular concentrations of Mg2þ and Kþ increased relative to controls within 10 min after the addition of 0.1 U/ml insulin and remained higher through 16 hr. There was no significant effect of insulin on total Ca2þ or Naþ at any time through 16 hr. It will be shown later that only the increases of intracellular Mg2þ induced by the insulin treatment had significant effects on the rate of protein synthesis, which were later translated into increases in DNA synthesis (Moscatelli et al., 1979). The externally bound cations were determined by washing the cells 5 times in CO2-free 0.25 M sucrose solution to remove unbound cations without displacing those that were bound (Sanui and Rubin, 1978). The surface-bound cations were removed for measurement by a 10-sec wash with carbonated (pH 4) 0.25 M sucrose to exchange Hþ for the cations (Sanui and Rubin, 1979b). Hþ are especially efficient in displacing bound cations because they are bound to membranes about 100 times more tightly than Ca2þ or Mg2þ, which, in turn, are bound about 100 times more tightly than Naþ and Kþ (Carvalho et al., 1963). There was a sharp decrease in externally bound Ca2þ of cells treated with 0.001 units of insulin and a smaller drop in Mg2þ. There was a more gradual decrease in externally bound Ca2þ and Mg2þ with higher doses of insulin up to a maximal decrease in 0.1 units of insulin of 34% externally bound Ca2þ and 45% externally bound Mg2þ. Data for externally bound Kþ and Naþ showed wide variation but the general trend was upward in exchange for the loss of externally bound Ca2þ and Mg2þ. It was estimated that about 105 divalent cations were displaced per bound insulin. This relation differs by 5 orders of magnitude from the stoichiometric

Mg2þ in Cell Growth Regulation and Transformation

17

changes in bound cations produced by cation exchange or by complexing agents like ATP or EDTA (Sanui and Pace, 1967). These results are consistent with the suggestion (Shlatz and Marinetti, 1972) that insulin induces a generalized conformational change in the membrane to which it binds and thereby causes the gross changes in bound cations. While Caþ is the major divalent cation bound to the external surface of intact cells, Mg2þ would be the dominant divalent cation bound to the inner surface of the membranes since there is so much more free Mg2þ than free Ca2þ in the cell. Any conformational change in the insulin-treated membrane would be expected to release enough Mg2þ to significantly raise the free Mg2þ concentration of the cytosol. Cortisol has effects on DNA synthesis and other elements of coordinate control in CEF that are antagonistic to the stimulatory effects of insulin (Fodge and Rubin, 1975a; Rubin, 1977). It inhibits the uptake of 2-deoxyglucose and uridine and the incorporation of uridine and thymidine into acid insoluble material. It has been estimated that 3000 additional divalent cations are bound to purified rat liver membranes for every dihydroxycortisone molecule bound, which indicates that it too produces a conformational membrane change (Shlatz and Marinetti, 1972). This would result in an increase in binding of cytosolic Mg2þ and a lowering of free Mg2þ. Lowering intracellular Mg2þ by depriving cells of the external supply reproduces the same coordinate inhibition of transport and metabolism as does the addition of cortisol or removal of serum (Rubin, 1976) and reinforces the idea that Mg2þ acts as the primary secondary messenger for the hormones. Although insulin is sufficient to stimulate DNA synthesis in CEF in the absence of serum, additional growth factors are needed to stimulate DNA synthesis in the established line of Balb/c 3T3 mouse fibroblasts, which have a much higher serum requirement for growth than CEF. The 3T3 cells were grown to confluence in 10% serum and incubated to a quiescent state for 1 day in 1% serum (Sanui and Rubin, 1982a). They were then shifted to media with 0 to 20% serum for 17 hr when measurements were made of the intracellular concentrations of the 4 cations and the rate of DNA synthesis. The rate of DNA synthesis increased 30-fold between 0 and 20% serum. The intracellular cation concentrations at 20% serum as compared with 0% serum were increased 14% for Mg2þ and 3% for Kþ, but were decreased 58% for Ca2þ and 9% for Naþ (Table II). Hence, the only significant increase in intracellular cation content was Mg2þ, with Ca2þ and Naþ showing large to minimal decreases in concentrations, respectively. The major increase in intracellular Mg2þ occurred between 2 and 5 hr with further increases to 10 hr and a slight increase to 15 hr of serum treatment. The major seruminduced increase in DNA synthesis of 3T3 cells occurred between 10 and 17 hr, unlike insulin-stimulated CEF, which began DNA synthesis in 4 to 5 hr after adding serum (Rubin and Steiner, 1975). The increase in Mg2þ and that

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of protein synthesis precedes that of DNA synthesis by several hours in stimulated CEF and 3T3 cells (Rubin and Sanui, 1979). Externally bound Mg2þ decreased gradually with increasing serum concentration to a level in 20% serum being 45% of that in the absence of serum (Sanui and Rubin, 1982a). Externally bound Ca2þ was much higher than Mg2þ and decreased sharply between 0 and 1% serum with a continued decrease to 20% serum down to 22% of the value of externally bound Ca2þ in the absence of serum. Hence, the use of serum as growth stimulant revealed the same picture with regard to release of externally bound cations as that found in the stimulation of CEF by insulin (Sanui and Rubin, 1978). Although no estimate was made of the number of binding sites for serum growth factors, some of which remain unknown, it is likely that the number of divalent cations released exceeds by far the number of receptor-growth factor interactions as was the case for insulin, and involves a conformational change in the membrane. As in the insulin case, the conformational change would release much more Mg2þ than Ca2þ from the inner side of the membrane and significantly raise the level of free Mg2þ in the cytosol by cation exchange, mainly with Kþ, the major intracellular cation. The increased free Mg2þ would propel increased protein synthesis and quicker transit through G1 to the onset of DNA synthesis. Although the release of Mg2þ from internal membranes would raise the cytosolic free Mg2þ, it would not account for the increase in total intracellular Mg2þ observed in cell stimulation by insulin or serum. That increase must come from increased uptake of extracellular Mg2þ into the cell. The increased total Mg in itself suggests that free Mg2þ also increases. It has, in fact, been reported that free Mg2þ rises disproportionately with increases in total Mg2þ (Corkey et al., 1986). That would imply that free Mg2þ would rise more than 20% after stimulation. Given the steep dependence of protein synthesis on Mg2þ concentration (Schreier and Staehelin, 1973) and of DNA synthesis as a function of protein synthesis, a sustained increase in Mg2þ would be expected to sharply increase DNA synthesis in cells. As will be described later, the increased Mg2þ operates on protein synthesis through an increase in the MgATP2, which activates a key protein kinase in a molecular pathway that regulates the initiation of translation.

VII. PROTEIN AND DNA SYNTHESIS IN VERY LOW Ca2þ WITH VARIATIONS IN Mg2þ CONCENTRATIONS Extreme reduction of Ca2þ in the medium from 1.5 mM to 0.02 mM reduces surface-bound Ca2þ by 75% and intracellular Ca2þ by 40% (Rubin et al., 1978). It increases the passive permeability of the cell as indicated by

Mg2þ in Cell Growth Regulation and Transformation

19

measured uptake of L-glucose (Bowen-Pope and Rubin, 1977), and inhibits the onset of DNA synthesis in crowded but not sparse cultures of 3T3 cells. The inhibition of DNA synthesis does not result from a reduction in total intracellular Mg2þ since it remains constant, but DNA synthesis is reversed by raising Mg2þ in the medium from 1.0 to 19 mM Mg2þ, which raises intracellular Mg2þ by about 40%. In contrast, the inhibition of DNA synthesis induced by drastic reduction of Mg2þ is not reversed by raising Ca2þ. These results suggest that the 40% reduction in intracellular Ca2þ induced by very low Mg2þ in the medium results in an increase of binding of intracellular Mg2þ to negatively charged membrane sites once occupied by Ca2þ, thereby bringing on a decrease in free Mg2þ which is compensated by raising external Mg2þ. The failure of high Ca2þ to reverse the inhibition produced by very low Mg2þ supports the primary role of Mg2þ in growth regulation. Increasing extracellular Mg2þ above 20 mM in very low Ca2þ inhibits DNA synthesis, giving a bell-shaped curve for DNA synthesis as a function of Mg2þ concentration that resembles the relation between Mg2þ concentration and protein synthesis in vitro (Fig. 2). The foregoing results prompted a study of the relation of protein synthesis to DNA synthesis as a function of Mg2þ concentration in very low Ca2þ (Rubin et al., 1979). Protein synthesis increased sharply with increasing external Mg2þ in the presence of 0.02 mM Ca2þ, and then decreased with very high concentrations of Mg2þ when measured at 3 hr after changing the cation concentrations (Fig. 5). The figure shows only the internal Mg2þ concentrations, but the corresponding external concentrations are in the figure legend. The peaks of synthesis of protein at 3 hr and DNA at 17 hr did not always coincide precisely with each other as they do in Fig. 5. However, both functions always exhibited a bell-shaped curve as a function of Mg2þ concentration. External Mg2þ of 35 mM in 0.02 Ca2þ was inhibitory for protein synthesis at 5 hr, but it began to rise at 10 hr and was at a maximum level at 22 hr (Fig. 4 in Rubin et al., 1979). The high level of intracellular Mg2þ at 3 hr in 30 mM external Mg2þ and low Ca2þ decreased in the following hours. These results indicated that the cells extruded their excess inhibitory Mg2þ and thereby increased the rate of protein synthesis, followed in about 5 hr by an increase in DNA synthesis. This behavior further supported a causal consecutive relationship between intracellular Mg2þ concentration, protein synthesis, and DNA synthesis. External Mg2þ of 48 mM in low Ca2þ reduced protein synthesis almost to zero at 3 hr with no recovery at a later time. Intracellular Mg2þ continued to rise with time, indicating the cells were unable to extrude a large excess of inhibitory Mg2þ. These results again confirmed a causal, tandem relationship between intracellular Mg2þ levels, protein synthesis, and DNA synthesis. At very low concentrations of both extracellular Ca2þ and Mg2þ, intracellular Naþ rose sharply and Kþ declined at 3 hr, but at higher extracellular

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Fig. 5 Rates of protein and DNA synthesis in Balb/c 3T3 cells as a function of intracellular Mg2þ concentration in very low external Ca2þ concentration. Quiescent cultures were stimulated with serum in medium with 0.02 mM Ca2þ and 1.0, 19, 30, and 48 mM Mg2þ. At 3 and 17 hr, the intracellular concentration of Mg2þ of each group was measured. At 3 hr, the rate of protein synthesis was determined by labeling with 3H-leucine, ~– –~, and at 17 hr, the rate of DNA synthesis was determined by labeling with 3H-thymidine, — . Controls in 1.7 mM Ca2þ and 1.0 mM Mg2þ were labeled with 3H-leucine m– –; or 3H-thymidine d—. The rates of incorporation are plotted against the intracellular concentrations of Mg2þ (Rubin et al., 1979).

 

Mg2þ beginning at 1.0 mM and extending through 48 mM, the intracellular Naþ and Kþ remained normal. The increased Naþ and reduced Kþ at 3 hr returned to normal values at 17 hr. Since the rates of protein and DNA synthesis remained at a markedly reduced level at 17 hr in the combination of very low Ca2þ and Mg2þ and in the combination of very low Ca2þ and very high Mg2þ, it is apparent that neither Kþ nor Naþ influenced the major macromolecular syntheses. Since intracellular Ca2þ remained constant under all the conditions, it is also evident that intracellular Mg2þ was the only cation whose concentration was correlated with protein synthesis, which later determined DNA synthesis. Uridine uptake, which depends on the rate of its phosphorylation, is stimulated within a few minutes of the addition to quiescent cells of 10%

Mg2þ in Cell Growth Regulation and Transformation

21

serum in physiological Ca2þ and Mg2þ (Rozengurt and Stein, 1977). The high serum fails to stimulate uridine uptake in low Ca2þ with low Mg2þ but does so if the Mg2þ level is raised to 10 or even to 40 mM (Bowen-Pope et al., 1979). Since 40 mM Mg2þ sharply depresses protein synthesis and DNA synthesis, it raises the question whether any of the early transport responses significantly affect the macromolecular syntheses required for growth.

VIII. KINETICS OF CELLULAR RESPONSIVENESS TO Mg2þ LIMITATION IN PHYSIOLOGICAL Ca2þ The wide variations in Mg2þ content of cells already described and their effects on protein and DNA synthesis were made possible by varying external Mg2þ over a wide range in very low external Ca2þ, which itself alters cellular cation content, although only temporarily. A transformed clone of 3T3 cells was found in which intracellular Mg2þ could be reduced to 50% of normal by severely lowering external Mg2þ for 12 hr in physiological concentrations of Ca2þ, and quickly restored to normal after 2.5 days by adding back Mg2þ (Fig. 6) (Terasaki and Rubin, 1985). This allowed observation of the dynamics of change in protein and DNA synthesis to determine how sensitive these functions are to intracellular Mg2þ in physiological Ca2þ. The deprivation of external Mg2þ for only 3 hr reduced cellular Mg2þ to 84% of control values and to 67% at 12 hr, beyond which it leveled off at 50% of control values (Fig. 6A). The rate of protein synthesis decreased to almost exactly the same extent and with the same kinetics as that of cellular Mg2þ (Fig. 6B). There was a 12-hr delay before the rate of DNA synthesis began to decline, but it reached a level 100-fold less than the control value between 36 and 48 hr (not shown), with only a minor decrease in cellular Kþ within a range known to have no effect on cellular activities (Moscatelli et al., 1979). It is important to note that there was no loss of total protein in the limited concentration of Mg2þ. In fact, there was a slight increase in total protein and the cells appeared in healthy condition after 60 hr with the only morphological change a flattening out so they appeared more like normal than transformed cells. Restoration of physiological Mg2þ to the deprived cells was followed by a coordinated return to normal of cellular Mg2þ and protein synthesis (Fig. 6C, D). Both parameters doubled within 1 hr and remained at that level thereafter. The rate of DNA synthesis showed its first increase between 8 and 12 hr after restoration of Mg2þ and rose rapidly to normal levels at 24 hr with a kinetics paralleling that of serum stimulation. The total protein

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Fig. 6 Parallel decreases with time in Mg2þ content and rates of protein synthesis in Balb/c 3T3 cells with deprivation of Mg2þ and their increase following restoration of Mg2þ. Medium with either 0.02 mM or 1.0 mM Mg2þ was added to Balb/c 3T3 cultures with 1.7 mM Ca2þ and the rate of protein synthesis (A), and intracellular Mg2þ content (B) determined at indicated intervals up to 48 hr. The medium was then changed in the remaining low-Mg2þ cultures to medium with 1.0 or 0.02 mM Mg2þ and rate of protein synthesis (C), and the Mg2þ content (D) determined at intervals up to 12 hr continuously in 1.0 mM Mg2þ d—d; 0.02 mM Mg2þ d– –d; 48 hr in 0.02 mM Mg2þ, then switched to 1.0 mM Mg2þ — (Terasaki and Rubin, 1985).

 

level in the Mg2þ-deprived cultures remained constant throughout while a detectable and steady increase with the restoration of Mg2þ began at 4 hr. The overall results of this experiment showed that the rate of protein synthesis correlated directly and proportionately with changes in cellular Mg2þ. The magnitude of the changes in protein synthesis were within the limits of those reported for the removal or restoration of growth factors in a variety of cell systems (Castor, 1977; Cohen and Stastny, 1968). The role of growth factors in altering rates of protein and DNA synthesis can therefore be fully accounted for by changes in total cellular Mg2þ, which is presumably reflected in free cytosolic Mg2þ.

Mg2þ in Cell Growth Regulation and Transformation

23

IX. MITOGEN-INDUCED INCREASES IN CYTOSOLIC FREE Mg2þ It was presumed in this work that the increases in total cellular Mg observed after treatment of CEF with insulin or of 3T3 cells with serum were associated with increases in free Mg2þ. This presumption was consistent with the observation that hepatocytes from streptozotocin-induced diabetic rats had 22% less total Mg2þ than those from normal rats and their free Mg2þ was 55% less as determined by null point titrations (Corkey et al., 1986). Total Mg2þ decreases in lymphoma cell lines as the cells enter the stationary phase (Hosseini and Elin, 1985). Somewhat surprisingly, the fraction of bound Mg2þ, as determined by specialized methods, increases as the total decreases, implying that free Mg2þ decreases disproportionately. It was reported that total cellular Mg2þ increased sevenfold with time after quiescent, confident mammary epithelia were subcultured at low density in correlation with DNA synthesis and returned to basal level when they became confluent again (Wolf et al., 2004). However, these findings did not establish that an increase in free Mg2þ occurred in cells treated with growth factors. The development of a Mg2þ-sensitive indicator mag-fura-2 (Murphy et al., 1989; Raju et al., 1989) facilitated the measurement of intracellular free Mg2þ. Epidermal growth factor (EGF) produced a 48-fold increase of DNA synthesis in a serum-starved line of muscle cells. Free Mg2þ increased after a 5-min lag period, rising gradually from an initial 0.32 mM to as high as 1.4 mM at 20 min after the addition of EGF to responsive cells and then leveling off (Grubbs, 1991). The dependence of EGF for increase in free Mg2þ was similar to that of DNA synthesis. There were no changes of pH or free Ca2þ during 20 min in the EGF-stimulated cells. The mag-fura-2 indicator was also used to monitor free Mg2þ in quiescent, confluent Swiss 3T3 cells stimulated by insulin or by EGF in combination with insulin (Ishijima et al., 1991). The combination led to a significant increase in free Mg2þ from basal 0.22 mM to 0.29 to 0.35 mM after 30 to 60 min. The free Mg2þ then began to decline, but the measurements became unreliable because the mag-fura-2 leakage after 20 to 30 min caused considerable error. There were also increases in free Ca2þ, but they were rapid and transient in contrast to the slow, long-lived increases in free Mg2þ. Further study revealed, however, that there was a rapid, early increase in free Mg2þ in bombesin-stimulated 3T3 cells (Ishijima and Tatibana, 1994). The free Mg2þ reached peak values in 15 sec in most cells and lasted for only 1 to 2 min. It was not dependent on external Mg2þ, but was partly dependent on external Ca2þ and the action of tyrosine kinase. The physiological role of this transient increase in free Mg2þ is not known, but it is obviously not involved in the long-term exposure through G1 to growth

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factors that are associated with the increase in protein and the onset of the S phase. We can learn more by taking a step away from the multicellular state in the strict sense and growth factors per se. The oocytes of Xenopus laevis are large cells—850 to 950 m in diameter, 350 to 500 g in weight—that can be isolated from their environment by immersion in paraffin oil. The paraffin oil allows the controlled alteration of the intracellular ionic state of the oocyte by injection of ions and measurement of amino acid incorporation in a single cell without a contribution from external cations (Horowitz and Lau, 1988). Exposure of the quiescent oocyte in its follicle to gonadotropin stimulates 3 H-leucine incorporation into protein and increases intraoocytic Kþ activity for at least 10 days (Lau et al., 1988). To mimic the gonadotropin effect on Kþ activity, the oocytes’ Kþ content was raised by injection of KCl into isolated oocytes in paraffin oil and the Kþ activity measured by microelectrodes. This treatment mimicked the influence of gonadotropin on both the rate of protein synthesis and the synthesis of specific polypeptides. The findings suggested that gonadotropin-stimulated oocyte growth is attributable largely to the hormone’s influence of transfollicular Kþ fluxes and supported the hypothesis that the changes in Kþ activity are critical for subsequent increases in protein synthesis and growth. Further analysis of the results, however, showed that there was a greater effect of Kþ on translation in vivo than was expected from its behavior on an in vitro translation system (Horowitz and Tluczek, 1989). It was also found that Naþ influences protein synthesis in the oocyte to the same extent as Kþ does, but Naþ has no significant effect in cell-free translation systems. This indicated that a mechanism exists through which either Kþ or Naþ can influence translation in the intact cell but is missing or degraded in cell-free systems. Experiments showed that both Kþ and Naþ are buffered in the cell, meaning they compete with other cations for high-affinity anions such as those associated with membranes. As a consequence, an increase in Kþ or Naþ will cause other cations to become less associated with fixed ionic sites and therefore increases the activity of the cations. If one cation among those newly displaced is more potent in its influence on translation than is Kþ, this exchange or buffering reaction could be the direct effector linking gonadotropin to its metabolic effects. Injection of Ca2þ into oocytes over a very wide range of concentrations had no significant effect on protein synthesis, nor did injection of EGTA, a chelating agent for Ca2þ, have any effect on the increase of protein synthesis due to Kþ or gonadotropin, indicating that Ca2þ is not the downstream effector for the activational increase in oocyte translation (Horowitz and Tluczek, 1989). In contrast to Ca2þ, however, injection of increasing concentrations of Mg2þ at first greatly stimulated and then at higher concentrations sharply inhibited protein synthesis. The function resembled those

Mg2þ in Cell Growth Regulation and Transformation

25

obtained by injecting Kþ and Na2þ except for the much lower and narrower range of Mg2þ concentrations required to obtain the full stimulatory and inhibitory response. The optimal concentration of free Mg2þ was about 4 mM, which is similar to the uncorrected Mg2þ requirement for in vitro synthesis of protein (Schreier and Staehelin, 1973). Isotherm data suggested that the oocyte contains exchange sites occupied chiefly by Kþ and Mg2þ. As mentioned earlier, the increase in protein synthesis from increasing Kþ is much greater than expected from the behavior of cell-free systems and the response to Mg2þ is more intense than that to Kþ. It was therefore hypothesized that increasing Kþ causes Mg2þ to dissociate from intracellular sites for which both compete, thereby increasing free Mg2þ and first stimulating, then inhibiting, translation with higher concentrations of Mg2þ. The hypothesis was put to the test by injecting EDTA, which chelates both Mg2þ and Ca2þ, to see if it prevents Kþ from stimulating protein synthesis. Injection of 0.3 mM EDTA reduced protein synthesis to the quiescent cell level. Since EGTA, which is a highly specific chelator of Ca2þ, had no effect on protein synthesis, it was concluded that Mg2þ is itself the intracellular effector controlling translation rates. Injection of EDTA also inhibited the gonadotropin-induced increase in protein synthesis. About 3 times more EDTA was required to inhibit the hormone-induced increase in protein synthesis than that induced by Kþ. The reason for the difference was proposed to be that gonadotropin stimulation was done in a salt medium that contained Mg2þ, which can then enter the oocyte, whereas Kþ stimulation was done in paraffin oil in which Mg2þ cannot enter the oocyte. Horowitz and Tluczek (1989) noted that this hypothesis had already been strongly argued as the basis for the regulation of protein synthesis and proliferation of somatic cells of vertebrates (Rubin, 1975a; Rubin et al., 1979; Sanui, 1970; Terasaki and Rubin, 1985). They also remarked that the Mg2þ activity of the dormant oocyte (0.3 mM) is 80 times less than expected at electrochemical equilibrium, assuming a membrane potential of 60 mV, which indicates that the oocyte, like other cells, has a transport system that actively extrudes Mg2þ. Since gonadotropin activation is followed by long-term growth and a continuous high rate of protein synthesis, translational control by Mg2þ implies that the activational increase in its activity must be maintained during postactivational growth. This suggests that the original increase of free Mg2þ brought about largely by Kþ-driven dissociation of Mg2þ from endogenous binding sites is likely to be accompanied by a down-regulation of Mg2þ active transport to maintain postactivational growth. How gonadotropin acts to bring about these changes remains to be determined, but it may have to do with cation exchanges initiated by perturbation of the cell membrane as suggested for stimulation of somatic cells by growth factors (Sanui and Rubin, 1978, 1982b).

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X. Mg2þ EFFECTS ON DIVERSE CELLULAR RESPONSES TO GROWTH FACTORS The addition of serum or other growth stimulatory agents to inhibited cell cultures accelerates a number of early responses (Rubin and Fodge, 1974; Rubin and Koide, 1975). These include the uptake of 2-deoxyglucose, uridine, amino acids, and choline as well as the production of lactic acid. Although the increased uptake of these materials is not required for the onset of DNA synthesis and does not require protein synthesis, they are considered characteristic features of the coordinate response of cells to growth factors. If Mg2þ is the primary intermediary in converting membrane perturbation into a growth response, it might be expected to stimulate the other associated reactions. The deprivation of Mg2þ by its chelation with sufficient inorganic pyrophosphate was found to inhibit the uptake of 2-deoxyglucose and production of lactic acid as well as the synthesis of protein, RNA, and DNA (Rubin, 1975a) and the uptake of uridine (Rubin, 1976). The rate of uptake of glucose analogs in cultured cells depends on the rate of transport rather than the rate of phosphorylation (Bowen-Pope and Rubin, 1977). Removal of serum from the medium or deprivation of Mg2þ reduces the transport of the glucose analogs and does so in both cases by reducing the Vmax but not the Km of the reaction. This is consistent with a serum-induced increase in cellular Mg2þ as the driving force in the increased transport of glucose into cells. The rate of uridine uptake by contrast depends on its phosphorylation by uridine kinase (Plagemann et al., 1978; Rozengurt et al., 1977). The serum stimulation of uridine is mimicked by increasing the cellular concentration of Mg2þ, and is blocked by depleting cells of their Mg2þ (Bowen-Pope and Rubin, 1977; Vidair and Rubin, 1981). Like changing the concentration of serum in the medium, directly altering the concentration of Mg2þ in cells affects the Vmax of the uridine uptake system with little change in the Km (Bowen-Pope and Rubin, 1977). Unlike uridine, however, the uptake of thymidine is unaffected by serum treatment (Vidair and Rubin, 1981). This can be explained by the observation in cell-free extracts that the requirement of thymidine kinase for Mg2þ is less than one-tenth that of uridine kinase. It indicates that the Mg2þ level in quiescent cells is high enough to support maximal activity of thymidine kinase, but uridine kinase requires a substantial increase of Mg2þ to effectively increase the uptake of uridine upon addition of serum (Vidair and Rubin, 1981). Neither Ca2þ, Kþ, or Naþ influences the increased uptake of glucose analogs or uridine by growth factors, which adds to the support of an increase of free Mg2þ as the mechanism of these effects, as well as other reactions of the coordinate response. Perhaps the most convincing demonstration of the role of Mg2þ in

Mg2þ in Cell Growth Regulation and Transformation

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regulating the phosphorylation of uridine in cells is that raising the intracellular concentration of Mg2þ high enough in the presence of the divalent cation ionophore A23187 and very high extracellular Mg2þ increases uridine uptake in Balb/c 3T3 cells to the same extent as addition of a growth factor (Vidair and Rubin, in press).

XI. POSSIBLE ROLES OF Kþ, Ca2þ, pH, AND Naþ IN GROWTH REGULATION A. Potassium Early changes induced by growth factors have been reported in the uptake and intracellular content of cations other than Mg2þ and their possible involvement in growth regulation. Serum or purified growth factors rapidly stimulate ouabain-sensitive Naþ/Kþ-ATPase activity in Swiss 3T3 cells or CEF as measured by the uptake of 86rubidium, or by enzyme assay (Rozengurt and Heppel, 1975; Smith, 1977). Treatment with ouabain, which inhibits the Naþ/Kþ-ATPase, interferes with the accumulation of Kþ and, if the reduction of Kþ in the cells is severe enough, prevents the onset of DNA synthesis. If intracellular Kþ in human fibroblasts is reduced to 60% of control, the rate of protein synthesis is decreased by no more than 20% from control values (Ledbetter and Lubin, 1975). As Kþ is further reduced to 30% of control, rates of DNA synthesis are maximally reduced without affecting RNA synthesis (Ledbetter and Lubin, 1977). The increase in Kþ uptake after serum stimulation of quiescent 3T3 cells resulted in a 75% increase in cell Kþ on a per mg protein basis, or a 40% increase on a per volume basis (Tupper et al., 1977). This increase peaked at 4 to 5 hr and declined steadily to initial levels at 10 to 14 hr, which is about the time the S period begins in the Balb/c 3T3 cells used. However, my laboratory found a rise of Kþ of only between 3 to 12% on a per mg protein basis in different experiments with serum stimulation of quiescent confluent Balb/c 3T3 cells (Sanui and Rubin, 1982a). In CEF stimulated by insulin, there was only a 14% increase in Kþ from quiescence to maximal response (Sanui and Rubin, 1978). The latter levels of change in Kþ per cell would, according to the earlier reports (Ledbetter and Lubin, 1975, 1977), have produced no change in protein synthesis. To further explore the role of Kþ in regulating protein and DNA synthesis, CEF were incubated in varying concentrations of Kþ, from the physiological level of 5.0 mM down to almost 0.4 mM for 16 hr in the presence of insulin (Moscatelli et al., 1979). An 80% reduction of intracellular Kþ in the CEF produced no change in the rate of DNA synthesis and a 90% reduction of

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intracellular Kþ produced less than a twofold reduction in DNA synthesis. Hence, changes in intracellular Kþ concentrations within the physiological range cannot account for changes in DNA synthesis from quiescence to stimulation by growth factors. Since the reduction in Kþ uptake and cellular content in most of the previous experiments (Ledbetter and Lubin, 1975, 1977; Rozengurt and Heppel, 1975; Smith, 1977; Tupper et al., 1977) was induced by ouabain to inhibit the Naþ/ Kþ pump, but these experiments did not include measurement of divalent cations, a study was undertaken of the effects of ouabain on the major intracellular cations and synthesis of protein and DNA synthesis in Balb/c 3T3 cells (Sanui and Rubin, 1979a). At 1 hr after serum stimulation, there was a large decrease in the ouabain-treated cultures of Kþ and an increase in Naþ, with no change in Mg2þ and no reduction of protein synthesis. Beyond 1 hr, the rate of protein synthesis was significantly depressed in the ouabaintreated cultures, the value at 17 hr being 40% of control. In addition to maintaining the large changes in Kþ and Naþ seen at 1 hr, however, there were significant reductions in Mg2þ, the average at 17 hr being 85% of control, and DNA synthesis was reduced more than 10-fold. Since there was no change in cellular Ca2þ, the implications were that the large reductions in Kþ as seen at 1 hr had no effect on protein synthesis, but the later reduction in Mg2þ did reduce protein synthesis and could account for the later reduction in DNA synthesis. In another laboratory using Balb/c 3T3 cells, serum growth factors stimulated equal rates of entry into the S phase in both the presence and the absence of 100 M ouabain (Frantz et al., 1981). Since there was a decrease of intracellular Kþ in the ouabain-treated cultures, it was concluded that an increase of intracellular Kþ is not required for entry into S phase, and serum growth factors do not regulate cell growth by altering intracellular Kþ. This interpretation was countered by experiments with Swiss 3T3 cells in which intracellular Kþ of quiescent cultures was altered by reducing extracellular Kþ and stimulating the cells with purified peptide growth factors instead of serum (Lopez-Rivas et al., 1982). In that serum-free medium, the intracellular Kþ content was close to the threshold required to allow a mitogenic response, which implies that any reduction in intracellular Kþ would reduce the rate of onset of DNA synthesis. After 20 hr in the medium with peptide growth factors, the rate of DNA synthesis was very low even in those cultures with a physiological concentration of 5 mM Kþ in the medium, at a time when DNA synthesis in serum-stimulated cultures would have reached a maximum (Frantz et al., 1981). By 40 hr, there was about a sixfold increase in DNA synthesis in all cultures in the serum-free medium with the 3 highest concentrations of extracellular Kþ, even though the intracellular Kþ concentrations were unchanged from 20 hr, indicating either that the cells were under stress in the serum-free medium even in a physiological concentration of Kþ, or that the peptide growth factors produced a much

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slower progression into the S period than did serum. One of the coauthors of the paper related that the combined growth factors would not support proliferation of the cells enough to form a colony (E. Adelberg, personal communication). Between 20 and 40 hr, the cells in the lower concentrations of extracellular and therefore lower intracellular Kþ not only increased their rates of DNA synthesis but their intracellular concentrations of Kþ as well. The cellular Kþ at all reduced extracellular concentrations of Kþ continued to increase to 60 hr, indicating that there was a time-based adaptation of the cells to low Kþ medium. Since the rate of protein and DNA synthesis at 40 hr decreased with intracellular Kþ, it was suggested that a small change in cellular Kþ can influence the ability of the cells to initiate DNA synthesis in a serum-free medium. It was also stated, however, that the 3T3 cells stimulated by a combination of serum and platelet-derived growth factor are much less sensitive to a reduction in cellular Kþ than cultures stimulated by the purified polypeptides in a serum-free medium. The implication of this statement as well as the results of the foregoing papers using serum (Frantz et al., 1981; Moscatelli et al., 1979; Sanui and Rubin, 1979a) is that the dependence of protein and DNA synthesis on cellular Kþ is only expressed under suboptimal conditions of growth in which the Kþ level of cells in physiological external Kþ was set close to the threshold for a mitogenic response. This condition may therefore resemble that of Xenopus oocytes, in which the synthesis of protein can be accelerated by the addition of Kþ, which displaces Mg2þ from bound sites such as those on membranes to increase free cytosolic Mg2þ and accelerates the synthesis of proteins (Horowitz and Tluczek, 1989). The rate of protein synthesis is far more sensitive to change in Mg2þ than to Kþ in vitro (Brendler et al., 1981; Ilan and Ilan, 1978; Schreier and Staehelin, 1973) and in vivo (Moscatelli et al., 1979; Rubin et al., 1979; Terasaki and Rubin, 1985). It is noteworthy that no measurement was made of cellular Mg2þ in the growth factor experiment in Swiss 3T3 cells (Lopez-Rivas et al., 1982) so the possibility cannot be ruled out that a reduction in cellular Mg2þ, not in cellular Kþ, accounts for the reductions in protein and DNA synthesis, as was apparently the case in ouabain-treated 3T3 cells (Sanui and Rubin, 1979a).

B. Calcium Very large reductions in extracellular Ca2þ (from 1.5 to less than 0.01 mM) failed to inhibit proliferation of CEF in serum, but complete omission of Ca2þ from the medium did so (Balk et al., 1973). CEF in plasma, which lacks the potent growth factor PDGF, multiply more slowly than in serum and are more sensitive to depletion of Ca2þ in the medium. CEF transformed by Rous sarcoma virus (RSV) were more resistant

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to omission of extracellular Ca2þ in either serum or plasma. Sensitivity to deprivation of extracellular Ca2þ varied greatly in an unspecified line of 3T3 cells grown in serum-containing medium (Boynton et al., 1974). Primary and secondary cultures of rat heart cells cultured in homologous plasma were arrested in the G1 phase in very low (0.02 mM) Ca2þ, but were not arrested in heterologous (calf) serum (Swierenga et al., 1976). From these and related experiments it was concluded that Ca2þ is a major regulator of cell proliferation in vertebrates. No measurement was made of intracellular Ca2þ in these experiments, so there was no evidence of its reduction when the extracellular concentration was drastically reduced. The addition of the specific Ca2þ-chelating agent EGTA in amounts beyond the concentration of Ca2þ in serum-containing medium inhibited the entry of CEF into the S phase of the cell cycle 5- to 10-fold, but actually increased the intracellular concentration of Ca (Moscatelli et al., 1979). The growth inhibitory concentrations of EGTA lowered intracellular Mg2þ about 10% and Kþ about 25% while it increased Naþ almost 300%. Simple decrease of Ca2þ in the medium, without chelation, from 1.7 to 0.01 mM Ca2þ did not significantly inhibit DNA synthesis. Further decreases in external Ca2þ decreased DNA synthesis threefold to fourfold but had no effect on intracellular Ca2þ; Mg2þ and Kþ were decreased and Naþ increased. Decreasing Mg2þ in the Ca2þ-deprived medium further reduced DNA synthesis as well as reducing intracellular Mg2þ and Kþ while further increasing Naþ. Stimulation of quiescent CEF with insulin or Balb/c 3T3 cells with serum slightly lowered intracellular Ca2þ and markedly lowered surface-bound Ca2þ (Sanui and Rubin, 1978, 1982a). As noted earlier, these treatments raised intracellular Mg2þ 22% in CEF and 15% in Balb/c 3T3 cells, but lowered externally bound Mg2þ to a lesser degree than externally bound Ca2þ. In later experiments with CEF in lower serum concentrations, 100-fold reduction of external Ca2þ inhibited cell proliferation but did not reduce DNA synthesis (Rubin and Chu, 1978). More severe Ca2þ depletion did reduce the rate of DNA synthesis in the CEF, but it also caused retraction of the cells from the dish and from each other, leading to a distinctly abnormal, rounded appearance. The results suggest that the effects produced by lowering Ca2þ in the medium are caused by its removal from the external surface of the cell. In contrast, reduction of Mg2þ in the medium below 0.8 mM reduced DNA synthesis and proliferation coordinately in CEF. The cells were flattened out in low Mg2þ resembling the cells in low serum concentration, and were quickly restored to a high rate of proliferation and DNA synthesis by replenishment of Mg2þ (Rubin and Chu, 1978). The strong inhibitory effect on DNA synthesis of reducing external Ca2þ to 0.03 mM in quiescent, confluent Balb/c 3T3 cells is reversed by

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raising external Mg2þ from 1 to 15 mM (Rubin et al., 1978). Raising the Mg2þ concentrations above 20 mM caused a marked inhibition of DNA synthesis in the Ca2þ-deprived cultures, giving a bell-shaped curve for DNA synthesis as a function of Mg2þ similar to that of protein synthesis in vitro (Brendler et al., 1981; Ilan and Ilan, 1978; Schreier and Staehelin, 1973). The inhibition of confluent 3T3 cells in very low Mg2þ was unaffected by large increases of Ca2þ. The results support the thesis that inhibitory effects of Ca2þ deprivation on cells are indirect and are caused by a reduction in the availability of Mg2þ. It is noteworthy that the deprivation of neither Ca2þnor Mg2þ in physiological concentrations of the other inhibits DNA synthesis in sparse cultures of 3T3 cells, which suggests that the free Mg2þ levels in exponentially growing cells is higher than that in quiescent confluent cells. Since DNA synthesis in quiescent confluent 3T3 cultures shows no response to variations in external Ca2þ or Mg2þ for about 10 hr after restoring physiological concentrations of the cations, their effect on DNA synthesis must be considered to be indirect. To determine what the direct influence of the cations on cell function is, a study was made on the uptake of uridine into the acid-soluble pool which responds immediately to the addition of serum growth factors (Bowen-Pope et al., 1979). Combined drastic reduction of both external Ca2þ and Mg2þ lowers uridine uptake to the same extent as the omission of serum from the medium. The rate-limiting step in the uptake of uridine is its phosphorylation and that is controlled by the availability of Mg2þ (Vidair and Rubin, 1981). The requirements for serum and for Mg2þ in uridine uptake are considerably less than are their requirements for the initiation of DNA synthesis. The very low rate of uridine uptake in cells deprived of Ca2þ and Mg2þ for 2 hr (Bowen-Pope et al., 1979) is unaffected by restoration of large amounts of Ca2þ. When hypernormal concentrations of Mg2þ are added, however, uridine uptake is quickly increased to control levels, indicating that Mg2þ is the regulatory agent for this reaction. The role of low Ca2þ in reducing uridine uptake is apparently to increase the permeability of the cells to Mg2þ so that its intracellular concentration is highly responsive to its external concentration. Further evidence of the increase in cell permeability in very low Ca2þ is the switch of intracellular concentrations of Kþ and Naþ, and particularly a marked increase in the rate of uptake of L-glucose which enters the cell only by unmediated diffusion. The increased uptake of L-glucose in low Ca2þ occurs even in physiological concentrations of Mg2þ, but is greatly reinforced by lowering external Mg2þ. In contrast, drastic lowering of Mg2þ in physiological Ca2þ has no effect on the uptake of L-glucose. Hence, Mg2þ acts directly as the regulator of uridine phosphorylation by uridine kinase (Vidair and Rubin, 1981) and, as at higher Mg2þ concentrations, acts indirectly as the regulator of the initiation of

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DNA synthesis. Since onset of DNA synthesis reflects the rate of protein synthesis through the G1 period, it can be assumed that the optimal Mg2þ requirement for the attachment of messenger RNA to ribosomes is much higher than it is for uridine phosphorylation. It might be said that the failure to detect an increase in total cellular Ca2þ in growth-stimulated cells does not rule out an increase in free Ca2þ since the basal level is 4 orders of magnitude lower than total Caþ. And indeed, the addition of serum to human fibroblasts produces an immediate rise of free Ca2þ that peaks at about a twofold increase in 30 sec, then rapidly declines and stabilizes at a new steady level in about 3 min (Moolenaar et al., 1984). However, several different growth factors fail to produce a transient rise of Ca2þ in human fibroblasts, and EGF produces no such rise in myocytes (Grubbs, 1991). In any case, no ‘‘trigger’’ reaction in the literal sense, as presumably represented by the early Ca2þ transient, can account for the requirement of the continuous presence of growth factors during most, if not all, of the G1 period to maintain the increased rate of protein synthesis that underlies a speedup of cell proliferation. Also relevant is the observation that the direct injection of a wide range of Ca2þ concentrations into Xenopus oocytes has no significant effect on protein synthesis, nor does the Ca2þ-chelating agent EGTA prevent the increase in protein synthesis induced by Kþ or hormone treatment (Horowitz and Tluczek, 1989). Therefore, the Ca2þ transient induced by some growth factors is no more essential for protein synthesis or the onset of DNA synthesis than are other early events such as increased transport of glucose or uptake of uridine. Those early events that are essential for growth are, of course, increased protein synthesis and the utilization of glucose in energy metabolism (Fodge and Rubin, 1975b; Rubin and Fodge, 1974). There is one final piece of evidence that has been cited to support a major role of Ca in regulation of DNA synthesis. The addition of hypernormal concentrations of Ca2þ to quiescent cultures of Balb/c 3T3 cells induces DNA replication (Boynton and Whitfield, 1976; Dulbecco and Elkington, 1975). However, the effect depended on the concentration of inorganic orthophosphate in the medium and was associated with the formation of insoluble complexes of Ca2þ with HPO2 (Rubin and 4 Sanui, 1977). It could be simulated by raising the concentration of HPO2 4 in normal concentrations of Ca2þ which resulted in precipitate formation. It could be prevented in hypernormal concentrations of Ca2þ by lowering HPO2 4 to levels that did not form precipitates. A more striking stimulation of DNA synthesis was produced by adding very small amounts of inorganic pyrophosphate to the medium, just enough to form floccules with Ca2þ which interacted with the cell surface (Bowen-Pope and Rubin, 1983; Rubin and Sanui, 1977). Strontium can partly replace Ca2þ in these effects. The increase of DNA synthesis in hypernormal Ca2þ therefore

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has nothing to do with an increase of intracellular Ca2þ but results from the action of Ca3(PO4)2 precipitate at the cell surface. Although intracellular Ca2þ plays no direct role in cell growth regulation, extracellular Ca2þ may do so because it is essential in forming adhesions between cells (Vleminckx and Kemler, 1999). The release of Ca2þ from the external surface of the cells by growth factors (Sanui and Rubin, 1978, 1982a) may therefore contribute to the stimulation of contact-inhibited cells by loosening intercellular contacts.

C. pH and Sodium The rate of cell proliferation in culture depends on the pH of the medium between pH 6.6 and 8.0 for CEF, with an optimum between pH 7.2 and 7.6 (Rubin, 1971b). The optimum pH varies with different cell lines (Ceccarini and Eagle, 1971). Cells are less susceptible to inhibition at pH below 7.0 at low population density than at high population density, and will reach a saturation density at pH 6.5 before they are fully confluent (Rubin, 1971b). It requires about 3 times as high a concentration of protons to inhibit a sparse population of cells as a dense population (Rubin, 1971a). There is estimated to be 2- to 10-fold higher proton concentration at the surface of the plasma membrane than in the bulk medium, and it increases with negativity of the electrostatic surface potential. Since the negative surface potential increases as the distance between cells decreases, the surface proton concentration is higher in a dense population than in a sparse population, and to that extent, lowered pH may contribute to density-dependent or contact inhibition of cells. The growth rate of normal cells is also proportional to their rate of migration which depends on membrane motility, which, in turn, reduces the local proton concentration (Rubin, 1971a,b). Surface proton concentration presumably influences intracellular pH and this, in turn, affects the rate of glycolysis in cells. A major enzymatic point of control of the glycolytic pathway is phosphofructokinase which is activated by increasing pH of the medium or the concentration of serum (Fodge and Rubin, 1973). Phosphofructokinase also exhibits a bell-shaped curve of dependence on Mg2þ in cell-free preparations, with an optimum in the same range as that of protein synthesis (Garner and Rosett, 1973). Metabolic inhibitors of glycolysis strongly and irreversibly inhibit the initiation of DNA synthesis, but the effect on ongoing DNA synthesis is minimal (Fodge and Rubin, 1975b; Rubin and Fodge, 1974). These observations indicate that intracellular pH might play an auxiliary role in the regulation of cell proliferation.

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The addition of a combination of growth factors (insulin, vasopressin, and PDGF) to quiescent Swiss 3T3 cells resulted in an average increase of cytoplasmic pH of 0.16 units (Schuldiner and Rozengurt, 1982). The increase required external Naþ or Liþ and could not be substituted by Kþ or choline. The half maximal effect of Naþ in the medium for the increase in pH was 38 mM and was attributed to Naþ/Hþ antiport in the cells since it was blocked by amiloride, an inhibitor of the Naþ/Hþ ATPase. The influx of Naþ was thought to be sufficient to activate the Naþ/Kþ pump and increase Kþ in the stimulated cells, thereby contributing to mitogenesis. Doubt was raised about this interpretation because amiloride also directly inhibits protein synthesis, which is essential for the onset of DNA synthesis (Lubin et al., 1982). Further investigation revealed that the decrease of Naþinhibited DNA synthesis by different mechanisms depended on the growth factor used to stimulate the cells and the monovalent cation used as an osmotic substitute for Naþ (Burns and Rozengurt, 1984). The decrease in DNA synthesis was correlated with a decrease in intracellular Kþ in some cases, and the blocking of cellular alkalinization in others. The latter effect occurred in the stimulation of early passage human fibroblasts (Moolenaar et al., 1983), and with a variety of mitogens in mouse thymocytes and Swiss 3T3 cells (Hesketh and Moore, 1985). The increase in cytoplasmic pH was, in most cases, preceded by a transient rise in Ca2þ, but the increased pH persisted for more than 25 min (Hesketh and Moore, 1985). It was thought likely that the activation of Naþ/Hþ exchange is necessary to proceed through the cell cycle but by itself is insufficient to ‘‘trigger cell division’’ (Glaser et al., 1984). As already noted, mention of a trigger for cell division raises questions of its own since it is generally agreed that the growth stimulus has to be maintained through most, if not all, of the G1 period in order to hasten DNA synthesis (Bolen and Smith, 1977; Brooks, 1977; Rubin and Steiner, 1975). The notion of necessity of elevated pH for mitogenesis has to be qualified because the substitution of 95% of Naþ in the medium by choline does not inhibit the increase of DNA synthesis in quiescent CEF stimulated by insulin, although substitution by Kþ profoundly suppresses DNA when external Naþ is decreased below 30 mM (Moscatelli et al., 1979). Another caveat is that the stimulation of myocytes by EGF does not raise cytosolic pH (Grubbs, 1991). It should also be borne in mind that decrease in Kþ of the medium suppresses the onset of DNA synthesis in Swiss 3T3 cells much less when they are stimulated by serum than by purified growth factors (Frantz et al., 1981; Lopez-Rivas et al., 1982), which illustrates the contingency of the effects of Kþ, Naþ, and pH on the cell cycle. This contrasts with the role of Mg2þ in mediating growth control since any alteration of intracellular Mg2þ alters the rate of protein synthesis (Terasaki and Rubin,

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1985) and thereby progression through G1 to DNA synthesis (Brooks, 1977; Castor, 1977).

XII. REGULATION OF PROTEIN SYNTHESIS BY THE PI 3-K AND mTOR PATHWAYS Most of what has been presented here defined the basic physiological parameters of growth regulation which were revealed in the period from the 1960s to 1985, with a few important accretions about the role of Mg2þ extending to 1991. This era did not include studies of molecular pathways of growth regulation, which began in earnest in the late 1980s and continues to this day. The most relevant of the molecular studies to stimulation by growth factors involved the response of protein synthesis to polypeptide growth factors, such as insulin, EGF, and PDGF (Alberts et al., 2002). Binding of the growth factors to the extracellular domain of their receptors (Fig. 7A) causes a conformational change which leads to their lateral movement in the membrane to form oligomers which then autophosphorylate on tyrosine in their cytoplasmic domain (Fig. 7B) (Schlessinger, 1988; Ullrich and Schlessinger, 1990). These phosphorylations entrain a cascade of serine/threonine kinases combined with inhibitory proteins in the PI 3-K pathway that lead to phosphorylations of a large protein, the mTOR kinase (Fig. 7C) (Richardson et al., 2004). mTOR phosphorylates S6 kinase which phosphorylates the S6 protein of 40S ribosomal subunit (Fig. 8A) to drive the initiation of protein synthesis on 50 TOP mRNAs (Fig. 8B) (Schmelzle and Hall, 2000). mTOR also phosphorylates the binding protein 4E-BP1 which dissociates from and activates the initiating factor eIF-4E (Fig. 8A) (Beretta et al., 1996). eIF-4E is the rate-limiting factor in formation of a larger complex, eIF–4F, which initiates protein synthesis on mRNA (Fig. 8B) (Richardson et al., 2004). The overall effect of mTOR phosphorylations of S6K and 4E-BP1 is an increase in the efficiency of translation initiation, which is a characteristic effect of the stimulation of protein synthesis by growth factors (Stanners and Becker, 1971), especially for the synthesis of ribosomal proteins (DePhilip et al., 1980) and elongation factors (Gingras et al., 2004; Terada et al., 1994). The previously stated considerations place the mTOR phosphorylations of S6 kinase and 4E-BP1 in a crucial position for the regulation of protein synthesis. The kinase activity of mTOR for these two targets is distinguished from most protein kinases analyzed to date by its high Km (Michaelis content) for ATP, i.e., slightly greater than 1.0 mM (Dennis et al., 2001), versus 10 to 20 M for the other protein kinases (Edelman et al., 1987). The marked reduction of ATP concentration by inhibiting glycolysis with

Fig. 7 Simplified model of the activation of the PI 3-K signaling pathway by binding of insulin to its receptors, accompanied by membrane perturbation and the partial release of membrane-bound Mg2þ. (A, B) The plasma membrane receptor for insulin is a tyrosine kinase. The binding of insulin to the external domain of the receptors causes their conformational change. They then move to form oligomers and phosphorylate each other on their intracellular domains. The conformational change and movement of the receptors perturb the membrane and weaken its binding for Mg2þ, which results in its partial release into the cytosol to increase the concentration of MgATP2. (C) The autophosphorylation of the receptors activates the PI 3-K pathway, which activates a kinase cascade leading to phosphorylation of mTOR (Rubin, in press).

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Fig. 8 Phosphorylation of mTOR and the increase in the initiation of protein synthesis. (A) Phosphorylation of mTOR and the increase in MgATP2 increases the phosphorylation of S6 kinase which activates the S6 protein of the 40S ribosomal subunit. mTOR also phosphorylates 4E-BP1 bound to eIF-4E initiation factor, which releases the latter. (B) The phosphorylated S6 and released eIF-4E (combined with other initiation factors) increase the frequency of initiation of protein synthesis on 50 TOP mRNA (Rubin, in press).

2-deoxyglucose, an analog of glucose, strongly inhibits key phosphorylations by mTOR of S6 kinases and 4E-BP1 (Dennis et al., 2001). It was therefore inferred that ATP is the regulatory factor that determines the initiation rate of ribosomal protein synthesis in cells stimulated by insulin. There are several inconsistencies in assigning such a regulatory role to ATP in the paper that made the proposal (Dennis et al., 2001). The omission of

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stimulation by insulin in control cultures reduced ATP by less than 10% but abolished detectable phosphorylations in S6 kinase and 4E-BP1; a mitochondrial inhibitor reduced ATP 28% but allowed considerable phosphorylation (Fig. 1 in Dennis et al., 2001). Stimulation of CEF with serum, which is a stronger mitogen than insulin, actually decreases ATP concentration (Fodge and Rubin, 1973). However, the true substrate of phosphoryl transfer reaction is MgATP2 rather than ATP itself (Lardy and Parks, 1956; Rose, 1968). The other forms of ATP such as ATP4, KATP3, and HATP3, which constitute a significant fraction of total ATP, are inhibitors of phosphoryl transfers (Achs et al., 1982, 1979). Variations in ratio of Mg2þ and ATP exert profound effects on the velocity of the kinase reactions of carbohydrate metabolism (Garner and Rosett, 1973) and are likely to act in a similar fashion in the mTOR phosphorylations since both have a similar Km for MgATP2. The Km of ATP for the mTOR phosphorylations was determined in vitro in a large excess (10 mM) of Mg2þ (Dennis et al., 2001) and is likely to be the Km for MgATP2. The free Mg2þ concentration in quiescent cultured mammalian cells is 0.2 to 0.4 mM (Grubbs, 1991; Ishijima et al., 1991), which is considerably lower than the 1 mM concentration of ATP (Gribble et al., 2000). Since free Mg2þ increases significantly in stimulated cells (Grubbs, 1991; Ishijima et al., 1991) in contrast to ATP (Fodge and Rubin, 1973), the level of MgATP2 would be determined by free Mg2þ. At the same time, it would also decrease the concentration of the inhibitory forms of ATP. The combined effect would increase mTOR phosphorylation of its two key substrates and thereby increase the frequency of the initiation of protein synthesis. In contrast, the other protein kinases of the PI 3-K pathway that have 50 to 100 times lower Km for MgATP2 would presumably be saturated with it in quiescent as well as stimulated cells, and their activity would be unaffected by changes in free Mg2þ, although stimulated by upstream phosphorylations of the PI 3-K pathway. The more general and more specific mechanisms would have to combine with each other in order to fully open the gates for increased protein synthesis.

XIII. ROLE OF CATIONS IN NEOPLASTIC TRANSFORMATION Efforts were made to determine whether neoplastic transformation changes the regulatory role of Mg2þ or Ca2þ in cells. The concentration of Mg2þ for half-maximal rate of proliferation of transformed human lung fibroblasts was about 20 times lower than the concentration required for normal diploid human lung fibroblasts (McKeehan and Ham, 1978). In contrast, there was no difference in the concentration of Ca2þ required for

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half-maximal rate of proliferation of the transformed and normal cells. The results indicated that for normal cells the role of Mg2þ is more proximal than Ca2þ to the intracellular events that determine maximal growth rate. The role of both cations for half-maximal growth rate of the normal cells was studied as a function of serum concentration and suggested that Ca2þ interacts more directly than Mg2þ with the serum molecules that stimulate proliferation of the normal cells. It was proposed that transformation causes a selective loss of the growth regulatory role of Mg2þ but not Ca2þ, and that the regulatory effect of Ca2þ in normal but not transformed cells is primarily mediated through Mg2þ-dependent processes. Hence, transformation is associated with the loss of ability to regulate to Mg2þ levels in the cell. Since the major process that regulates the onset of DNA synthesis is protein synthesis, and there is no indication that ribosomes of transformed cells differ from those of normal cells in their requirement for Mg2þ, it would appear that the transformed cells have lost their capacity to regulate Mg2þ, which depends, at least to some extent, on the binding capacity of membranes. The cellular content of the 4 major cations was measured in normal epidermis of control mice and in epidermis painted repeatedly with methylcholanthrene, a powerful carcinogen (Carruthers and Suntzeff, 1943). The Mg2þ content of the methylcholanthrene-treated epidermis was 20% higher than that of the controls, with no change in Naþ and Kþ but there was a 60% decrease in Ca2þ. There is an increase in Kþ in many tumors but that is related, at least in part, to an increase in the rate of proliferation as seen in normal tissue (deLong et al., 1950). Squamous cell carcinomas induced in mice by methylcholanthrene had only 20% of the Ca2þ of normal epidermis (Lansing et al., 1948). Three spontaneously transformed clones of Balb/c 3T3 fibroblasts had on average 15 to 20% more total Mg2þ than nontransformed fibroblasts, although there was widespread variation among the clones in degree of transformation and in Mg2þ content (Terasaki, 1983). The Ca2þ content of 12 human intestinal cancers was reduced an average of 44% compared with adjacent normal mucosa (deLong et al., 1950). The Ca2þ content of spontaneously transformed Balb/c 3T3 cells was only onethird that of nontransformed cells (Rubin et al., 1981). It appears then that a wide variety of neoplastic cells have a much lower capacity to retain Ca2þ than non-neoplastic cells. Ca2þ plays a major role in the mutual adhesion of cells to one another mainly through binding to proteins known as cadherins (Vleminckx and Kemler, 1999). The adhesiveness of cells to one another was long ago ascribed to mediation between cell surface proteins by Ca2þ and to a lesser extent by Mg2þ (Lansing et al., 1948; Zeidman, 1947). The adhesiveness of cells from human squamous cell carcinomas and a variety of adenocarcinomas is much lower than that of their normal counterparts (Coman, 1944; McCutcheon et al., 1948). The reduced adhesiveness may

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result from a reduction in a major cadherin in some cases (Shimoyama et al., 1992; Umbas et al., 1992) but in others it could result from other causes like the destabilization of membranes and their loss of Ca2þ in response to oncogenes or other factors from within genetically altered cells. In view of the evidence that transformed cells have a diminished capacity to regulate their free Mg2þ, the effects of Mg2þ deprivation on their behavior were examined. Nontransformed and spontaneously transformed clones of Balb/c 3T3 cells were isolated and their quantitative requirement for Mg2þ to support proliferation was determined (Rubin, 1981). As was the case with normal and transformed human lung cells (McKeehan and Ham, 1978), the transformed Balb/c 3T3 fibroblasts had a much lower requirement of Mg2þ for proliferation than did the nontransformed cells. The transformed cells in physiological concentrations of Mg2þ did not have a true saturation density due to contact inhibition in conventional cultures because they depleted the medium of essential constituents at high population density even with daily medium changes. However, when external Mg2þ was reduced 60-fold, the rate of exponential growth of the cells was reduced slightly and they developed a true saturation density, i.e., the result of intercellular contact rather than depletion of essential medium components. After 3 days at saturation density, the cells resumed exponential growth. If subcultured at lower density before that happened, the cells flattened on the dish and simulated the appearance and regular arrangement of normal cells (Fig. 9). However, when they resumed multiplying, they

Fig. 9 Normalization of transformed cells by deprivation of Mg2þ. Growing cultures of a nontransformed and a transformed clone of Balb/c 3T3 mouse cells were maintained in medium with 10% calf serum and 1.0 mM Mg2þ. The medium was exchanged for fresh medium with (A) 1.0 mM Mg2þ for the nontransformed cells, and (B) 1.0 mM Mg2þ or (C) 0.01 mM Mg2þ for the transformed cells for 2 days. The nontransformed cells in (A) were well spread, flat, and tended to line up with each other. The transformed cells in 1.0 mM Mg2þ of (B) were retracted, thin, and randomly arranged. The transformed cells in 0.01 mM Mg2þ flattened out and lined up with each other in (C), resembling the nontransformed cells in (A) (Rubin, 1981).

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reverted to the thin, spiky appearance of transformed cells. The normal cells at low saturation density in very low Mg2þ also resumed multiplication after a week or so. In both cases, the resumption may have occurred by the adaptation to low Mg2þ through increasing their intracellular Mg2þ content (Rubin et al., 1979). Just as the transformed clone required much less Mg2þ for multiplication than the nontransformed cell, it also required much less serum (Rubin et al., 1981). Deprivation of Mg2þ raised the serum requirement of the transformed clone for DNA synthesis, especially at higher population densities, resulting in a behavior similar to that of nontransformed cells. None of these normalizing effects of lowering Mg2þ concentration could be reproduced by lowering Kþ or Ca2þ concentration, nor could either of the latter restore the normal appearance or arrangement of the cells. Addition of dibutyryl cyclic AMP partly flattened the transformed cells but did not induce either serum or density dependence. The Ca2þ content of the transformed cells was only one-third that of the nontransformed cells, but it was raised to the same level as the nontransformed cells by deprivation of Mg2þ. Another indication that Mg2þ deprivation restores the normalized phenotype of transformed cells is that it is much more effective in inhibiting DNA synthesis in crowded cultures than in sparse cultures (Rubin, 1981, 1982). In contrast, deprivation of Kþ or Ca2þ or treatment with cyclic AMP is more effective in sparse than in crowded, transformed cultures. Taken together, these observations support the view that a defect in Mg2þ regulation is a basic feature of neoplastic transformation and argue against a direct role for a defect in regulation by Kþ, Ca2þ, or by cyclic AMP. Highly transformed cells do not reach a saturation density at confluence under ordinary conditions of culture because they deplete the medium of essential constituents even when the medium is replenished daily. If they are attached to a small coverslip which is then placed in a dish with a large volume of medium, they achieve a multilayered saturation density, depending on serum concentration, that does not deplete the essential components of the medium (Rubin and Chu, 1982). At saturation density in physiological Mg2þ, the transformed cells took on the morphology and orderly arrangement of normal cells. Their rate of DNA synthesis was markedly decreased and total cellular Mg2þ was reduced by 33%. When they were subcultured at low densities, they at first exhibited the flattened appearance of isolated normal cells followed by a lag period of 10 to 13 hr before an increase in DNA synthesis began. The rate of DNA synthesis and percentage of cells labeled with 3H-thymidine were highly dependent on serum concentration, as would be expected of stimulated normal cells. The capacity of the density-inhibited transformed cells to produce colonies when trypsinized and suspended in agar was reduced 10-fold. Within 3 days after subculture, the cells resumed their transformed appearance and underwent a fivefold

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increase of colony formation in agar. The self-normalization of the transformed cells at very high saturation density may be related to the normalization of individual, spontaneously transformed cells by contact with confluent normal cells (Stoker, 1964; Stoker et al., 1966; Weiss, 1970). It suggests that the inhibition of membrane activity in transformed cells at very high population density lowers free Mg2þ activity and restores normal appearance and behavior. It is important to note that the inhibition of protein synthesis in transformed cells by cycloheximide does not reverse the transformed phenotype (Terasaki and Rubin, 1981). Apparently, the fully transformed phenotype, including its characteristic morphology, depends on membrane/ Mg2þ activation of metabolic pathways in addition to protein and DNA synthesis. Those additional pathways would include energy metabolism in the form of glycolysis and the Krebs cycle (Achs et al., 1979, 1982; Garfinkel et al., 1979).

XIV. CONCLUSIONS Several biological features of cell growth regulation have been established that provide guidelines for critically analyzing the mechanism of the process. The first of these has to do with the specificity of the treatments used to stimulate or inhibit cell proliferation. Cells need growth factors, most commonly animal serum or certain polypeptide hormones, in order to stimulate multiplication. A considerable body of evidence shows that these act through combination with specific receptors on the plasma membrane. But there are also a number of nonspecific ways to stimulate cell proliferation that have mainly been demonstrated in confluent, contact-inhibited cultures. The most graphic demonstration of surface action is seen after scraping away part of the confluent, contact-inhibited layer, thereby allowing individual cells to migrate from the confluent region on the denuded surface, which allows them to multiply at a maximal rate. Other nonspecific agencies include treatment of CEF with trypsin and certain other proteases; subtoxic concentrations of heavier metals, i.e., Zn2þ, Cd2þ, Hg2þ, Mn2þ, and Pb2þ, on CEF; and addition to the medium of Balb/c 3T3 cells of 5 mM of Ca2þ, which forms precipitates with inorganic phosphate, or addition of 0.15 mM inorganic pyrophosphate which forms floccules with Ca2þ (Rubin and Sanui, 1977). The precipitates presumably interact with the lipid bilayer of Balb/c 3T3 plasma membranes to stimulate their proliferation (Bowen-Pope and Rubin, 1983) but they do not stimulate CEF. (When the pyrophosphate concentration exceeds that of Mg2þ, it is inhibitory to growth due to drastic lowering of Mg2þ by chelation.) Proliferation of CEF is inhibited by glucocorticoids (Fodge and Rubin, 1975a) or by suspending them in a semi-solid or viscous liquid medium.

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The converse of suspension is the requirement for normal cells to attach and spread on a solid substratum in order to multiply. Accumulated evidence indicates most, if not all, of stimulatory treatments perturb the cell membrane, and the inhibitory treatments stabilize it. A second biological feature of growth stimulation is the crucial relationship between increased protein synthesis and the acceleration of cells through the G1 period into the S period. The increase in protein synthesis begins immediately after the addition of serum or other growth factors and their action must be continued for hours through much or, in some cases, all of the G1 period to initiate DNA synthesis among a large population of cells. Indeed, removal of serum and lowering pH of CEF cultures during the exponential rise of DNA synthesis allows initiated cells to continue DNA synthesis but inhibits cells still in G1 from entering the S phase (Rubin and Steiner, 1975). Furthermore, a strong shutdown of protein synthesis with cycloheximide during the S period will shut down DNA synthesis (Kim et al., 1968). Thus, there is a continuing need for protein synthesis to maintain DNA synthesis. There are many other early responses to growth factors, such as increased uptake of hexoses and other metabolites, and accelerated transport of Kþ, Ca2þ, and Naþ but few, if any, of these are consistently required for accelerated onset of DNA synthesis. Exceptions are blockage of glycolysis or oxidative phosphorylation by metabolic inhibitors which inhibit the initiation but not the continuation of DNA synthesis (Rubin and Fodge, 1974). The most acute relation of an early response to later DNA synthesis is elevation of protein synthesis but, as stated, that must be maintained throughout the G1 phase to be effective. The primary problem of stimulating DNA synthesis then resolves itself into the manner by which perturbation of the cell membrane by growth factors or by nonspecific treatments translates into the stimulation of protein synthesis. It is common knowledge that protein synthesis depends on the availability of Mg2þ. The initiation of protein synthesis by attachment of messenger RNA to the small subunit of ribosomes requires a higher concentration of Mg2þ than elongation of the polypeptide chain (Revel and Hiatt, 1965). The concentration of free Mg2þ in the cell has been estimated as 1.0 mM or less (Rink et al., 1982), with most recent estimates in quiescent cells running between 0.2 and 0.4 mM (Grubbs, 1991; Ishijima et al., 1991). The optimal free Mg2þ concentration for initiation of protein synthesis in vitro is 2 to 4 mM, which ensures that any increase in free Mg2þ in cells will increase the rate of protein synthesis. Inoculation of rabbits with insulin more than doubles the fraction of a phosphoglucomutase in the active Mg2þ form in skeletal muscle and lowers the inactive Zn2þ form, indicating that the action of insulin on muscle cell membrane increases the level of free Mg2þ in the cytosol (Peck and Ray, 1971). Continuous treatment of confluent CEF or Balb/c 3T3 cells with insulin or serum, respectively, sharply increases the rate

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of DNA synthesis after a lag of 4 or 10 hr (Sanui and Rubin, 1978, 1982a) in which the rate of protein synthesis remains at an elevated level. The total Mg2þ content of the cells increases 14 to 22% within an hour or two after treatment with growth factors and is maintained at the elevated level throughout the G1 and S periods (Sanui and Rubin, 1978, 1982a). It was assumed that the increase in total Mg2þ was accompanied by an increase in free Mg2þ. This assumption is supported by the finding of large changes in free Mg2þ with small variations in total cell Mg2þ in the hepatocytes of streptozotocin diabetic rats (Corkey et al., 1986). Direct evidence for a physiological role of increasing Mg2þ in initiating protein synthesis came from injection of Mg2þ into single Xenopus oocytes, which reproduced the physiological action of gonadotropin (Horowitz and Tluczek, 1989). Confirmatory evidence of a prolonged rise in free Mg2þ in somatic mammalian cells treated with growth factors was obtained with the presence in the cells of the Mg2þ-sensitive fluorescent indicator mag-fura 2 (Grubbs, 1991; Ishijima and Tatibana, 1994; Ishijima et al., 1991). An estimate can be made of the intracellular rise in Mg2þ resulting from its release from binding sites on the internal surface of the plasma membrane after insulin stimulation, based on the measurement of externally bound divalent cations released by insulin (Sanui and Rubin, 1978). CEF were repeatedly washed with a 0.25-M sucrose solution. The externally bound cations are displaced by Hþ in a 10-sec rinse with pH 4.0 medium. The results show that treatment of CEF with 0.1 unit of insulin reduces externally bound Mg2þ by 45% and externally bound Ca2þ by 34% (Sanui and Rubin, 1978). The insulin treatment displaces 1.37 nmoles of Mg2þ and 4.02 nmoles of Ca2þ per mg of cell protein. Using values for CEF of about 104 insulin binding sites per cell (Raizada and Perdue, 1975) and 4  106 cells per mg protein, about 5  105 nmoles of insulin can be bound per mg cell protein. From these figures, it is estimated about 105 divalent cations are displaced per insulin bound, which is orders of magnitude higher than the stoichiometric exchange with other cations or with EDTA or ATP (Sanui and Pace, 1967). Such large cation displacements indicate that insulin induces a conformational change of the membrane to which it binds. Shlatz and Marinetti had come to the same conclusion after finding that insulin markedly reduces the binding of Ca2þ to isolated rat liver plasma membranes and, conversely, that one molecule of hydrocortisone leads to the additional binding of 3000 atoms of Ca2þ (Shlatz and Marinetti, 1972). Significantly, insulin and cortisol, an analog of hydrocortisone, have opposite effects on DNA synthesis and other reactions of coordinate control in CEF (Fodge and Rubin, 1975a), and Mg2þ deprivation simulates quantitatively the addition of cortisol to CEF (Rubin, 1976). Accepting that insulin induces a conformational change in the plasma membrane, it should release approximately the same number of divalent

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cations from the inner surface of the plasma membrane as it does from the external surface of the cell. Since the association constant of Mg2þ to anionic sites on membranes is the same as that of Ca2þ (Sanui and Pace, 1967), and free Mg2þ is 3 to 4 orders of magnitude higher in concentration in the cytosol than Ca2þ, most of the divalent cations bound to the internal surface of the plasma membrane would be Mg2þ. Calculating the approximate number of Mg2þ cations released into the cytosol to be about 108 per CEF cell with an average volume of 1000 3 (Rubin and Hatie´ , 1968), there would be an approximate increase of 1.0 mM free Mg2þ per cell. This is within the range of the values reported from measurement of free Mg2þ by mag-fura 2 in cells stimulated by growth factors (Grubbs, 1991; Ishijima et al., 1991), and would account for the increase in protein synthesis needed to drive DNA synthesis and mitosis. The mag-fura 2 indicator was only reliable for 1 hr as it leaked from the cells, but in that time, it recorded a constant elevation in free Mg2þ, so it is plausible that the increase persisted through G1 and into S period in keeping with the increase in total Mg2þ that remained constant for at least 17 hr after the addition of insulin (Sanui and Rubin, 1978). More than 300 enzymes use Mg2þ as a cofactor and their activity displays the same bell-shaped curve for dependence on Mg2þ as protein synthesis does with a similar optimum (Ebel and Gu¨ nther, 1980; Garner and Rosett, 1973). That large catalog of enzymes includes phosphofructokinase which is a major control point in glycolysis and operates as such in serum-stimulated CEF (Fodge and Rubin, 1973). This parallelism implies that energy metabolism is coordinately controlled by Mg2þ to a similar extent as protein synthesis. It, of course, makes sense that the energy generation and synthetic activities of cells rise coordinately in response to growth stimulation to keep up with the increased demand for energy and maintain balanced growth. Similarly, early responses which are not needed for a single round of DNA synthesis may be part of a reserve for repeated rounds. That would be true for increased uptake of hexoses and other metabolites which are regulated by Mg2þ availability (Bowen-Pope and Rubin, 1977; Rubin, 1977), and cation pumps which are driven by Mg2þ-dependent ATPases (Lostroh and Krahl, 1973, 1974). A simplified model for the internal release of Mg2þ from membranes by mitogens and consequent effects on cellular metabolism and growth is shown in Fig. 10. It has been designated the MMM (Membrane, Magnesium, Mitosis) model of cell proliferation control (Rubin, in press). Transformed cells are much less subject to inhibition of growth by deprivation of Mg2þ than are nontransformed cells (McKeehan and Ham, 1978; Rubin, 1981). In very low Mg2þ, the transformed cells take on the appearance and regulatory behavior of normal cells, which includes increased sensitivity to contact inhibition, higher requirement for serum, and reduced capacity to form colonies when suspended in agar (Rubin et al., 1981). When grown to very high density on a coverslip in a large volume of

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Fig. 10 Major features of a model for coordinate control of intermediary metabolism and growth by the availability of Mg2þ. Intracellular cations are bound to the internal surface of the plasma membrane. The relative amount of each ion that is bound depends on its abundance and affinity for fixed anionic groups in phospholipids, like phosphotidyl serine, and to a lesser extent to proteins. The affinity for divalent cations is determined in part by the closeness of the fixed anionic groups to allow two-point attachment of the divalent cation. An external stimulus like insulin that perturbs the membrane (Shlatz and Marinetti, 1972) extends the distance between the anionic groups and reduces affinity for the divalent cations (Dawson and Hauser, 1970), of which Mg2þ is the predominant intracellular species. The Mg2þ can also be displaced by heavy metals, such as Zn2þ, Cd2þ, Hg2þ, or Pb2þ, which stimulate cells at subtoxic doses (Rubin and Koide, 1973; Sanui and Rubin, 1984). The increase in availability of Mg2þ speeds up intermediary metabolism, which increases the supply of substrates, including nucleotides for biosynthetic reactions and other cellular activities, by changing the inhibitory K-ATP to Mg-ATP, which is the substrate for phosphorylation reactions. The increased free Mg2þ also increases the frequency of the initiation step for protein synthesis which requires higher [Mg2þ] than elongation of the polypeptide chain (Revel and Hiatt, 1965). The increase of protein synthesis accelerates progress through G1 to the S phase of the cell cycle and then to mitosis (Rubin and Sanui, 1979).

medium with normal Mg2þ, their intracellular content of Mg2þ is reduced by one-third, and they adopt a normal, flattened morphology which is retained for a few days on subculture at low density (Rubin and Chu, 1982). These observations suggest that phenotypically transformed cells have less control of their free Mg2þ content than do nontransformed cells, and they therefore constitutively maintain a higher activity of Mg2þ. Many transformed cells have higher proteolytic activity at their surface than do normal cells (Rubin, 2001), reduced adhesiveness to the substratum, and more random movement of the cell surface (Abercrombie and Ambrose, 1962). This implies either some defect in the cell membrane, as in the case of APC mutations in human colorectal cancer (Kinzler and Vogelstein, 1996), or metabolic change that perturbs the membrane from the inside just as growth factors do from the outside.

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Transformation by retroviruses or transfection of their oncogenes requires the action of strong promoter-enhancer regions that drive overexpression of the oncogenes (Chakraborty et al., 1991; Hua et al., 1997). Even normal protooncogenes can transform cells if they are coupled to a strong promoter and are heavily overexpressed (Blair et al., 1981; Chakraborty et al., 1991; Chang et al., 1982; Miller et al., 1984; Zhou and Duesberg, 1990). The src gene product of RSV is a 60,000 molecular weight protein pp60v-src that is responsible for the transforming activity of the virus (Brugge and Erikson, 1977). It is a protein kinase (Collett and Erikson, 1978) that specifically phosphorylates tyrosine residues (Hunter and Sefton, 1980). The pp60v-src is found mainly in the plasma membrane of RSV-transformed cells (Courtneidge et al., 1980; Krueger et al., 1980), where its substrates are 115- to 120k-Da proteins (Linder and Burr, 1988; Reynolds et al., 1989). Linkage of the src protein to myristic acid is necessary for its association with membranes and for transformation (Cross et al., 1984; Kamps et al., 1985). Oncogenic P3K retroviruses code for mutated homologs of the 110-kDa catalytic unit of the PI 3-Kinase fused to Gag (G-antigen of oncogenic retroviruses) sequences (Aoki et al., 2000; Aoki and Vogt, 2004). The mutations are not necessary for transformation since fusion of the cellular PI 3-K to Gag causes transformation. The Gag proteins localize the PI 3-kinase at the plasma membrane, and can be substituted by myristylation or farnesylation for this purpose. Both the membrane localization and the kinase activity are required for cell transformation, as is the activity of mTOR (Aoki et al., 2001). Since deprivation of Mg2þ reverses the transformed phenotype (Rubin et al., 1981), it suggests that the extensive phosphorylation of membrane components by overexpressed pp60v-src or PI 3-kinase may sufficiently alter membrane structure to release bound Mg2þ to the cytosol and continuously activate the coordinate response of the cell. In that sense, these oncogenes would be acting on the internal surface of the plasma membrane in a manner similar to that of growth factors on the external surface, only the activation would have to be constitutive and more intense in order to generate the transformed phenotype. It would therefore be of interest to compare the affinity for Mg2þ of the plasma membrane obtained from normal and transformed cells. Alternatively, perturbation of the membrane by heritable changes in the intracellular milieu might greatly influence the binding of Mg2þ (Dawson and Hauser, 1970) but not affect the affinity of isolated membranes. Given the weight of experimental evidence for a primary role of free Mg2þ in regulating the rate of protein synthesis and cell proliferation, much of it more than 20 years old, it might be asked why it has not received more attention in the literature on cell growth regulation. For example, a major review of early signals in the mitogenic response gives prominent attention to Naþ, Kþ, Ca2þ, and Hþ but there is no mention of Mg2þ (Rozengurt,

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1986). More surprisingly, there is no mention in the review of the widely acknowledged, early, and persistent increase in protein synthesis induced by growth factors in normal cells and its essential relation to DNA synthesis (Brooks, 1977; Castor, 1977; Kim et al., 1968; Levine et al., 1965; Stanners and Becker, 1971). Once the role of protein synthesis is acknowledged, its sensitivity to small changes in Mg2þ, as demonstrated in vitro (Brendler et al., 1981; Ilan and Ilan, 1978; Schreier and Staehelin, 1973), would merit its consideration as a candidate for in vivo regulation of protein synthesis. However, a common attitude seems to be that intracellular Mg2þ is very well buffered and that the cell goes to ‘‘some pains to hold [it] steady’’ because it influences so many reactions (Rink et al., 1982). Mg2þ was therefore not considered a likely candidate for cellular regulation. It was contrasted with Ca2þ ‘‘which is well set up as a regulatory ion: translocation of small amounts producing large proportional changes in free concentration’’ (Rink et al., 1982). To bolster this argument, the authors found ‘‘rapid changes in T-cell [Ca2þ]i in response to plant lectins but no measurable change in cell Mg2þ.’’ However, insulin stimulation of large increases in the Mg2þ form of phosphoglucomutase have long been known (Peck and Ray, 1971) and probably reflect an increase in free Mg2þ. It was also known that insulin markedly reduces the binding of Ca2þ to the plasma membrane of rat liver cells (Shlatz and Marinetti, 1972) and that Mg2þ has the same binding properties to membranes as Ca2þ (Carvalho et al., 1963). In addition, my own laboratory had reported that insulin treatment of quiescent CEF, which markedly raised DNA synthesis and led to cell division, consistently raised total Mg2þ and presumably free Mg2þ, but was without effect on total intracellular Ca2þ (Sanui and Rubin, 1978). Of course, when a fluorescent indicator for Mg2þ became available, it was shown that mitogens do induce a sustained increase in free Mg2þ (Grubbs, 1991; Ishijima and Tatibana, 1994; Ishijima et al., 1991). The fact that Mg2þ influences so many cellular processes is an argument for rather than against its primary role in growth regulation because all cellular structures have to be reproduced in a coordinate fashion and energy metabolism has to be raised to meet the demand, a need which is ideally met by Mg2þ. In contrast to early and fleeting changes in Ca2þ of mitogen-stimulated cells, the increase of Mg2þ persists for hours (Sanui and Rubin, 1978, 1982a), as would be expected of a second messenger for proliferation since the mitogen itself must remain on the cells for most, if not all, of the G1 period in order to produce its full effect on the initiation of DNA synthesis (Bolen and Smith, 1977; Brooks, 1977; Rubin and Steiner, 1975). Given the acute sensitivity of protein synthesis to free Mg2þ, its increase need not be great but must be tightly controlled by its extensive buffering. Another critic states that growth-stimulated cells do not exhibit a steep surge of Mg2þ, as they do of Ca2þ, so they are ‘‘not set up to use Mg2þ as a natural trigger’’ (Whitfield, 1982). In a similar vein, this critic

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endorses the growth regulatory role of Ca2þ because it tends to arrest cells near the G1/S border while Mg2þ deprivation affects cells in all parts of G1 (Whitfield, 1982). In both those respects, however, Mg2þ mimics the effects of growth factor application and removal while the ‘‘trigger’’ action of Ca2þ does not. In any case, the evidence for a primary role of Mg2þ in cell growth regulation is strong enough to invalidate the objections and to raise questions about the role of the other cations. The role of Mg2þ in regulation of protein synthesis and thereby of DNA synthesis and mitosis receives strong support from current molecular studies of the PI 3-K pathway of response to growth factors (Richardson et al., 2004). This pathway leads through a protein kinase cascade to mTOR kinase, which is considered a central regulator of protein synthesis (Schmelzle and Hall, 2000). mTOR accomplishes this role by phosphorylating serines and threonines on two protein substrates which then drive further steps that initiate translation, mainly of ribosomal proteins and elongation factors (Terada et al., 1994). The mTOR phosphorylations have a high Km for what was thought to be ATP (Dennis et al., 2001; Jaeschke et al., 2004) but must, in fact, be MgATP2, which is the true substrate for all phosphoryl transfer reactions in the cell. Further considerations indicate that the level of MgATP2 is dependent on the availability of Mg2þ which is increased by stimulation with growth factors (Grubbs, 1991; Horowitz and Tluczek, 1989; Ishijima et al., 1991; Sanui and Rubin, 1978, 1982a). Hence, the molecular studies confirm a central role for Mg2þ in growth regulation that had already been inferred from the more physiological earlier studies. The molecular studies, however, indicate that the role of Mg2þ in protein synthesis is exerted mainly through its effect on the concentration of MgATP2 as substrate for mTOR kinase with its high Km for this substrate. It may be significant for the coordination of protein synthesis with energy production that the kinases of carbohydrate metabolism, which phosphorylate low molecular weight substrates, have Km for MgATP2 similar to that for mTOR (Edelman et al., 1987; Garner and Rosett, 1973). Mg2þ and its chelation with ATP may therefore play as central a role in metabolic and growth regulation as ATP does as the source of energy.

ACKNOWLEDGMENTS I am grateful for the manuscript preparation and editing by Dorothy M. Rubin and the creation of Figs. 7 and 8 by Joel Ou. This paper is dedicated to the memory of Dr. Hisashi Sanui whose ingenuity and skill in measuring intracellular and surface-bound cations by atomic absorption spectrophotometry added immeasurably to understanding the role of cations in growth regulation. The effort for the paper was supported by the National Institutes of Health grant G13LM07483-03.

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Weiss, R. A. (1970). The influence of normal cells on the proliferation of tumor cells in culture. Exp. Cell Res. 63, 1–18. Whitfield, J. F. (1982). The roles of calcium and magnesium in cell proliferation: An overview. In ‘‘Ions, Proliferation, and Cancer’’ (A. Boynton, W. L. McKeehan, and J. F. Whitfield, Eds.), pp. 283–294. Academic Press, New York. Wolf, F. I., Fasanella, S., Tedesco, B., Torsello, A., Sgambto, A., Faraglia, B., Palozza, P., Boninsegna, A., and Cittadini, A. (2004). Regulation of magnesium content during proliferation of mammary epithelial cells (HC-11). Front. Biosci. 9, 2056–2062. Won, K.-A., Xiong, Y., Beach, D., and Gilman, M. Z. (1992). Growth-regulated expression of D-type cyclin genes in human diploid fibroblasts. Proc. Natl. Acad. Sci. USA 89, 9910–9914. Zeidman, I. (1947). Chemical factors in the mutual adhesiveness of epithelial cells. Cancer Res. 7, 386–389. Zetterberg, A., and Engstro¨ m, W. (1983). Induction of DNA synthesis and mitosis in the absence of cellular enlargement. Exp. Cell Res. 144, 199–207. Zetterberg, A., and Killander, D. (1965). Quantitative cytophotometric and autoradiographic studies on the rate of protein synthesis during interphase in mouse fibroblasts in vitro. Exp. Cell Res. 40, 1–11. Zhou, H., and Duesberg, P. (1990). A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 87, 9128–9132.

Presence and Influence of Human Papillomaviruses (HPV) in Tonsillar Cancer Hanna Mellin Dahlstrand and Tina Dalianis Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, 171 76, Stockholm, Sweden

I. II. III. IV.

Introduction Tonsillar Cancer Human Papillomavirus (HPV) Human Papillomavirus (HPV) in Tonsillar Cancer A. Frequency and Type of HPV in Tonsillar Cancer B. HPV and Tonsillar Cancer Patient Features C. HPV and Prognosis in Tonsillar Cancer D. HPV and Radiosensitivity in Tonsillar Cancer E. HPV and Correlation to Cell-Cycle Proteins in Tonsillar Cancer F. HPV and Genetic Instability in Tonsillar Cancer V. HPV and Other Tumors of the Head and Neck VI. HPV Vaccines VII. Conclusions References

Tonsillar cancer is the most common of the oropharyngeal carcinomas and human papillomavirus (HPV) has been found to be present in approximately half of all cases. Patients with HPV-positive tonsillar cancer have been observed to have a better clinical outcome than patients with HPV-negative tonsillar cancer. Moreover, patients with tonsillar cancer and a high viral load have been shown to have a better clinical outcome, including increased survival, compared to patients with a lower HPV load in their tumors. Recent findings show that HPV-positive tumors are not more radiosensitive and do not have fewer chromosomal aberrations than HPV-negative tumors, although some chromosomal differences may exist between HPV-positive and -negative tonsillar tumors. Current experimental and clinical data indicate that an active antiviral cellular immune response may contribute to this better clinical outcome. These data are also in line with the findings that the frequency of tonsillar cancer is increased in patients with an impaired cellular immune system. Thus, therapeutic and preventive HPV-16 antiviral immune vaccination trials may be worthwhile, not only in cervical cancer, but also in tonsillar cancer. ß 2005 Elsevier Inc.

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Copyright 2005, Elsevier Inc. All rights reserved

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I. INTRODUCTION When zur Hausen (zur Hausen, 1976) proposed that cervical cancer might be caused by human papillomavirus (HPV), the scientific community accepted that HPV could potentially be involved in the development of some but definitely not all cervical cancers. Today, it is fully accepted that different types of HPVs are present and instrumental in the induction of almost all cervical cancers as well as some other types of human cancer (zur Hausen, 1996, 1999). In addition, several mechanisms by which HPVs exhibit their oncogenic potential have also been revealed (zur Hausen, 1996, 1999). The possibility that preventive vaccines against HPV may soon reach clinical practice (Koutsky et al., 2002; Lehtinen and Dillner, 2002) has drawn even more attention to the association of HPV with other types of cancer. However, HPV is only present in a proportion of other types of cancer, e.g., head and neck cancer, anogenital cancer, and nonmelanoma skin cancer, and much less is known about the role of HPV in these tumors (Alani and Munger, 1998; de Villiers, 1991, 1997; Gillison et al., 1999; Licitra et al., 2002; Mork et al., 2001; Snijders et al., 1992). Nonetheless, tonsillar carcinoma is of particular interest, since it is the head and neck cancer where HPV is most commonly found (Gillison et al., 2000; Mork et al., 2001; Paz et al., 1997; Snijders et al., 1996). Approximately half of all tonsillar cancers are HPV positive (Andl et al., 1998; Klussmann et al., 2001; Mellin et al., 2000; Paz et al., 1997). In addition, recent reports suggest that patients with HPV-positive tonsillar tumors have a lower risk of relapse and longer survival compared to patients with HPV-negative tonsillar tumors (Gillison et al., 2000; Mellin et al., 2000). These data motivate further comparisons between HPV-positive and HPV-negative tonsillar tumors with regard to clinical outcome, sensitivity to radiotherapy, biology of the tumor, and genetic stability in order to better understand possible options for treatment and vaccination studies. The purpose of this article is to review current knowledge on the status and significance of HPV in tonsillar cancer.

II. TONSILLAR CANCER Cancer of the palatine tonsil, in the lymphoid region called the Waldeyer’s ring, is usually referred to as tonsillar cancer. Tonsillar cancer is the most common of the oropharyngeal malignancies, and 75% of all tonsillar carcinomas are squamous cell carcinomas (Genden et al., 2003). As with all head and neck squamous cell carcinomas (HNSCC), smoking and alcohol abuse are regarded as the main etiological factors for tonsillar

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cancer and are known to account for 80 to 90% of all HNSCC (Decker and Golstein, 1982; Licitra et al., 2002). However, HNSCC and tonsillar cancer also occur in some 15 to 20% of patients without these risk factors (Gillison et al., 2001; Licitra et al., 2002). In many of these instances, viruses are most likely to be involved in the development of HNSCC, and data now indicate that high-risk types of HPV, similar to those observed in cervical cancer, are associated with a subset of HNSCC (Alani and Munger, 1998; de Villiers, 1991; Gillison et al., 2000, 2001; Gissmann et al., 1982; Mork et al., 2001; Naghashfar et al., 1985; Snijders et al., 1992; Syrjanen et al., 1983). Patients with tonsillar cancer do not normally seek health care until the tumor is fairly large and presents symptoms like swallowing-related pain or difficulties in swallowing (Mashberg and Samit, 1995). This is because small tumors generally do not cause any discomfort. Other common first symptoms are pain in the ear or a lump in the neck due to the tumor spreading to the lymph nodes (Mashberg and Samit, 1995). In advanced cases, vital functions such as breathing, eating, and speaking may be significantly affected. Further suffering may be caused by cancer growth in the face and the neck. Later on, the curative treatments of surgery and radiotherapy (Genden et al., 2003; Mellin et al., 2000) can be disabling and disfiguring. Tonsillar cancer is generally treated with (pre-operative or post-operative) full-dose radiotherapy (64 Gy) against the primary tumor and the neck. The extent of the surgical intervention depends on the size of the primary tumor, the presence of metastases in the neck lymph nodes, and the response to the radiotherapy given. Overall survival for patients with oropharyngeal cancer is 67% with stage I, 46% with stage II, 31% with stage III, and 32% with stage IV (Pugliano et al., 1997). However, the overall survival for patients with oropharyngeal cancer is only about 38% (Pugliano et al., 1997). Despite similar histology and stage, as well as standardized treatment, it is not easy to predict the outcome of each individual case. Hence, both predictive and prognostic markers would be of significant clinical value in order to tailor treatment for individual tonsillar cancer patients. This would allow for optimization of therapy to give the most efficient treatment with minimal impact on function and form. Moreover, given the high proportion of HPV-16 associated with tonsillar cancer (see following text), patients could obtain substantial benefit from the use of the same prophylactic and adjuvant therapeutic strategies that are being developed to prevent and/or treat HPV-associated anogenital cancers (for reviews, see Devaraj et al., 2003; Ling et al., 2000). However, before using such treatment, it is important to investigate in which cases this could be an option.

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III. HUMAN PAPILLOMAVIRUS (HPV) There are more than 100 HPV types, and for general reviews on HPV and cancer, as well as, more specifically, head, neck, and oral cancer, see, for example, Zur Hausen (1996), de Villiers (1997), Syrjanen (2003), and Scully (2002). Some HPV types are associated with common warts, while others are associated with chondylomas and papillomas. Finally, there are HPV types such as HPV 16, 18, 31, 33, and others that are associated with malignant tumors. Nevertheless, the genomes of all HPVs are similar and consist of double-stranded circular DNA with a size of 7 to 8 Kb. The genome is enclosed in a 52- to 55-nm viral capsid, and is arbitrarily divided into a noncoding region and two coding regions, the early and late regions. The early region encodes for the early proteins E1-E2 and E4-E7, which are important for pathogenesis and transformation, while the late region encodes for L1 and L2, the two capsid proteins. Of particular interest in this context is that among the HPV types associated with malignant tumors, E6 and E7 are classified as oncogenes. E6 binds to the cellular protein p53 and degrades it, while E7 binds to pRB and abrogates its function (Dyson et al., 1989, 1992; Scheffner et al., 1990). Under these conditions, the intracellular levels of normal p53 and pRB are reduced and this combination results in the inhibition of cell cycle control and facilitation of tumor development (for reviews, see Hanahan and Weinberg, 2000; zur Hausen, 1996). Also of interest is that the L1, the major capsid protein, can self assemble and form viruslike particles (Kirnbauer et al., 1993), which are useful for vaccination against HPV infections.

IV. HUMAN PAPILLOMAVIRUS (HPV) IN TONSILLAR CANCER A. Frequency and Type of HPV in Tonsillar Cancer HPV DNA has been shown to be present in 45 to 100% of all tonsillar tumors (Andl et al., 1998; Dahlgren et al., 2003; Gillison et al., 2000; Koskinen et al., 2003; Mellin et al., 2000; Ringstro¨ m et al., 2002; Snijders et al., 1992). The variation depends mainly on the type of material that is available for analysis and the methodology used for detection (as has been discussed) (Mellin, 2002; Mellin et al., 2002). In general, it is easier to  detect HPV in fresh-frozen (
70 C) tumors compared to formalin-fixed and paraffin-embedded tumors, where the DNA is degraded and where

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degradation progresses even after storage for decades (Mellin, 2002; Mellin et al., 2000, 2002). The most common and sensitive technique for the detection of HPV is based on polymerase chain reaction (PCR) technology (Mellin, 2002; Mellin et al., 2002). In the past, less sensitive methods such as Southern blots or in situ hybridization techniques have been used (Mellin, 2002; Syrjanen, 1990). Screening for HPV by PCR analysis is usually initially performed using HPV consensus/general primers (e.g., GP5þ/6þ, My9/10, CPI/IIG, FAP59/61) (de Roda Husman et al., 1995; Forslund et al., 1999; Manos et al., 1989; Tieben et al., 1993). These primer sets allow for the amplification of a wide range of HPV types and are useful for screening. Alternatively, they can be type-specific and identify only one specific HPV type (Hagmar et al., 1992). General primers are complementary to sequences (often in L1) in HPV that are highly conserved among many HPV types, while HPV type-specific primers bind to a sequence found in a single HPV type (often in E6 or E7) and do not cross-bind to other HPV types. Instead of an HPV type-specific PCR, HPV typing can also be performed by sequencing the PCR product obtained by a PCR run with general primers (Mellin et al., 2002). For HPV typing, HPV type-specific oligonucleotide probes using either enzyme immunoassays or Southern blot hybridizations are also commonly used (Herrero et al., 2003; van Houten et al., 2001). Without doubt, HPV type 16 is the type predominant in tonsillar cancer (Andl et al., 1998; Gillison et al., 2000; Klussmann et al., 2001; Koskinen et al., 2003; Mork et al., 2001; Paz et al., 1997; Snijders et al., 1992; Strome et al., 2002; Wilczynski et al., 1998). In most reports of HPV-positive tonsillar cancer biopsies, 85 to 100% contain HPV-16 followed by 0 to 7% containing HPV-33. HPV-31, HPV-59, or non-typeable HPVs are found even more rarely. In addition, when using DNA as well as RNA in situ hybridization, the viral genome and its transcription products (performed on HPV-16) have been located in cancer cells and nodal metastases but not to the surrounding stroma of the primary tonsillar tumor or the nodal metastases (Demetrick et al., 1990; Niedobitek et al., 1990; Snijders et al., 1992; Strome et al., 2002; Wilczynski et al., 1998).

B. HPV and Tonsillar Cancer Patient Features Patients with HPV-positive tonsillar tumors are less likely to be heavy smokers and drinkers, although it has been reported that HPV may have a synergistic effect with regard to tumor development in smokers (Gillison et al., 2000; Haraf et al., 1996; Herrero et al., 2003; Koch et al., 1999; Ringstro¨ m et al., 2002; Schwartz et al., 1998). A current concern regards the possible sexual transmission of HPV in oral and oropharyngeal

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squamous cell carcinoma (OSCC) (Herrero et al., 2003; Scully, 2002). This is suggested by the significant increase in tonsillar cancer reported among men in the United States from 1973 to 1995 (Frisch et al., 2000b). This may be explained by changes in sexual habits resulting in the increased transmission of HPV (Devaraj et al., 2003). It is also known that individuals with HPV-associated anogenital malignancies have an increased risk for a second primary cancer in the tonsils and oral cavity (Boice et al., 1985; Frisch and Biggar, 1999; Rabkin et al., 1992). In one of these studies, the increased risk was estimated to be 4.3-fold (Frisch and Biggar, 1999). Moreover, a similar study showed an increase in tonsillar cancer in women aged >50 years with a history of in situ cervical cancer, as well as an increased incidence of both tonsillar and tongue cancer in the husbands of cervical cancer patients (Hemminki et al., 2000). In contrast, patients with an HPV-unrelated cancer, e.g., colon cancer or breast cancer, had no increased risk of developing tonsillar cancer (Frisch and Biggar, 1999). Since the histology of the oral mucosa resembles that of the uterine cervix and other lower genital localizations, one can anticipate similar HPV infection patterns in the oral cavity as described for the genital tract (Syrjanen, 2003). HPV infection of the cervix is transmitted by sexual contact and there is a correlation between the prevalence of HPV, the number of sexual partners, and a low age at sexual debut (Oriel, 1971; Schiffman and Brinton, 1995; Syrjanen and Syrjanen, 1990). Orogenital contact has also been suggested to lead to HPV infections (Devaraj et al., 2003; Maden et al., 1992; Schwartz et al., 1998; Scully, 2002; Smith et al., 1998). In cervical cancer, which has been studied in more detail, HPV-involved cancer progression has been shown to be a multistep process. This process includes E6 and E7 transcription (of ‘‘oncogenic’’ HPV types), modification of cellular genes, and possibly also genetic susceptibility, an impaired cellmediated immunity, and co-factors such as smoking (Beskow and Gyllensten, 2002; Hanahan and Weinberg, 2000; Schiffman and Brinton, 1995; zur Hausen, 1996, 1999). Less is known about HPV-induced carcinogenesis at other tumor sites. However, it is reasonable to assume that the pathways are similar but not necessarily identical for tonsillar cancer. In the cervix, the immune system usually clears HPV infections within months or years. Only rarely after 10 to 30 years of latency do HPV infections progress to cervical carcinoma (Beskow and Gyllensten, 2002; Rome et al., 1987; Schiffman and Brinton, 1995; Syrjanen and Syrjanen, 1990). It is possible that this is also true for tonsillar cancer; however, the latency period is still unknown. An impaired cellular-mediated immune system (such as in HIV-infected or transplanted patients) results in an increase in HPV-induced lesions as well as an increase in HPV-associated cancers, including tonsillar cancer

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(Berkhout et al., 1995; Demetrick et al., 1990; de Villiers, 1997; Frisch et al., 2000a; Swoboda and Fabrizii, 1993). Evasion of the cell-mediated immune system is critical for HPV-transformed tumor cells. In cervical cancer, the expression of HLA class I antigens and accessory molecules is often downregulated (Cromme et al., 1994; Stanley, 2001; Stern, 1996). It is possible that similar mechanisms will also be found in tonsillar cancer. The role of humoral immunity in HPV infection is not well understood. In HPV-infected women, an IgG response to HPV-16 and HPV-18 is often observed 4 to 12 months after HPV DNA detection (Carter et al., 1996; Lehtinen and Paavonen, 2001). In the sera of patients with HNSCC, the presence of antibodies against HPV-16 is significantly more frequently observed compared to individuals not having HNSCC (Mork et al., 2001). However, while antibodies against the viral capsid proteins are markers of past or present infection, antibodies against E6 and E7 are markers more clearly associated with malignant disease (Herrero et al., 2003; Lehtinen and Paavonen, 2001).

C. HPV and Prognosis in Tonsillar Cancer In 1998, a survival analysis of 31 patients with tonsillar cancer using tumor pRB expression demonstrated a significantly better survival for patients with pRB-negative tumors (Andl et al., 1998). HPV presence and survival were not analyzed separately. However, there was an indication of a significant correlation between lack of pRB expression and presence of HPV (Andl et al., 1998). It was first reported in 2000 that HPV is a favorable prognostic factor in tonsillar cancer (Gillison et al., 2000; Mellin et al., 2000). In one of these studies (Mellin et al., 2000) on 60 patients with tonsillar cancer, it was found that 52% of the patients with HPV-positive tumors were tumor-free 3 years after diagnosis, as compared to 21% of patients with HPV-negative tumors (Fig. 1). Patients with HPV-positive tumors also exhibited a significantly longer 5-year survival compared to patients with HPV-negative tumors (53.5 compared to 31.5%, p ¼ 0.047, log-rank test). HPV was a favorable prognostic factor independent of tumor stage, age, gender, and grade of differentiation (Mellin et al., 2000). In the study by Gillison et al. (2000) on 253 head and neck cancer patients, 60 had oropharyngeal cancers (mostly tonsillar cancers) and disease-specific survival was significantly improved for the HPV-positive oropharyngeal cancer group compared to the HPV-negative group. However, among patients with non-oropharyngeal cancers, the disease-specific survival was similarly independent of HPV status (Gillison et al., 2000). Accordingly, the prognostic value of HPV did not seem to hold for head and neck cancer in general, but only for tonsillar

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Fig. 1 Number and percent of disease-free patients at 3 years after diagnosis for each stage and HPV status. Reproduced from Mellin et al., 2000, with permission from Wiley.

cancer specifically (Gillison et al., 2000; Paz et al., 1997; Riethdorf et al., 1997; Snijders et al., 1996). Additional reports on HPV as a favorable factor for tonsillar cancer have subsequently been reported (Dahlgren et al., 2003; Friesland et al., 2001; Mellin et al., 2002; Ringstro¨ m et al., 2002; Strome et al., 2002). Moreover, in a recent study on the prognostic value of HPV in HNSCC, HPV was not found to have any prognostic value for HNSCC as a whole (Koskinen et al., 2003). However, in this study, all tonsillar tumors were HPV positive and all patients with tonsillar cancer remained alive during the observation period (which for all HNSCC ranged between 1.4 and 89.6 months, mean 24.5 months) (Koskinen et al., 2003). In subsequent studies, the possible importance of the viral load and physical status of HPV on the clinical outcome was evaluated for tonsillar cancer by Mellin et al. (2002) and for HNSCC in general by Koskinen et al. (2003). Mellin et al. (2002) analyzed the presence of HPV in 22 fresh-frozen pretreatment tonsillar samples by general and type-specific PCR using a quantitative PCR. Eleven of the 22 analyzed patients had HPV-16 positive tonsillar cancer; the viral load ranged from 10 to 15,500 HPV-16 copies/cell with a mean of 190 copies/cell. The estimation of viral load in tonsillar cancer was thus in line with the previous study of Klussman et al. (2001). In the six tonsillar cancers and their metastases that were analyzed, the viral copy number per -actin varied between 5.8 and 152.6. Interestingly, Mellin et al. (2002) found that patients with >190 HPV-16 copies in their tumor cells had a significantly longer survival rate than did patients with