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Annual Review of Immunology Volume 5 1987 Before and After Elvin A. Kabat. Vol. 6: 1–25 Transgenic Mice and Oncogenesi...

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Annual Review of Immunology Volume 5 1987

Before and After Elvin A. Kabat. Vol. 6: 1–25

Transgenic Mice and Oncogenesis Suzanne Cory and Jerry M. Adams. Vol. 6: 25–48

Kinin Formation: Mechanisms and Role in Inflammatory Disorders D Proud, and A P Kaplan. Vol. 6: 49–83

Immunobiology of CR2, the B Lymphocyte Receptor for Epstein-Barr Virus and the C3d Complement Fragment N R Cooper, M D Moore, and G R Nemerow. Vol. 6: 85–113

Veto Cells Pamela J. Fink, Richard P. Shimonkevitz, and Michael J. Bevan.Vol. 6: 115–137

Antigenic Variation in Lentiviral Diseases Janice E. Clements, Susan L. Gdovin, Ronald C. Montelaro, and Opendra Narayan.Vol. 6: 139–159

Structure, Organization, and Regulation of the Complement Genes R D Campbell, S K Alex Law, K B M Reid, and R B Sim. Vol. 6: 161–195

Normal, Autoimmune, and Malignant CD5+ B Cells: The LY-1 B Lineage? K Hayakawa, and R R Hardy. Vol. 6: 197–218

Opioid Peptides and Opioid Receptors in Cells of the Immune System N E S Sibinga, and A Goldstein. Vol. 6: 219–249

Structure and Function of Human and Murine Receptors for IgG J C Unkeless, E Scigliano, and V H Freedman. Vol. 6: 251–281

Melanoma Antigens: Immunological and Biological Characterization and Clinical Significance M Herlyn, and H Koprowski. Vol. 6: 283–308

The Developmental Biology of T Lymphocytes H V Boehmer. Vol. 6: 309–326

V Genes Encoding Autoantibodies: Molecular and Phenotypic Characteristics C A Bona. Vol. 6: 327–358

Role of the Major Histocompatibility Complex Class I Antigens in Tumor Growth and Metastasis K Tanaka, T Yoshioka, C Bieberich, and G Jay. Vol. 6: 359–380

The Immunoglobulin Superfamily—Domains for Cell Surface Recognition A F Williams, and A N Barclay. Vol. 6: 381–405

Lymphotoxin N L Paul, and N H Ruddle. Vol. 6: 407–438

Regulation of Cytokine Gene Expression T Taniguchi. Vol. 6: 439–464

Unique Tumor-Specific Antigens H Schreiber, P L Ward, D A Rowley, and H J Stauss. Vol. 6: 465–483

Molecular Regulation of B Lymphocyte Response T Kishimoto, and T Hirano. Vol. 6: 485–512

IgE-Binding Factors and Regulation of the IgE Antibody Response K Ishizaka. Vol. 6: 513–534

Nonprecipitating Asymmetric Antibodies R A Margni, and R A Binaghi. Vol. 6: 535–554

Three-Dimensional Structure of Antibodies P M Alzari, M B Lascombe, and R J Poljak. Vol. 6: 555–580

Prospects for Gene Therapy for Immunodeficiency Diseases P W Kantoff, S M Freeman, and W F Anderson. Vol. 6: 581–594

C1 Inhibitor and Hereditary Angioneurotic Edema A E Davis, III. Vol. 6: 595–628

The T Cell Receptor/CD3 Complex: A Dynamic Protein Ensemble H Clevers, B Alarcon, T Wileman, and C Terhorst. Vol. 6: 629–662

To the Malaria Circumsporozoite Protein: An Immunological Approach to Vaccine Development M F Good, J A Berzofsky, and L H Miller. Vol. 6: 663–688

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews

Annual Reviews Ann.Rev. Immunol.1988.6: 1-24

IBEFORE AND AFTER


Elvin A. Kabat Departmentsof Microbiology, Genetics and Developmentand the Cancer Center/Institute for Cancer Research, ColumbiaUniversity College of Physicians and Surgeons, NewYork, NewYork 10032, and the National Institute of Allergyand InfectiousDiseases,NationalInstitutes of Health, Bethesda, Maryland20892 Introduction Theearlier portion of myautobiography(1) dealt with the period from the time I started to work with MichaelHeidelberger as a laboratory helpe~.on January 2, 1933 until mygrants from the USPublic Health Service weresummarilycancelled in 1952.This chapter continues mystory from that point. First though, I wouldlike to add a few notes about my origins andearliest days. Beginnings Mymother and father had married in 1913 and I was born on September 1, 1914, Both of myparents had cometo the United States as small children. Myfather, Harris Kabatchnick,wasborn in 1872and emigrated to the UnitedStates fromLithuaniaas a small boy. His first recollection after landing in the United States by boat from Hamburg was that the flags wereat half mastbecausePresidentGarfield hadjust died. His family settled on the lower East Side. Mymother, DoreenOtesky, cameto the UnitedStates from Kievin 1893at the age of seven. Myfather completed elementaryschool, went to work, and with his two brothers, Samueland Joseph, started manufacturing women’sdresses. In 1908 they changed their nameto Kabat and the firm wascalled KabatBros. I knowlittle of mymaternal grandfather. Hewaskilled in an accident before I wasborn. Mygrandmotherand the children lived with his brother Morris. It seemsthat the generationthat emigratesto a foreign country, andespeciallythe first generationbornin the UnitedStates, tends to ignore ~ The USGovernmenthas the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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2 KABAT its history and background until a complete story becomes difficult or impossible to get. Mymother completed high school and played the piano very well. Her mother lived with us until her death, whenI was 11 or 12 years old. We had a five-room apartment on the fourth floor at 24 West 11 lth Street. Both my mother’s and father’s families were very close. Manylived in Harlem within four or five blocks of our home; others lived in the Bronx or in Brooklyn. Aunts, uncles, and their children visited one another frequently. Mycousin Saul Meylackson, a physician who had been a captain in the Medical Corps, US Army, during World War I, became my role model. Myinterest in medicine grew largely from him, and when he visited, as he and his wife Pearl did frequently, I constantly plied him with questions about his patients and about medical research. I used to visit his office frequently on Saturdays, especially during myhigh school and college years, and looked at the smears he made of urethral exudates to diagnose gonorrhea. Pearl’s brother was Dr. Murray Peshkin, a wellknownallergist at Mount Sinai hospital, whomI also questioned incessantly about medicine. One grewup in the 1910s and 1920s keenly aware of the role of infectious disease. I lost a brother whodied of pneumoniaat a few weeks of age in 1918; a cousin died of polio in the 1918 epidemic; myfather was very sick in the influenza pandemic of 1917; a friend in our apartment house died of diphtheria, and manyfamilies lost a child or youngrelative. Epidemics of whoopingcough, chicken pox, scarlet fever, measles, and diphtheria were frequent. Whenthe Schick test and immunization with diphtheria toxin-antitoxin were first introduced in NewYork City Schools in 1924, I was Schick negative, an early indication of mypotentiality as an antibody former. Myparents were very devoted to me and to mysister Harriet, born May 8, 1920. I had everything I wanted for the first 12 years of mylife. My mother tended to be somewhatover-protective. At the age of 10 or 11 I went to a school on 117th Street, and had to cross Lenoxand St. Nicholas Avenues on the way. She wanted to accompany me, but I absolutely refused. She then followed me at some discrete distance. WhenI turned around and saw her, I laid down in the middle of the road and motioned to her to go back before I would stand up. WhenI was seven or eight I went through a religious phase and asked mymother to get me a Hebrewteacher; I studied with him until I became Bar Mitzvah at 13. Unfortunately, I was taught to read and memorizebut never to knowthe structure of the language. This knowledgewould have been of great value in myextensive contacts with the WeizmannInstitute and Israel.

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Myinterests in chemistry began early. Oneof myolder friends who lived on the samefloor wasgiven a chemistryset and let mehelp him. I soongot myownset and was alwaysexperimenting.Manyof the chemicals in the sets kids used in those days are nowhighly restricted even for laboratories. Myfather wouldstop off at Macy’son Saturday afternoon to buy mesomescience books.I wasgreatly influenced by Paul de Kruif’s MicrobeHunters. WhenI wasthirteen life becamevery difficult for us. Thefamily firm had prospered during the second and muchof the third decade of the century, manufacturingdresses selling for $24.95 to $89.95 wholesale. Thencheapdresses beganto flood the market, and the firm went bankrupt in 1927. They tried to get started again with moneyborrowed from relatives, but this enterprise also failed. From1929to 1933myfather did odd jobs, our incomedeclined continuously, and we were dispossessed from one apartment after another because we could not pay the rent. Whenwe movedinto a small apartment, wehad to put goods in storage and these were lost whenwe could not pay the monthlycharges. They containedmostof the papersof myearly life. Onelandlord turned off our electricity so we were in the dark. Wehad no food but a small piece of butter whichwekept from getting rancid by wrappingit and letting cold water run over it. Myfather and I went to court and the judge ordered the electricity turned on. Oneapartmenthotel held on to our furniture, so wethen had to find a furnished apartment. Twocousins whowere dress manufacturers,Arthurand Dick Shill, gave myfather a small job, helping to wait on customersin their showroom. Hestarted in 1932at five dollars per week, but as things improvedduring the NewDeal he continued to work and ended up with a more respectable salary--perhaps $40 to $50 per week. Mysurvival and ability to continue myeducation were largely consequencesof the educational concepts of the early 1920s. LewisM. Terman at Stanford during WorldWarI had emphasizedthe use of intelligence testing to select gifted children for special attention. TheNewYorkPublic SchoolSystemwasallowing bright children to skip grades (half years) they felt that they could do the work. WhenI entered elementaryschool in September1920, I wasalready able to read and to do arithmetic. This was in part becausethe kids in our apartmenthouse played school, with someof the older girls as teachers. Byskipping four times in elementary school I saved two years. WhenI entered DeWittClinton HighSchool at the age of 12, Terman’sextensive study of 1000gifted children whowere to be followedfor a goodportion of their lives waswell underway (2). The Termanwavehad reached high school. Bygetting goodgrades and taking somesummerclasses, I completedhigh school in three years instead of

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four. Thus, in the fall of 1929, at the age of 15 I entered City College. I had applied for a Pulitzer Scholarship to Columbiabut was not selected. At City College I also did well. Since I received extra credits for A and B grades, applied toward the 128 required for the bachelors degree, and since I took one or two summercourses, I was again able to complete the four year course in three. By June of 1932, I was graduated from City College with a B.S. degree. I applied to two medical schools, Cornell and Columbia, whose catalogs said that they had scholarships. Indeed, Columbia had a scholarship specifically for a City College graduate, but it was not being awarded in those days. (It is of interest that I was subsequently on the medical faculty of both these institutions.) Regardless of the extensive subsequent changes in psychologists’ ideas about the limitations and validity of IQ tests, the Termanideas had a very crucial influence on what happened to me. From 1931 to the time I was graduated, the relatives who were helping to support us applied substantial pressure on meto quit college and get a job. I was picking up small amounts of moneyby working during college registration, and I worked as an usher in the old Loew’s NewYork movie theatre during one summer. However, the pressure kept growing for me to quit college. I finally told one of mymother’s brothers to "get the hell out and don’t come back." It was many years before we made up. Twogood friends, Joseph Silagy and Joel Hartley, graduated in 1931 and went to NewYork University Medical School. I used to visit them and sit in on their classes on Saturday mornings. I heard lectures by Homer Smith and R. Keith Carman. I later became very friendly with Cannan while he was at NewYork University as chairman of the Biochemistry Department and at the National Research Council. Wealso met during summers at Woods Hole. While at City College, I had the opportunity to work with Professor Leo Lehrmanin analytical chemistry. Later I started working with Michael Heidelberger, but I continued at City College on Tuesday and Thursday evenings, when Leo taught evening classes. Myearliest papers were published with him. I also becamevery friendly with the professor of organic chemistry, W. L. Prager, and with a biology instructor, Alexander S. Chaikelis. I took his very popular course in physiology during one summer. As a way of trying to help me financially, we constructed a large flow chart of synthetic reactions of aromatic compoundsand tried, unsuccessfully, to interest publishers in it. I did showit to MichaelHeidelberger while I was looking for a job; he liked it and pointed out sections that represented his earlier work. It probably influenced his decision to take me. Charts of this type later becamepopular teaching tools. I was a good Germanstudent and became very friendly with my German teacher, Mark Waldman. Leo

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Lehrman tried, also unsuccessfully, for us to get the rights to translate Fritz Feigl’s bookon spot tests into English. I visited the Feigls many yearslater in Brazil. As a freshmanat City College, I brashly decided to volunteer for the curriculum committee, a student group that madesuggestions to the faculty. Theywere generally juniors and seniors; they had never had a freshmanon the committee,but they took me. I enjoyedtheir meetingsand did makesomesuggestions. DavidRittenberg was also on the committee duringmyfirst year. The Story Continues I will nowtake up mystory wherethe first chapter concluded. Onlyin 1981did I learn, underthe Freedomof InformationAct (Figures 1, 2), that the CriminalInvestigationDivisionof the Department of Justice had placed meon a "list for the apprehensionand detention of prominent individuals considered dangerousto the security of the UnitedStates." The FBI documentimplies that by 1954they were at least looking for information that might lead to myremovalfrom this Security Index. I include this since most Americansare probablyunawareof the existence of such a list. Considering the admittedinjustice of the forcedresettlement of Japanese Americansduring WorldWarII, one wonders howuseful sucha list actually is. Needlessto say, I wasneverapprehended or detained. The last monthsof 1953 and 1954were spent trying to continue work without support from the Public Health Service. Thestudies on cerebrospinal fluid gammaglobulin had proven very useful as an aid in the diagnosis of multiple sclerosis, and HoustonMerritt arranged for the Presbyterian Hospital to makeit a routine clinical laboratory deterruination. Theyprovidedmewith funds for a technician and someparttime help in washingglassware, drawnfromthe fees chargedfor the tests. My laboratory continued to do this until the late 1970s whenautomated methodsweresubstituted. I received a small grant from United Cerebral Palsy, whichhelped. I activated myONRcontract, whichallowed us to continue work on the blood group and dextran problems. The NSFgrant in 1954madeit possible to keepmytechnicians, graduateand postdoctoral students. The University took responsibility for mysalary (1). It was impossible, however,to support the monkeycolony; the allergic encephalomyelitis work was discontinued, just whenwe were planning to isolate the encephalitogenicantigen. Fortunately, this problemwastaken up by manyother workers. Blood Group Substances Weresumedworkon the structure of the BloodGroupA, B, H, Le", b, Le I and i glycoproteins. Mybook Blood GroupSubstances(3) was written

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6

Figure 1

during several summers at WoodsHole and was published in 1956. Oligosaccharides were isolated, after mild acid hydrolysis, and later by alkaline borohydride degradation. The use of periodate oxidation followed by Smith degradation made it possible to propose (with Kenneth O. Lloyd)

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BEFORE

Figure 2

a composite structure for the carbohydrate moiety of the blood group substances. Later, DonCarlson developed improvedconditions that prevented peeling after alkaline elimination and madepossible isolation of oligosaccharides with reducedN-acetylgalactosaminitol, so that we could

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isolate intact reduced oligosaccharide chains. Walter Morganand Winifred Watkins were isolating oligosaccharides and determining blood group structures independently during the same period. Our results were in excellent accord, and we becamevery close friends. Our oligosaccharides isolated by 3H-alkaline borotritiide differed extensively; manyside chains resulted from incomplete biosynthesis, and this revealed extensive heterogeneity. The findings up to 1982 have been extensively described in a more personalized account (4). Later studies with Albert M. Wu, David Zopf, and Bo Nilsson, Flavio Gruezo & Jerry Liao on six grams of a humanovarian cyst blood group A substance subjected to Smith & Carlson degradations yielded many more oligosaccharide structures from the interior of the blood group substances and made possible extension of the composite structure. This also permitted placing on the compositestructure the oligosaccharides isolated earlier from various intact blood group substances. These were done by Kenneth Lloyd, Luciana Rovis, Byron Anderson, Marilynn Etzler, and by Sherman Beychok,who also studied their optical rotatory dispersion and circular dichroism. The development of the hybridoma technique by Kohler & Milstein led many groups to produce monoclonal anti-A, anti-B, and other blood group reagents. Dr. H. T. Chen and I mappedthe fine structures of the combining sites of monoclonal anti-A and anti-B produced commercially in Canada, England, and Sweden. All were equivalent as blood grouping reagents by the usual hemagglutination tests, but their fine structures differed substantially. We have made cDNAsand have cloned and sequenced them to correlate sequence differences with variation in site structure. Interest in the blood group Ii system began with the studies of Donald Marcus and Richard Rosenfield in the early 1960s. These showed that enzymesof Clostridium tertium destroyed I activity of red ceils, liberating galactose and N-acetyl-glucosamine. Subsequent studies, with Ten Feizi, classified I and i antigens into groups. Anti-IMa(group 1) specificity was shown to involve DGalfl(l ~ 4)DGlcNAcfl(1 ~ OCH2-. With Ray Lemieux’s laboratory we have mapped the fine structure of monoclonal anti-IMa sites more extensively. Weused a substantial number of synthetic oligosaccharides, each modifying a distinct portion of the antigenic determinant, to evaluate its contribution to binding. Mostingenious was Lemieux’s replacement of one of the two H in the -OCH2moiety by deuterium to give two optical isomers, one of which was highly active and the other inactive. After leaving our laboratory, Dr. Feizi continued studies on other I and i determinants, and Dr. Marcusclarified the nature of the determinants of the blood group P system.

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Interlude The Josiah MacyFoundation ran a series of conferences on Polysaccharides in Biology during 1955-1959.Weall exchangedviews, using the Frank Fremont-Smithprinciple, "Don’t speak while I am interrupting." Oneamusingincident in 1955comesto mind. K. G. Stern and I differed on the question of whetherthe microheterogeneity of proteins wasascribable to mistakesin synthesis. I took the position that this could not be unequivocallyestablished since small amountsof impurities mightgive the appearanceof microheterogeneity. Stemreplied, "I cannothelp but feel that nature cannotworkso precisely that it can reproducea moleculeof a million or twentymillion molecularweightexactly in its own imageas, for example,the imprint of a gramophone record. Bywayof example,if two typewriters of the samemakeand the sametype are used, and the sameletters are writtenwiththem,at first glance,it will be said that the imprintis identical, but it is a well-knownfact that criminological investigation can distinguish betweendifferent individual typewriters by microscopicinvestigation, becausethere exist alwaysminute irregularities; so I wouldsay this is a microheterogeneity,somethingwhichat first approximationlooks homogeneous but cannotstand up to the mostsearching criteria." Boyd:"Youmeanthat nature does not always produce homogeneity,or can never produce homogeneity?" Stem:"I wouldsay that at the level of the maeromolecule, it is too muchto expect of nature that such structures shouldbe absolutelyidentical. This is a carry-overfrom the experiencewith small molecules." Kabat:"I think I wasvery careful to say that the evidencedid not permitus to infer whether or not nature produced a homogeneousmacromolceule.However,for the record I amperfectly willing to state that Dr. Stemhas completelyconvincedmeof the microheterogeneity of typewriters."

GeorgSpringerwhowasediting the conference,indexedthis under: "Typewriters, microheterogeneity of, p 163." Lectins I had beeninterested in lectins since the studies on ricin duringWorldWar II carried out with MichaelHeidelberger and AdaBezer. WhileI was in Swedenin 1967, Sten Hammarstrrm,from Peter Perlmann’s laboratory, proposed to becomea postdoctoral student. He mentionedthat he had isolated a lectin fromHelix pomatiawhichagglutinated A red cells, so I told himto bring somealong. Westudied its combiningsite and determined its association constant by equilibriumdialysis. MarilynnEtzler studied the site specificity of Dolichusbiflorus. ArneLundbladcamefromUpsala, bringing someof his blood group oligosaccharides fromurine whichwere of great help in mappingA, B, Hspecific combiningsites of lectins and antibodies.

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WhenMiercio Pereira, with an M.D. degree, came to the laboratory from Brazil, he expandedthe lectin studies to purify and characterize the combiningsites of blood group specific lectins from Lotus, Ulex, soybean, and peanut. Hagen Bretting studied sponge lectins from Axinella; Jerzy Petryniak and Pereira studied Evonymuseuropeus; Shunji Sugii did l, Vistariafloribunda; and Paul Kaladas studied l~icia villosa with Kimuraand Erson from Hans Wigzell’s laboratory in Sweden. Hagen Bretting and Stephanie Phillips studied the mutageniceffects of,4xinella lectin. Charles Woodwith S. Ebisu, L. A. Murphy,and Irwin Goldstein investigated the lectins of Bandeireae simplicifolia A4, B4, and BSlI. Santosh Sikder with C. J. Steer and Gil Ashwellstudied the chicken hepatic lectin, and he did lima bean lectin with Goldstein and Dave Roberts. Wewere very fortunate to have collaborations with Nathan Sharon of the WeizmannInstitute and Irwin Goldstein at Ann Arbor and their colleagues on many of these studies. Manysimilarities exist between the fine structures of antibody combining sites to A, B, H glycoproteins and those of the blood group specific lectins as determined by immunochemicalmapping. Of special interest is the finding that lima bean lectin (5) and a hybridoma antiblood group (6) are very similar in reactivity with a monofucosylhexasaccharide from humanurine but behave entirely differently whena second fucose is substituted on the third sugar from the nonreducing end. The comparative mapping of the fine structures of lectin and antibody sites of similar specificities and correlating these differences with amino acid sequences and X-ray crystallographic structures should throw much light on the evolutionary emergence and functions of these two very important and diverse groups of molecules. Studies

on Antipolysaccharides

The immunochemical mapping of the combining sites and later recombinant DNAstudies on antibodies to ~(1 ~ 6)dextran became the predominant problem of the laboratory, with ancillary studies on anti-~(1 3)~(1 -~ 6)dextran (B1355S), antilevan, and anti-SIII. Rose Magestudied rabbit anti-or(1 ~ 6)dextran and could also showthat the rabbit anti-SIII site was complementaryto a hexasaccharide. Gerald Edelman studied the various antibodies I produced in myself--antidextran, antilevan, anti-A-as well as other humanantidextrans, after reduction and alkylation, using starch gel electrophoresis. These were studied in acrylamide, by Bill Yount and Marianne Dorner, and their subgroup compositions and genetic factors were determined by James Allen, Bill Yount, Marianne Dorner, and Henry Kunkel. Myantilevan turned out to be a monoclonal IgG; the others were pauciclonal. Aminoacid sequencing of antibodies was just

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beginning. Hopingto be able to get enough of myantibodies to sequence, Dick Rosenfield kindly took 15 one-liter plasmaphereses from me, each a week apart, so that eventually I had about 7.5 liters of my serum to fractionate. Stuart Schlossman, Justus Gelzer, and Marianne Dorner tried to fractionate my antidextran to obtain monoclonal populations. While some fractionation into populations with smaller and larger antidextran sites was achieved, monoclonal populations were not obtained, and the amounts produced by fractionation were too small for the sequencing methods of the 1960s. Michael Potter at the National Institutes of Health and Melvin Cohn and Martin Weigert at the Salk Institute had isolated mouse monoclonal anti-~(1 -~ 6)dextrans. The fine structures of the combiningsites of three IgAs were studied with John Cisar, Jerry Liao, Marianne Dorner, and Michael Potter. In 1970 John Cisar had taken a summercourse that I gave at OregonState University at Corvallis, and he had cometo the lab after completing his PhDdegree. Weshowed that the combining sites of W3129 and QUPC52differed. W3129 and W3434were complementary to five ~(1 -~ 6) linked glucoses, whereas QUPC52was complementary to six. Despite this, the binding constant of QUPC52was only 1/30 that of W3129.By competition assays by equilibrium dialysis, methyl ~-D-glucoside and isomaltose contributed 60%of the binding energy of the pentasaccharide with W3129,but only 5%of the binding energy of the hexasaccharide with QUPC52.This indicated that the specificity of W3129 was directed toward the terminal nonreducing end of the ~(1 -* 6) linked oligosaccharide, whereas QUPC52 was specific for the internal chain of ¯ (1 -~ 6) linked glucoses, loosely termed cavity-type but also called end binders and groove-type sites, respectively. This distinction could be readily demonstrated with a synthetic linear dextran synthesized by Ruckel & Schuerch. Since the linear dextran had but one nonreducing end, it inhibited precipitation of W3129by dextran as well as the pentasaccharide; toward QUPC52,however, it was multivalent and so formed a precipitate with hybridomaascitic fluids. This provides a rapid screening methodfor classifying the two kinds of monoclonalanti-~(1 -~ 6)dextrans. Human Monoclonal

Antibodies

The demonstration by Waldenstr6m that macroglobulins and gamma globulins could occur as monoclonal homogeneousproteins in the serum of individuals with various blood dyscrasias, and by Edelmanand Gally that Bence Jones proteins were the light chains of immunoglobulins, led manyinvestigators to search for antibody activities..A numberwere soon reported. Several laboratories acquired large collections of humanmonoclonal proteins, but unlike the mousegammopathies, relatively few of

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12 KABAT the humanmonoclonalshad identifiable antibody activities. Myinterest in screening such humanmonoclonalsarose from two directions. Elliott Ossermanhad a large collection of sera from individuals with multiple myelomaand Waldenstrrm macroglobulinemia. Wedecided to screen these serums with mycollection of polysaccharides, blood group substances, etc. EdwardFranklin at NewYorkUniversity also had a large collection of humansera that had been screened for various antibody activities, but without success. MarianKoshlandhad asked him for a humanIgM;in an attempt to purify it over a Sepharosecolumn,it did not comeoff. It seemedto her that somemight be eluted with a number of sugars, so she thoughtI wouldbe interested. In screeningby gel diffusion we found that the IgMreacted strongly with a water extract of agar preparedat 20°C.Since agar contained4,6-pyruvylatedgalactose, I asked MichaelHeidelberger, whowas studying rabbit antisera to Klebsiella polysaccharides, for a numberof samples. The purified Franklin monowEAreacted muchmore strongly with Klebsiella K21than it clonal IgM did with agar in quantitative precipitin assays; only about one twentieth as muchof these Klebsiella K21polysaccharideswasneeded. At the same time we had found another humanmonoclonalIgM~AYin the Osserman collection that also reacted with agar and with the sameKlebsiella polysaccharide. Unlike IgMwEAMAy which was studied as purified IgM, IgM was studied using whole serum. Wetested other Klebsiella K polysaccharides, and K30and K33reacted in both sera in a wayidentical to K21.Onthe basis of these studies, whichhadbeen acceptedby the Journal of Experimental Medicine (7), the AmericanCancer Society gave me research development grant for a postdoctoral fellow, Dr. Arapalli S. Rao, whocamefromCalcutta. Since he wasto learn the quantitative precipitin methodto continue the work, I thought it wouldbe logical for him to MAy.Wehad run out of the first repeat the studies on IgMwEAand IgM sample of K21and madeup a solution from another sample. He learned the techniquerapidly and wasable to confirmall of our data except that K21gave no precipitate with IgMWEA; he becameconvincedthat our curve with K21waswrong.By this time the proof of the JEMpaper was due to arrive, and I had to decide whether to take out the curve of K21with wEA.In worrying about this I remembered IgM that MichaelHeidelberger had first sent mesix samples, of whichonly K21,whichhad the same4,6pyruvylatedgalactoseas agar, reacted, thus giving us our first clue as to wEA.Onlylater did we get K30and K33whichhad the specificity of IgM 3,4-pyruvylatedgalactose and precipitated equally well per unit weight. I told Raothat there must be someother explanation for the difference betweenthe two K21samples, since if the reaction with 1(21 had not providedthe initial clue to the specificities of the twoIgM,I wouldnever

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have been able to apply for the grant and he would not have come to the laboratory. The explanation finally emerged. It turned out that the first K21 was the neutral sodium salt, but the second K21 preparation had been isolated as the free acid and was progressively lowering the pHof the unbuffered purified IgMwEAbut was not altering the pH of the serum of IgMs~gv. Rao carried out a study of the effect of pH on IgMTM and IgMMAyand found that IgMwEggave identical precipitin curves with K21, sfgv K30, and K33 at pH 4, but K21 did not react at higher pH. With IgM all three polysaccharides reacted identically from pH7.0 to 4.0, indicating differences in the structures of the two antibody combiningsites. Subsequent screening of the Ossermancollection revealed a number of other humanIgM monoclonals with various anticarbohydrate specificities including antibodies specific for the interior of the water soluble blood group A and B substances and for chondroitin sulfates. Most exciting was the discovery of IgMN°v (8), an antibody specific for poly-~(2 ~ 8)Nacetylneuraminic acid, the specific capsular polysaccharide of the group B meningococcusand of E. coli K1, against which vaccination has not been successful in humans. This antibody is as protective per #g antibody in rats infected with E. coli as is a standard horse antigroup B meningococcal serum. IgM~°v also reacts with poly A, poly I, and with denatured DNA probably ascribable to similar oligomeric patterns of the carboxyls in the group B polysaccharide and the phosphates in the polynucleotides and in DNA,permitting reactivity in the IgMN°v antibody combining site. Michael Heidelberger in 1983 put forward a similar explanation of the cross-reaction of type 8 and 19 pneumococcalpolysaccharides; he suggested it was due to the ability of negatively charged phosphoryl-fl-D-Nacetylmannosamineto enter the type 8 site specific for cellobiuronic acid with its negatively charged COO-and vice versa. IgMN°v has potential as adjunct serotherapy together with standard antibiotic therapy for group B meningococcalmeningitis, a possibility suggested by studies in the first three decades of this century of reductions in the death rate due to intrathecal horse antimeningococcal serum. GiampaoloMerlini, Steve Birken, and Marian Gawinowiczare determining the amino acid sequence of the Vn and VL regions of IgMr~°v so that the antibody can be synthesized by recombinant DNAtechniques. Stanley Hoffman and Gerald Edelman have shown that IgMN°v is antibody to N-CAM,and Karl Pfenninger has found it to show the histochemical distribution of N-CAM. The Variability

Plot

Careful amino acid composition analyses of antibodies of different specificities by Marian Koshland had revealed differences. In 1965 Hilschmann and Craig presented the first amino acid sequences of two humanBence

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14 KABAT Jones proteins and showedthat they differed extensively in the first 107 residues fromthe aminoterminusbut wereessentially identical throughout the C-terminal half of the molecule. Theseare nowtermed variable and constant regions. At an antibody workshop(9) at the Weizmann Institute in 1965Dreyer & Bennettsuggestedthat these two domainswere codedby separate genes; additional sequences were presented by Putnam, Milstein, Hood, Gray, Dreyer, and their colleagues. I becameinterested in these developments and in 1966-1967began a Sabbatical in Pierre Grabar’s laboratory at the Institut Pasteur, writing the first edition of "Structural Conceptsin Immunology and Immunochemistry."I thought it would be important to include the sequencedata. At that time two mousex and several human k BenceJones sequenceshad been published. Onaligning the data it was evident that in variable regions of humanand mousevery few aminoacid differencesdistinguish mousefromhuman,e.g. there werevery few speciesspecific residues (nowtermed"species-associatedresidues," as later suggested by Capra). The constant region, however,contained43 differences between humanand mouse; this was comparable to findings with hemoglobins, cytochromes,etc, from different species. A sequenceof a human 2 Bence Jones protein by Titani, Wikler, and Putnamappeared, which further reducedthe numberof species-associated residues and was cited in a "note added in proof." Francois Jacob and VernonIngram both thought the finding important, so I sent it to the Proceedingsof the NationalAcademy of Sciences. Shortly thereafter, in comparingthe V and C regions, I noted the occurrence of moreinvariant glycines in the Vregion, including those at residues 99 and 101 whichhavenowbeenfound in homologouspositions of V-regionsin essentially all immunoglobulin light and heavychains, in the a and fl chains of the T-cell receptor for antigen, andin T cell surface antigens T4andT8. I suggestedthat the role of these two variant glycines mightbe to provide flexibility and permit movementof the rest of the V-region to makemaximum contact with an antigenic determinant. Cesar Milstein, Niall & Edman, and the Hood group published sequencesof several V~chains whichindicated that there weresubgroups largely based on the first 23 aminoacids from the N-terminus. It then becameevident fromthe frequencyof identical repeats that this segment of the chain did not showthe variability neededfor it to be involved in generating antibody complementarityand diversity. This led to my suggesting that the first 23 aminoacids were involved only in threedimensionalfolding. Also, Milstein had noticed that beyondaminoacid 94 he could no longer define subgroupsand suggestedthat frequent crossing over mightbe occurring beyondresidue 94, a finding prophetic of V-

Annual Reviews


BEFORE AND AFTER

15

J joining, which was largely ignored. The 1967 Cold Spring Harbor and Nobel Symposia provided forums for these exciting developments on aminoacid sequences of antibodies. In 1969 I received a letter from a Dr. Tai Te Wu, then at Cornell University Medical College and Sloan-Kettering in NewYork, asking if he could spend some time in the laboratory to learn quantitative immunochemistry. Whenhe visited the laboratory I asked about his background. He said he was a mathematical biophysicist and had some computer experience. I proposed that we collaborate on the sequence variability. I had previously been entering and aligning the sequence data by hand using pencil and paper. Webegan to meet one day a week to evaluate and discuss results. By this time more complete sequences were beginning to pour into the literature from the laboratories of Capra, Edelman, Hilschmann, Hood, Metzger, Potter, Milstein, and their coworkers. In June 1969 at a symposiumin Prague, Edelman, Franek, and I discussed positions containing more substitutions than were seen in the subgroups. Franek had tabulated dissimilarity in the subgroups, whereas Wu and I had searched for maximumvariability, and our data were in good agreement for three hypervariable regions. In June 1970 (10) we introduced an equation measuringvariability at each position in a set of VLsequences aligned for maximumhomology as follows: Numberof different aminoacids at a given position Variability = Frequencyof the most common aminoacid at that position" At this time some 77 complete and partial sequences of VLregions of humanV~ and V~ and of mouse V~ chains were available. This led to the recognition of three linear stretches of hypervariability, whichwe predicted would fold to form the antibody combiningsite. Whena sufficient number of VHsequences were examined, they too had three hypervariable regions (11). X-ray crystallographic studies on Fab fragments, BenceJones dimers, and more recently on the lysozyme-antilysozyme site by the Poljak group at the Institut Pasteur have all confirmed this prediction almost on a residue-by-residue basis. These hypervariable regions are nowtermed complementarity-determining regions (CDR). Wuand I proposed (10) in an insertional mechanismby which nucleotides coding for the CDRswould be recombined into the frameworkresidues of the various subgroups. The variability plot was based on the assumptionthat, since the antibody forming system was universal among vertebrates, combining data from different subgroups, species, etc, was justifiable, and the combiningsites would be in one place rather than in different portions of the variable regions. WhenMartin Weigert and Melvin Cohn with Cesari and Yon-

Annual Reviews


16 KABAT kevich described the mouseV~chains, they found that most of their sequenceswere identical throughoutthe Vxregion. Theypostulated that these werethe productof a single germ-linegene. Thosechains that showed variation from the germ-line sequence were localized to the CDRsand could have been formedby point mutations. This providedthe first evidence for somatic mutation in immunoglobulinV~regions. The assumption is still valid for the very large numberof VLand VHchains now available. Variability plots havesince beenusedto define better allelisms in the MHC class I and II systemsand also to locate sites of variability in AIDS viruses. Variability plots for the T cell receptor require further analysis. Variability plots for other proteins, such as cytochromes,did not show hypervariable regions. A statistical examinationof each residue in the CDRsmadeit possible to designate certain positions as structural and others as probablyin contact with the antigenic determinant.

Compilationof Sequencesof ImmunoglobulinChains In 1973Harry Rose retired as chairmanof the MicrobiologyDepartment, and Harold S. Ginsburg whomI knew very well from WoodsHole was appointed.Since he wasjust getting started, I delayedmySabbatical one year so that I could participate in the teaching. I waschosenas a Fogarty Scholar and spent 1974-1975 at the NationalInstitutes of Health, writing the second edition of Structural Concepts in Immunologyand Immunochemistry. As it is customaryfor Fogarty Scholars to meet with the director of NIH,I was given an appointmentwith Dr. Robert S. Stone, whoinquired as to myresearch interests. WhenI replied that I was interested in the structure of antibody combiningsites and had been tabulating variable region sequences, he suggestedthat NIHhad the PROPr~Tcomputersystem and that this might help me. DeWittStettin, Jr., then DeputyDirector for Science at NIH, introduced me to WilliamF. Raub, then with the Division of Research Resources. NowDeputyDirector of NIH,he took a great interest in myactivity. To evaluate PROPrmT, HowardBilofsky came downfrom Bolt Beranek and Newman to help me in preparingthe tables of sequencesto be used in Structural Concepts.It soonbecameclear that PROPI-IEX had greatly superior potentialities for tabulating and keepingtrack of variable region sequences. Whenmyterm as a Fogarty Scholar was up, I wasasked to serve as an Expert, first to the NationalCancerInstitute andlater to the NationalInstitute of Allergy and Infectious Disease, spendingtwo days a weekat NIHto organize and maintain a data bank of variable region sequences.I rented an apartment in Building 20 at NIH. T. T. Wucame from Northwestern and Howard Bilofsky from Cambridgeto spend two days a monthkeeping track of and

Annual Reviews


BEFORE AND AFTER

17

checkingthe data that I collectedfromthe literature. Thefirst edition (12) appeared in 1976 and contained only aminoacid sequences of variable regions; the secondedition (13) wasexpandedto include constant regions; the third (14) also containednucleotide sequencesand derived aminoacid sequences and included other membersof the immunoglobulinsuperfamily. Thefourth edition (15) appearedin April 1987, included T cell receptors, and had grownto 804 pages; Wuand I, along with Harold M. Perry and KayGottesman,are nowworkingon a fifth edition. Keeping track of exponentiallyincreasingdata is extraordinarilydifficult. Thecompendiumhas been distributed free to workers in the field and has been consideredvery useful. WhileI amvery happythat so manycolleagues find it valuable, I would not have undertaken it had I not felt it was essential to myresearch interests. In addition to providing an enormousbodyof data supporting our prediction (10, 11) that the CDRs in the light and heavychains would fold up to form the antibody combiningsite, wewere able to demonstrate independentassortment of framework(FR) aminoacids (16). This led to formulate the minigenehypothesis, in whichnucleotides coding for the three CDRswere assorted by recombination or insertion into the frameworknucleotides. This hypothesis was put forwardjust as Tonegawa et al. (17) had shownthat, in 12-day-old mouseembryoDNA,the region nucleotides only codedthrough aminoacid 95. Since adult myeloma coded through amino acid 107, we postulated a mechanismof somatic joining of a minigenethat codedfor the remainingaminoacid to give the intact V gene. The J minigene coding for amino acids 96 to 107 was found shortly thereafter by Tonegawa in 12-day-old embryoDNA.In the expressed assembledgene from adult myelomaDNA,it had been joined to the V-region(18). D and J minigenescoding for segmentsof the genewerefoundshortly thereafter (19). In the T-cell receptor for antigen, J minigenesare also present in e, fl, and ~ chains and Dminigenesin the fl chains. Thesefindingshold for all species examined. Since genescodingfor the rest of the VLand VHoccurredin the germline as single stretches of nucleotides, considerablecontroversyexisted as to whetherthe minigenehypothesisapplied to the V-region. DavidBaltimore and RichardEgel proposedthat geneconversionscould explain our assortmentdata. The Rajewskylaboratory (20) described a double recombinant in which CDR1of one germline gene was recombined with CDR2and 3 from another. H. G. Zachau’s laboratory (21) showedby nucleotide sequencingand comparisonsof V-regionsthat our assortment data could be accounted for by gene conversions in the DNAcoding for the FR segments.Mostrelevant are the studies of Reynaudet al (22) in J. Weill’s laboratoryat the Institut Pasteuron chickenVachains. Thechicken

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18 KABAT


has only a single functional germline V~gene and a large numberof pseudogenes;thus diversity andthe antibody repertoire must be generated by rescue of functional CDRsof pseudogenes by gene conversions-essentially our original minigenehypothesis(16). Work with WHO In 1963, the WorldHealth Organizationbecameinterested in research and training in immunology. Theyorganizedfive meetingsof scientific groups. I wasinvited to participate at one of these at Ibadanin December 1964. Niels Jerne was one of the prime movers.I met Professor Ian Mclntyre, then Deanof the Faculty of Veterinary Science at University College, Nairobi, and Professor and later Dean in the Veterinary School at Glasgow.Webecomeclose friends. Ahigh priority wasassigned to setting up research and training centers in immunology in developingcountries in various parts of the world. Thesewereto be coordinatedwith centers in Geneva and elsewhere. HowardGoodman,Zdenek Trnka, and I went to Nigeria, Uganda,and Gambia.Weselected Ibadanas the first center. Ada E. Bezer, whohad been a technician with me for manyyears, and Dr. Ivan Riha fromPraguestaffed the center, whichwasin the Department of ChemicalPathology.A course in Immunology wasgiven for six to eight monthsfollowedby opportunities for students to do research. Thecenters initially took students fromall of WestAfrica and a numberof students completed PhDs. In 1966 HowardGoodmanand I went around the world and selected Singaporeas another center. Centerswereultimately selected in Sao Paolo, MexicoCity, Nairobi, Beirut, Teheran,and NewDelhi, with support centers in Lausanneand Geneva.From1968to 1971, I spent two weekseachyear (the first three years) with Dr. Otto G. Bier, andthe last year with lvam Mota, directors at the Sao Paolo center located at the Escola Paolista and later movedto the Institute Butantan. I conducteda reviewof the coursework, gavethe final exam,and tried to get a regular research seminarstarted. It wasvery difficult to maintaina critical mass and sustained enthusiasm for continuing the seminars whenI was not there. Dr. VasekHoubaand AdaBezer staffed the Nairobi center for several years. Later, regional centers tended to drawstudents moreexclusively from the country in whichthey were located, and Thailandset up its own immunological center. Dr. GeorgioTorrigiani is currently in chargeof the program,but WHO has difficulties supportingthe centers (23). I continue as a memberof the WHO Expert Advisory Panel on Immunologyand have recently been involved with work on immunizationwith bacterial polysaeeharides. A further developmentwas the setting up of ILRAD (International Laboratoryfor Researchin AnimalDiseases) in Nairobi.

Annual Reviews BEFORE

AND AFTER

19

This began work in 1978 under the auspices of the WorldBank, the Rockefeller Foundation, and the Agencyfor International Development, to develop vaccines against east coast fever and trypanosomiasis. I was involvedin the early planning.


Interlude OnDecember26, 1970, the front page of the NewYork Times contained an appealfrom 14 Soviet scientists to President Nixonto see that Angela Davisreceiveda fair trial. I thought:"Wehavea free judicial system,the trial will be open to the public; President Nixonoughtto invite themto comeand see for themselves."I reflected on howone mightget himto do this. It became clear that onecould not write to the Presidentdirectly; he wouldnever see the letter. I decidedthat one hadto addressthe suggestion to someone on his staff, whoat least could bring it to his attention if he thoughtit a goodidea. I sent the followingtelegram: Dec. 26, 1970 John Ehrlichman The White House Suggest President Nixon reply to fourteen Soviet scientists’ appeal about Angela Davis by inviting them or any Soviet lawyers they nominate to attend her trial. Stop. Also call attention to request of five NewYork City District Attorneys to attend trials of Jews accused by Soviet Union of plotting highjacking. Elvin A. Kabat Member, National Academyof Sciences

Theidea evidently was favorably received. Figure 3 is the reply from John Ehrlichman.On Sunday,January 3, 1971, the six o’clock evening newsannouncedthat President Nixonhad invited the 14 Soviet scientists to attend AngelaDavis’s trial. Figure 4 showsa portion of Monday’s New YorkTimesarticle andits editorial of January8. It still seemsremarkable that a suggestionfroma private individual could reach the highest levels and be acted uponso rapidly. Current Activities and Future Plans AlthoughI becameEmerituson December31, 1984, I have been permitted to keep mylaboratories and to maintain mygroup at about the samesize. I decideda few years backthat the laboratory had to go into cloning and sequencingif wewereever to be able to formulatea detailed conceptof the interactions in the CDRsof antibodycombiningsites to explain antibody diversity. AccordinglyI spent a short time in MarkDavis’ laboratory, then at NIH, learning to makeand done cDNA.This was followed by about 10 days at the Weizmann Institut learning nucleotide sequencingin DavidGivol’s laboratory. I workedclosely with the carbohydratechemists

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20 KABAT

1970


Dear M3:. Kabob: Thanks very much for your telegram in which ~’ou put fo~h your thoughts Presidenttsreply to the appeal ~de So%e% se~tis~s ~ be~lf Of ~gela

of December Z7th regarding ~e by fourteen Da~s.

i am sendln~ your wire to Henry Kissingerfor his

Thanks again

for letting

me have your thoughts.

7{) i{ave~Avenue New York 10032

Fiyure 3

in the laboratory,SantoshSikderandPradipAkolkar,to establish the making,cloning, andsequencingof cDNAs on a routine basis. Theythen taughtthe otherpost-doctorals,graduatestudents,andtechnicians. Weset ourselvesthe goal of trying to probethe repertoireof antibody sites formedto a single antigenicdeterminant, the epitope of a(1 -, 6)linked glucoses fromdextranB512.Graduatestudents JacquelineSharon and BarbaraNewman had been engaged in mappingthe antibody combiningsites ofhybridoma antibodiespreparedby injecting ~(1 -~ 6)dextran

Annual Reviews


BEFORE ANDAFTER 21

Fiyure4 into BALB/cand C57BL/6mice. Eric Lai had carried out similar studies of C57BL/6hybridom~is from mice immunizedwith stearylisomaltosyl oligosaccharides. H. T. Chenand D. Makovercharacterized hybridomas to the stearylisomaltosyl oligosaccharides in a nude mouseand in C58 mice. Thus, we had a sampling of hybridomas with groove-type sites complementaryto six and seven ~(1 ~ 6)-linked glucoses. These were about equally divided between IgM and IgA, but two IgG3 hybridomas

Annual Reviews


22 r,~d~Ar were obtained. Both antigens used for immunizationyielded T-independent hybridomas. Flavio Gruezo prepared mRNA and Drs. Sikder, Akolkar, Bhattacharya, and Jerry Liao have been cloning and sequencing them. Sherie Morrisonis collaborating. She and her colleagues pioneered in preparing transfectomassubstituting C-regiongenes of other species onto mouseVngenes. WithAmeliaBlack, a graduate student, Sherie and I are trying to express the transfection products in E. coli and in mammaliancells. TsukasaMatsudais studyingthe repertoire by the use of the isomaltose oligosaccharides coupledto BSAor to KLHto obtain a set of hybridomasto T-dependentantigens with an ~(1 ~ 6) epitope sufficiently large to fill the antibodycombiningsite. Paula Bordenhas preparedantiidiotypic hybridomasand is studying the idiotypic determinants of the various anti-a(1 ~ 6)dextrans. Oneimportant finding is that the T-independenthybridomashave Via chains belonging to three major germline gene families of Brodeurand Riblet, J558, J606, and J36-60and that the light chains also belong to several germline gene families although their combiningsites are very similar. This wouldtend to indicate that the antibody-formingsystemis very well protected against germlinegeneloss. Since mostother epitopes prepared by coupling small moleculesto a protein create heterogeneous populationsof antibodies, the findingthat different germlinegenefamilies were used could not be interpreted unambiguously. Paula Bordenfound that IgG3antiidiotypes to an anti-a(1 ~ 6)dextran hybridomaprecipitated with antidextran. The reaction differs from the usual precipitin reactions in that it is highly dissociated, and its role in idiotype antiidiotype reactions requires further study. H.-T. Chenhas mappedthe combiningsites of various antiblood group A and B hybridomas and, with Arne Lundblad and R. M. Ratcliffe, has cloned and sequencedseveral. These blood group epitopes are more complicatedstructurally and should also provide an intimate picture of those antibody combiningsites. Wesorely need high resolution X-ray studies of one or more antibody combiningsites to carbohydrate epitopes to be able to correlate immunochemicalmappingdata with the interactions of the dextran or blood groupoligosaccharidesin their respective sites. Wealso intend to study anti-antiidiotypes in this systemin relation to the "internal image"antibodyto a carbohydrate.I intend to stay in the laboratoryfor as long as I feel I can do creative work. Mylife at Columbiahas alwaysbeen most satisfying. Overthe years I have been able to collaborate with manycolleagues at Columbiaand throughout the world. The University provided mewith the opportunity to do as I wishedscientifically and alwaysdefendedacademicfreedomin

Annual Reviews BEFORE AND AFTER

23

general and mine in particular. I am indebted to the Office of Naval Research for seventeen years of support; to the molecular biology section of the National Science Foundation for 36 years of continuous support; and since 1974 to the National Institutes of Health for providing the opportunity for me to pursue my interests there as well as at Columbia. I thank Mr. Darryl J. Guinyard for typing the manuscript.


Literature Cited I. Kabat, E. A. 1983. Getting started 50 years ago--Experiences,prospectives, andproblemsof the first 21 years. Ann. Rev. Immunol.l: 1-32 2. Boring, E. G. 1956. LewisMadisonTerman.FromBiographical Memoirsof the NationalAcademy of Sciences1959, Vol. 33: 414-61. NewYork: ColumbiaUniv. Press 3. Kabat, E. A. 1956. Blood GroupSubstances--Their Chemistry and Immunochemistry. NewYork: Academic 4. Kabat, E. A. 1982. Contributions of quantitative immunochemistry to knowledge of blood groupA, B, H, Le, I and i antigens. Am.J. Clin. Pathol.78: 28192 5. Sikder, S. K., Kabat,E. A., Roberts,D. D., Goldstein, I. J. 1986. Immunochemicalstudies on the combiningsite of the blood groupA-specific lima bean leetin. Carbohyd.Res. 151:247~i0 6. Chen,H.-T., Kabat, E. A. 1985. Immunochemical studies on blood groups. Thecombining site specificities of mouse monoclonalhybrldomaanti-A and antiB. J. Biol. Chem.260:13208-17 7. Kabat, E. A., Liao, J., Bretting, H., Franklin,E. C., Geltner, D., Frangione, B., Koshland,M.E., Shyong,J., Osserman, E. F. 1980. Humanmonoclonal maerogiobulins withspecificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6pyruvylated-D-galactose.J. Exp. Med. 152:979-95 8. Kabat, E. A., Niekerson, K. G., Liao, J., Grossbard, L., Osserman,E. F., Glickman,E., Chess,L., Robbins,J. B., Schneerson, R., Yang, Y. 1986. A humanmonoclonalmacroglobulin with specificity for ~z(1 ~ 8)-linked poly-Nacetyl neuraminic acid, the capsular polysaccharideof groupB meningococci and Escherichiacoli K1, whichcrossreacts with polynucleotides and with denatured DNA.J. Exp. Med.164: 64254 9. Porter, R. R. 1986. Antibodystructure

and the antibody workshop1958-1965. Symposiumon The Role and Significance of International Cooperationin the BiomedicalSciences,eds. G. Salvatore, H. K. Schachman,in Perspectives in Biology and Medicine29: Part 2, S161-S165 10. Wu,T. T., Kabat,E. A. 1970. Ananalysis of the sequences of the variable regions of Bence Jones proteins and myelomalight chains and their implications for antibody complementarity. J. Exp. Med. 132:211-50 11. Kabat, E. A., Wu,T. T. 1972. Attempts to locate complementarity-determining residuesin the variablepositionsof light and heavy chains. Ann. N.Y. Acad. Sci. 190:382-93 12. Kabat, E. A., Wu,T. T., Bilofsky, H. 1976. Variable regions of immunoglobulin chains. MedicalComputerSystems. Cambridge,MA:Bolt Beranek & Newman 13. Kabat, E. A., Wu,T. T.~ Bilofsky, H. 1979. Sequences of Immunoglobulin Chains. NIHPublication 80-2008. National Institutes of Health, Bethesda, Md. 14. Kabat,E. A., Wu,T. T., Bilofsky, H., Reid-Miller, M., Perry, H. 1983. Sequences of Proteins of Immunological Interest. NationalInstitutes of Health, Bethesda, Md. 15. Kabat, E. A., Wu,T. T., Reid-Miller, M., Perry, H. M., Gottesman, K. S. 1987. Sequencesof Proteins of Immunological Interest. U.S. Departmentof Health and HumanServices, Public Health Service, National Institutes of Health, No. 165-462,pp. 1-804. Washington, DC: USGPO 16. Kabat, E. A., Wu,T. T., Bilofsky, H. 1978. Variable region genes for the immunoglobulinframeworkare assembled from small segments of DNA--A hypothesis. Proc. Natl. Acad.Sci. USA 75:2429-33 17. Tonegawa,S., Maxam,A. M., Tizard, R., Bernard, O., Gilbert, W. 1978.

Annual Reviews


24 roa~,a" Sequenceof a mousegerm-linegene for version. Proc.Natl. Acad.Sci. USA80: a variable region of an immunoglobulin 4997-5001 light chain. Proc. Natl. Acad.Sci. USA 21. Jaenichen, H.-R., Pech, M., Linden75:1485-89 maier, W., Wildgruber,N., Zachau,H. 18. Bernard, O., Hozumi,N., Tonegawa,S. G. 1984. CompositehumanVkgenes and 1978. Sequences of mouse immunoa modelof their evolution.NucleicAcids globulin light chain genes before and Res. 12:5249-63 after somatic changes. Cell 15: 1133- 22. Reynaud, C.-A., Anquez,V., Grimal, 40 H., Weill, J.-C. 1987. Ahyperconversion 19. Early, P., Huang, H., Davis, M., mechanism generates the chicken light chain preimmunerepertoire. Cell 48: Calame, K., Hood, L. 1980. An immunoglobulinheavychain variable region 379-88 geneis generatedfromthree segmentsof 23. Kabat,E. A. 1986.A tradition of interDNA:V~, D, and J~. Cell 19:981-92 national cooperation in immunology. 20. Krawinkel, U., Zoebelein, G., BrugProc. Int. Symp. "The Role and Si#gemann,M., Radbruch, A., Rajewsky, nifieance of lnternationalCooperation in K. 1983. Recombinationbetween antithe Biomedical Sciences,"" ed. G.Salvabody heavy-chain V and variable V tore, H. K. Schachman.Perspect. Biol. region genes: Evidencefor gene conMed. 29:S159-S160



Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol. 1988. 6 : 25-48 Copyright © 1988 by Annual Reviews Inc. All rights reserved


TRANSGENIC MICE AND ONCOGENESIS Suzanne Cory and Jerry M. Adams TheWalterand Eliza Hall Institute of MedicalResearch,Post Office Royal MelbourneHospital, Victoria, 3050, Australia INTRODUCTION Thecreation of transgenic mice carrying specific cancer-promotinggenes has openedan exciting newera in oncology.Thebiological effects of an individual oncogeneon diverse cell types can nowbe assessed directly within the living animal. Whiletransgenic animals bear the introduced oncogenein every tissue, expressionof that genemayeither be widespread or directed to a particular cell lineage, dependinguponthe regulatory sequenceschosen, Thetransgene shouldbehaveidentically in every animal of an establishedlineageand, indeed, perhapsin everycell of a giventype. Thus, a well-characterized transgenic line becomesa permanentresource. Perhapsthe most significant opportunity providedby these transgenic animalsis the possibility of exploring the pre-neoplastic state. Onecan attempt to assess whetheran oncogenehas perturbeddifferentiation within particular lineages. The perturbations mayhelp to delineate early maturation stages and to clarify howdifferentiation is controlled. Thus,new insights mayemergeregarding the normalbiological functions of protooncogenes. The rules for oncogenecooperativity can also be evaluated within diverse cell types. For example,onecan isolate the relevant cells from a pre-neoplastic animal bearing an oncogeneand attempt to transformthemfully in vitro with retroviruses carrying other oncogenes.Alternatively, the second oncogenecould be introduced simply by breeding mice of two independenttransgenic lines. Whilethe studyof transgeniconcogenes is still in its infancy, majornew insights have already been gained. This chapter briefly summarizeshow transgenic animalsare produced,and then considers our current state of 25 0732~582/88/04104)025502.00

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CORY & ADAMS

knowledgeconcerningthe effects of the different classes of oncogenesin transgenic mice. PRODUCTION

OF TRANSGENIC

MICE


Gene Transfer to the Germline by Microinjection Introduction of newgenes into the mousegermlineis currently performed most efficiently by microinjection of DNA.Onlya brief outline of the technologyis presented here, since detailed reviews haveappearedelsewhere(1-5). Pronucleiof recently fertilized eggs are injected with a few hundredcopies of the cloned gene of interest, and the embryosare then transferred into the oviducts of pseudo-pregnantfemales and allowedto develop to term. In skilled hands, some25%of the mice that are born carry one or morecopies of the foreign gene. If integration occursprior to the first cell division,all cells in a transgenicpup,includingthe germcells, will carry the "transgene."In practice, a significant proportion(~ 30%) the transgenic pups are mosaic, indicating that integration often occurs subsequentto the first round of replication. Since the same degree of mosaicismis usually exhibited in somatic and germcells, lines reliably transmitting the transgene can usually be established even from mosaic foundersby breedingtheir transgenic offspring. The mechanismof integration is not known.Presumably on random breakage of a mousechromosome,the injected DNA is incorporated by the normalcellular repair enzymes(for a discussion, see 4). Typically, some1-50 copies of the injected gene are inserted in a tandem,head-totail array at a single chromosomalsite. On subsequent breeding, the transgenic array is inherited as a simple Mendeliantrait, usually in a remarkablystable manner.Morerarely, insertion occursat two positions, and these loci segregate amongthe progeny.Since the transgeneis inserted into only one homologueof the relevant chromosome, the founder mouse is heterozygousat that allele. Homozygous lines can be established by breedingonly if insertion has not interrupted a genenecessaryfor development(6). Expression of an inserted gene depends primarily on the regulatory regions that it bears. A majordeterminantis "strength" of the promoter/ enhancercombinationused, but the level and tissue distribution of expression mayalso be influencedby the site of chromosomal insertion and perhaps by unknownelements within the transgene, such as attachment sites for the nuclear matrix. Theexpressionof certain transgenes(but not others) is strongly inhibited by linked prokaryoticsequences(2). Manyof the problems associated with transgenic mouseproduction wouldbe alleviated by the developmentof vectors that can be maintained

Annual Reviews


TRANSGENIC MICE AND ONCOGENESIS

27

as autonomously replicating episomes.In principle, all injected eggs developing to term wouldbe transgenic, transmission to offspring wouldbe quantitative, problemsof insertional mutagenesisnonexistent, and most important, variation in expressionimposedby variable integration sites wouldbe absent. Attemptsto use bovine papillomavirus as an episomal vector have not been successful so far, however. In one study, many primarymice contained the vector in episomalform but weremosaic, and BPVDNA was apparently diluted further on subsequent breeding (7). two other studies, only integrated BPVDNA wasdetected (8). A promising advancehas been the serendipitous creation of autonomouslyreplicating elements containing defined segments of mousechromosomalDNAby injection of fertilized mouseeggs with a polyoma-based vector (9) together with trace levels of another plasmid(F. Cuzin, personal communication). Significantly, the incorporated mousesequences are homologousto conserved yeast centromeric sequences. Whetherthe propensity of the episomesto recombinationcan be overcomeremainsto be established. Retroviral

Vectors for Gene Delivery to the Germline

Considerableinterest exists in usingrecombinantretroviruses to introduce additional genes (including oncogenes)into the mousegermline. Amajor advantageof retroviral delivery is that it is technically less demanding, oncea suitable virus-producingline is available. Moreover,only a single gene is inserted per site, instead of a concatamer,whichmayadversely affect neighboringchromatinand transcriptional units (10). Severe problemsremain to be overcomewith the retroviral approach, however.To date, successful infection protocols use eight-cell stage embryos(11, 12)i hence, all founderanimalsare mosaic.Moreimportantly, however,expressionof the integrated provirus is usually suppressed,and this suppression is maintainedduring subsequentdevelopment(13). The mechanism of suppressionis not clear but mayinvolve methylation (14), inability of the long terminal repeat (LTR)to promotetranscription (1517), and/or transacting negativeregulatoryfactors (18). Twoapproaches have been taken to overcome suppression. One involved modifyingthe retroviral LTRby replacing the enhancerwith one knownto function within embryonalcarcinoma(EC) cell lines (12, 19). Theother involved redesign of the retrovirus to allow expressionof the geneof interest froman internal, cellular promoter(20-22). Despitepreliminarysuccesses,it is not yet clear howgenerallyusefulsuchvectorswill be. A potentially exciting use of recombinantretroviruses is to infect embryo-derived (ES) pluripotential cell lines capable of forminggermline chimeras(23). Since the viruses can be engineeredto express a selectable

Annual Reviews 28

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markergeneas well as the geneof interest, infected EScells can be selected in vitro prior to either injection into blastocystsor aggregationwith eightcell embryos.Preliminaryexperimentsindicate that provirusesselected in .vitro for expressionin ECand EScells are stably expressedthroughout development and within~all tissues (22, 23). Amajor probleminherent this approach,however,is the tendencyof the ESlines to becomeaneuploid duringthe in vitro culture period and henceto lose their ability to form chimeras. Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Choice of Regulatory Sequences In attempting to target expression, a numberof different promoter/ enhancersequencescan be attached to the gene of interest (see 4 for detailed discussion). Theexpressionpattern of the engineeredhybridgene often conformsto that of the gene from whichthe regulatory region was derived. Novel patterns are sometimes observed, however, perhaps becauseof the unforeseen creation of a "recombinantenhancer," and/or the influence of flanking DNA. Thebest studied regulator for generalized expression is that of the metallothionein (MT)gene. The endogenous MTgene is expressed virtually all cells and is responsiveto several regulators, includingglucocorticoids and metals. Whilemost MTfusion genes have been expressed wellin liver, intestine, kidney,heart, pancreas,andtestes, unusualpatterns of expressionhavealso beenobserved(4). Threetissue-specific regulators havebeenparticularly successful: the immunoglobulin enhancersfor expressionin B lymphoidcells; the 5’ region of the rat elastase-I gene, for expressionin the exocrinepancreas;and the 5’ region of the rat insulin II gene, for expressionin the//cells of the endocrinepancreas. Theidentification of "strong" tissue-specific regulators for other cell lineages will greatly increase the versatility of the transgenic approach. ONCOGENES AND THE PROGRESSION MALIGNANCY

TO

Cancerhas long been perceived to be a multistage process. In molecular terms, this progressionis currently believedto involvethe stepwiseacquisition of activated oncogenes(24). Theexperimentalevidencesupporting this view derives froman analysis of the transforminggenesin DNA tumor viruses (25, 26) and fromin vitro transfection studies with primarycells, principally fibroblasts (27-29). Transformationwas foundto require the concertedaction of two genes. Significantly, effective collaboration was usually achievedonly if one of the pair of oncogenescamefrom the class

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expressed in the nucleus and the other from the class expressed in the cytoplasm(24). Thecytoplasmicclass includes proteins related to growth factor receptors (erbB, fms), as well as proteins (e.g. ras) thought to regulate levels of critical second messengerssignalling interaction of growthfactors with their receptors. Thefunction of the nuclear class (e.g. myc, N-myc,fos) is unknownbut seemslikely to involve regulation of transcription. In markedcontrast to primaryfibroblasts, established but nontumorigenic lines such as NIH3T3can be readily transformed by a cytoplasmic oncogenewithout the mediation of a nuclear oncogene.This has led to the concept that the nuclear oncogeneprovides an "immortalization" function, while the cytoplasmiconcogenecompletesthe transformationprocess. Theconceptis an oversimplification, however,because transfection with a nuclear oncogenedoes not necessarily permit primary cells to growindefinitelyin vitro. Studieswith transgenicmicepermitdetailed investigationof the validity of the complementing oncogenemodelfor different cell lineages. Will a particular oncogeneprecipitate transformation as soon as expression is turned on, or does the disease progress in multiple stages? Are some transgenic strains essentially tumor-freeuntil crossed with a strain harboringa different oncogene?Cancells frompretumorigenictransgenic mice be readily grownas immortallines in vitro, or are they only able to do so if supplied with another activated oncogene?Are certain mousestrains moreprone than others to developingtumors from the action of a particular oncogene,and if so, do they harbor a susceptible proto-oncogene?

VIRAL ONCOGENES Introduction of a viral oncogeneinto the mousegermline opens up the possibility of testing its potential for transforming cell typesnot accessible to infection by the virus. Moreover,oncogenesisolated from viruses of other speciescan readily be investigated.

SV40 T Antigen Thefirst geneshownto exhibit oncogenicpotential in transgenic micewas the T antigen of the monkeypapovavirus SV40(30). The SV40virus transforms rodent and humancells in culture and induces tumors in hamsters but only rarely in mice. Newbornhamsters usually develop fibrosarcomasor, after intracerebral inoculation, choroid plexus papillomas, but adult animals can develop a wide range of tumors (30). The completetransformingpotential of SV40has been shownto be due to the large T antigen encodedby the early region. Since T antigen is expressed

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at the plasmamembraneas well as within the nucleus, it mayplay two different roles in the transformationprocess. Transgenicmice that carry the SV40early region linked to its natural regulator invariably developchoroid plexus tumorsat 3-5 monthsof age (30, 31). Thetumorsarise fromepithelial cells lining the ventricles of the brain. Tumordevelopmentdependson the presence of the SV40enhancer and the large T antigen codingsequencew~thinthe transgene, but not the small t antigen (31). Since most foundermicedevelopedthe samedisease, the chromosomallocation of the SV40antigen gene did not in general influencethe onset of disease (see below). T antigen wasreadily detected in tumorsbut not in unaffected tissues or in susceptible tissue prior to overt pathology.Withinthe tumors, the transgenic DNAwas usually amplified or rearranged. Nontumorigenic changes in SV40T antigen mice included thymic hypertrophyand kidney pathology;these tissues exhibited lowerlevels ofTantigen expressionthan did the choroid plexus tumors. It is not clear whetheramplified expression is sufficient to provoke tumorsor merelya prerequisite. Noris it knownwhychoroid plexustissue is permissiveto T antigen activation. TheSV40enhanceris thought to be inactivated during early development,and its reactivation in vivo maybe a rare event, dependentuponcell type. Reactivation mayreadily occur in vitro, however,becausemost apparently normaltissues from the SV40T antigen micereadily producedimmortalizedcell lines whenput into culture (30). Oneline of mice bearing an SV40T antigen gene remainedessentially free of tumors(32, 33). Significantly the level of expressionof T antigen in these micewas muchlowerthan in the other lines, both in vivo and in vitro (33), presumablydue to the chromosomal location of the transgene. Thecrucial check to tumor formation in this line may,however,be the immune response (32). Withthe low-incidencestrain, in whichonly 1 57 micedevelopeda choroidplexustumorwithin the first year of life, most mice mounta strong cytolytic T cell response after immunizationwith SV40virus. The developmentof cellular immunitypresumablyoccurs in response to chronic stimulation by the low levels of endogenousSV40T antigen expressedat the surface of cells. In markedcontrast, micewithin a strain with the normalhigh susceptibility to tumors failed to mount either a humoralor a cytotoxic response whenimmunized with virus, even though their response to vaccinia virus was normal. Suchstrains thus appearto be tolerant of T antigen, presumablybecauseantigen is expressed early in development.The high incidence of choroid plexus tumors thus appears to dependon the inability of mosttransgenic SV40T antigen mice to carry out immune surveillance.

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TRANSGENIC ~tCE A~qDON¢OGENESIS31


SV40 T Antigen Driven by Tissue-Specific Regulators TheSV40T antigen appears to be a potent tumorigenicagent for manycell types. Attachmentof a different regulatory sequenceavoids the negative regulation imposedby the S¥40 enhancer on transmission through the germlineand enablesexpressionto be directed to specific cell types. ~T/a" ANTIGEN Underthe control of the metallothionein enhancer, 13 of 16 primary transgenic mice exhibited a demyelinatingperipheral neuropathy associated with excessive numbersof Schwanncells (34). Myelination in the central nervoussystem wasapparently normal.Manyof the transgenic mice developedhepatocellular carcinomas(11 of 16) and islet cell adenomas (8 of 16), but only one exhibiteda choroidplexuspapilloma. Thosemice having a mild form of neuropathy and living long enoughto producelitters provedto mosaic.Their transgenic offspring all exhibited the severe peripheral neuropathy, all had hepatocellular carcinomasand 3 of 6 hadislet cell adenomas. ~t-CRYSTALLIN/T ANTIGEN Underthe

control of the murine ~-crystallin promoter/enhancer, transgenic SV40T antigen induced highly invasive lens tumors(35), eventhoughthis tissue has never beenobservedto become malignant under "natural" circumstances. T antigen expression and dysplasia wereobservedas early as midgestation, but it generally took 2 to 3 monthsfor invasive growthto occur outside the lens capsule. Whether the onset of malignancyrequired additional genetic changeis not clear. rNSULrN/T ANTIGEN Underthe control of the insulin regulatory region, S¥40T antigen transgene expression occurred exclusively in the fl pancreatic cells and culminatedin fl-cell tumors (36). On a normaldiet, transgenic mice frequently died suddenlyfrom hypoglycemia precipitated by hyperplasia of pancreatic fl cells. Thosespared by a high sugar diet succumbedlater (10-20 weeks)to pancreatic tumors. Presumablya second genetic alteration had occurred in such tumors, becauseno morethan 4 or 5 of the ~ 100 hyperplastic islets in a pancreas becameneoplastic. Lines developing "autoantibodies" against T antigen developed tumors considerablylater than did a line with no "autoantibodies." Again, this suggestsa role for immune surveillance (37). ELASTASE/T ANTIGEN Underthe

control of the elastase promoter region, SV40T antigen producedtumors of the exocrine pancreas (38). A marked hyperplasia wasevident in the pancreas within 17 days of gestation, but newbornmice retained pancreatic function and the DNAcontent of the cells wasdiploid. Onlyone monthlater, however,most cells had become tetraploid. The pancreas was soon riddled with nodules containing

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aneuploid cells, and the chromosome numbervaried betweennodules. The cells within these apparently independentclones were tumorigenicwhen transplanted. Theseresults suggesta role for SV40T antigen in the induction of karyotypicinstability via tetraploidization. Tumoronset maybe provoked by the subsequent loss of specific chr.omosome(s),perhaps becausecritical anti-oncogeneproducts are then either reducedin concentration or absent. T Antigen of Human Papovaviruses JCV and BKV The ubiquitous humanpapovaviruses JCV and BKVare structurally related to SV40.Themost divergent region of the three genomesis that encompassingthe region of replication and the enhancersequences, but homologybetween the T antigen genes is considerable (70-80%). The pathologyinducedby these viruses has beenreviewedby Smallet al (39). BKVreplicates preferentially in the kidney, producinga subclinical infection. Wheninjected into newbornhamsters, this virus causes brain tumors,insulinomas,and osteosarcomas. It is significant that in transgenic mice, the early region of BKVinducedrenal and liver tumors after 8-10 months(39). In humans,JCVhas beenlinked to the fatal demyelinatingdisease found in certain immunodepressed patients, someof whomalso develop glial tumors.Whilethe virus has not beenprovento cause these tumors, it does induce tumors in tissues of neural origin wheninjected into newborn hamsters. Significantly, transgenic mice mosaicfor the JCVearly region were found to develop metastasizing adrenal neuroblastomas(39). Those that were not mosaicdid not live long enoughto developtumors, because they developeda severe shaking disorder causedby dysmyelinationin the central nervoussystem(40). Thus, for both viruses, the pathologyinducedin transgenic mice by T antigen closely paralleled that thought to occur in immunosuppressed humansas a result of viral infection. Transgenicmice maythus provide excellent modelsfor humanvirus-induced disease. Bovine Papillomavirus Bovinepapillomavirus(BPV)(8) causes benignfibropapillomasin cattle. Ahigh proportionof infected animalsgo on to developtumorsif they graze on brackenfern, whichmayprovide a synergistic factor for oncogenesis. Certain other species, including C3HeB mice, developfibroblastic tumors wheninfected with the virus. Thegene(s) responsible for the transforming potential of the virus have not been fully characterized but are known from in vitro studies to reside within a fragmentcomprising69%of the genome.

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Aline of transgenic micehas beenestablished from an animalinjected with a plasmidcarrying a partial tandemduplication of the completeBPV1 genome(8). Themice display a dramaticpropensity to developmultiple skin tumors, starting at about 8 monthsof age. Thetumors, whicharise frequently in areas prone to abrasion and wounding,are characterized by disorganizeddermallayers. In addition, the skin is abnormallythickened over wide areas. Bycontrast, 15 lines of transgenic mice harboring the BPV-69%-transforming region failed to develop tumors whenthe mice weremonitoredfor 10 months(Palmiter& Brinster, cited in 8), so the 31% region presumablycontributes an essential function for transformationin vivo. The long latency prior to transformation is interpreted to mean that additional genetic change(s)are required to precipitate oncogenesis. Extrachromosomal replication is probably a crucial factor because the tumors harbor BPVDNA in episomalform, as well as an amplified number of integrated copies. Theintegrated transgenewasunchanged in unaffected tissue. Studies like these with humanpapillomavirusDNA mightprovide valuable animalmodelsfor cervical cancer.

CYTOPLASMIC

CELLULAR

ONCOGENES

Ras The family of ras proto-oncogenesencodes membrane-bound cytoplasmic proteins believedto transducethe proliferative signals deliveredto the cell membrane by growthfactors and mitogens(for a recent review, see 41). Mutationof a c-ras gene at certain critical positions converts it into a potent transforminggene for NIH3T3 fibroblasts. Similarly mutatedgenes have been identified within manyhumanand animal tumors. Recent transgertic mousestudies have dramatically underlined the transforming potential of mutatedras genes. ~LASrASE/c-Ha-RAS Transgenicmice were producedcarrying either a normal or a mutated c-Ha-ras gene driven by the elastase promoter (42). Expressionof the transgenesprovedto be specific to acinar cells, but the level wasonly a fewfold higher than that of the endogenous Ha-rasgenes. Withthe unmutatedHa-rastransgene, no tumorsdeveloped.Nevertheless, anaplastic changeswereobservedin the pancreas of adult animals after about 11 months. Withthe mutatedgeneencodingvaline at position 12 instead of glycine, the results weredramatic. Theonly transgenic pupsto survive (5 of 19) were probably mosaic--the other 14 were dead or moribundat birth. The surviving mice all eventually developedpancreatic tumors. Whenanother

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20 transgenic micewereexaminedas fetuses, 18 had an abnormalpancreas. Dysplasiawasapparentas early as 14 days of gestation, the time expected for onset of elagtase-drivenexpression.Byday20, large-scale disruption of pancreatic architecture had ensued:Islets wereno longer recognizable, and insulin-containing cells werescattered throughoutthe cellular mass. Differentiation of the acinar cells appearedto be blocked.Whilethe pathology resembledthat of a carcinoma,the acinar cells maynot have been malignant:There wasno evidenceof aneuploidy,invasiveness, or metastasis, and the cells did not initiate tumors in syngeneic or nude mice. Thus, the disease maymore properly be regarded as a lethal polyclonal proliferation than a malignancy. If the mosaicelastase/ras micecontainedevena fewtransgenic cells in the pancreas, howdid they survive for so long without developing a pancreatic carcinoma? One obvious possibility is that the level of expression mayhave been lower. Anotherpossibility is that ras has a muchmoreprofoundeffect in embryonicthan adult cells. A third, very intriguing, suggestion (42) is that the transformedphenotypewas suppressedin the presenceof an excessof normalcells, as postulated by Land et al (29) for fibroblast transformation. WAP/c-Ha-RAS TOinvestigate the consequencesof expressionof a ras oncogene in mammary epithelial cells in response to lactogenic hormones, transgenic lines were constructed bearing a mutatedHa-rasgene driven by the promoterregion of the gene encodingmurinewheyacidic protein (Wap)(43). Twolines exhibited no expression, but two others derived from female founders showedtissue-specific expression. A low level of Wap-rasRNAwas detected in the mammary glands (and brain) of lactating females, and no expression could be detected after lactation terminated.Maleswithin these lineages did not express the transgene. After 11 months, one lactating founder female developedmammary tumors that expressed elevated levels of Wap-rasRNA. In one unusual line, expression was obtained in the salivary gland of males, presumablydue to the unusual location of the transgene (the chromosome).Five animals developed adenocarcinomasof that gland, but only after 9 months; this suggests the need for additional genetic change. Again, Wap-RasRNAlevels were higher in the tumors than in nonmalignantsalivary tissue from the sameanimals. The transgene was neither amplifiednor rearrangedin the tumors,and its transcription rate was unchanged.Increased expression maythus have beendue to an alteration in mRNA stability. MMTV/v-Ha-RAS Thirteen strains of mice wereproducedcarrying the v-Ha-

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ras oncogene under the control of the mousemammarytumor virus (MMTV) promoter (78). As expected, the transgene was predominantly expressedin the mammary tissue and in the salivary glands, with variable expression in spleen, thymus, lung, seminal vesicle and the Harderian gland. Expression in the Harderian gland provokedmassive though nonmalignant hyperplasia, resulting in severe secondary bilateral exophthalmia. Most (10) strains developed adenocarcinomasof the mammary and salivary glands and, occasionally, lymphomas. The stochastic nature of tumor onset and the apparent monoclonality of the tumors strongly suggestedthat additional genetic changeshad occurred prior to the onset of malignancy. In summary,a transgenic mutant ras gene can provokeabnormallevels of proliferation in several different cell types. Theseverity of disease apparently dependson the level of expressionand cell type. Theonset of true neoplasia usually (perhapsalways)requires additional change. Transgenic Hernopoietic

Growth Factor Gene

Theproliferation, differentiation, and functionalactivation of the myeloid hemopoieticcell lineagesis controlled by a groupof glycoproteinsreferred to as colonystimulating factors (CSFs). The granulocyte/macrophage CSF (GM-CSF) stimulates proliferation and differentiation of granulocytes, macrophages,and eosinophils (44). Autocrinestimulation of factor-dependent but nontumorigenicimmortalizedcell lines renders themmalignant (45-47).Thus,it is of the greatest interest to ask whethertransgenicmice programmedfor constitutive CSFproduction are predisposed to tumor development. Twotransgenic mouselines were generated that contained the murine GM-CSF gene expressed from a retroviral LTR(48). The transgenic mice had elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. Striking pathogeniceffects ensued. Theeyes of newborntransgenic pups were opaque, due to an abnormalaccumulationof activated macrophages;the associated retinal damageled to blindness. Theperitoneal and pleural cavities accumulatedactivated macrophages in large numbers.The mice died at 2-4 months, and a high percentage exhibited progressive muscle wasting. Histological examination revealed macrophage-containing lesions in their striated muscletissue. Expressionof the transgene wasdetected in the peritoneal cell populationand in striated muscleand eye tissue harboring macrophagelesions. No tumors have yet been detected, but the severity of disease maynot allow sufficient time for the accumulationof additional genetic change(s).

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NUCLEAR

& ADAMS

CELLULAR

ONCOGENES

Fos The c-los proto-oncogene encodes a nuclear polypeptide implicated in the control of cell proliferation. One of the earliest responses of manycell types to growth factor or mitogenstimulation is a transient burst of c-fos synthesis. The gene mayhave other roles, however, since high constitutive levels have been found in extra-embryonic tissues and in macrophages(for a review, see 49). The gene was first identified as a viral oncogene. Two independent isolates of murine osteosarcoma viruses carry sequences homologous to c-fos: FBRmurine sarcoma virus, in which the transforming protein differs from c-fos mainly by a frameshift mutation at the C-terminus, and the more aggressive FBJ-murine sarcoma virus, which encodes a greatly altered version of the protein. Both viruses induce chondro-osseoussarcomas when injected into newborn mice. A mutation within the evolutionarily conserved central portion is necessary for immortalization, while COOHterminal changesare critical to their transforming properties (50). Transgenic mice have been produced bearing a murine c-fos gene controlled by the humanmetallothionein promoter/enhancer (51). Twoconstructs were used. Onehad the natural 3’ untranslated region of the c-fos gene, which contains a mRNA-destabilizing sequence. This region was replaced in the second construct by the 3’ LTRfrom the FBJ virus. In eight transgenic strains involving the natural 3’ end, very little expression could be detected, except after treatment with cycloheximide, which is thought to inhibit degradation of c-fos mRNA. In contrast, two of the five transgenic lines carrying the 3’ LTR-modifiedgene exhibited high levels of expression in pancreas, kidney, brain, heart, muscle, and lower levels in lung and salivary gland. Liver expression had been expected but was absent. While none of the animals developed tumors within the first 8 months, a marked abnormality in bone formation became apparent soon after birth. Swollen tibiae were evident in some2-3 weekold mice. Bonelesions were subsequently found in other mice by X-ray analysis. The swellings did not increase after 4 weeks of age. Histological analysis revealed a disturbance in the normal process involved in bone remodelling, with much more bone formation than bone resorption. The level of c-fos expression probably determines the severity of the disease, because a c-fos gene linked b promoter induced multiple swellings in all leg bones, not just to the H-2K the tibiae. These results point to a significant role for the normalfos gene in regulating bone morphogenesis. In the MT/c-fos mice, none of the other tissues expressing the transgene

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exhibited detectable abnormalities. However,H-2/c-fos miceexhibit perturbations of lymphopoiesis (E. Wagner,personal communication), quantitative effects maybe involved.ThethymicT cells exhibiteda marked depressionin the proportionof cells bearingboth L3T4and Lyt-2 markers. Dueto a defect in T cell maturation, these mice are immunologically incompetent. Thevery early onset of the bonelesions inducedby the c-fos transgenes, togetherwith their intriguing similarity to the frank osteosarcomas induced by v-fos bearingretroviruses, suggeststhat the lesions can be regardedas a hyperplastic state from which tumors could arise should additional genetic changetake place. Indeed,tumorshavearisen in someof the older c-fos mice. The ready induction of tumors by the retroviruses probably reflects the structural differences betweenv-fos and c-fos and/or a higher expressionlevel. Myc The c-mye proto-oncogeneis a nuclear phosphoproteinwith DNA binding activity. Its function remainsunclear but almostcertainly involves regulation of cell proliferation (52). Arapid increasein my¢expressionoccurs whenresting (Go)cells of several lineages are stimulatedto enter the cell cycle by mitogensor growthfactors. Recentevidencesuggests that c-myc protein plays a role in DNA synthesis (53). The fundamentalmechanism releasing the oncogenicpotential of c-myeis believed to be deregulation of its expression (54). Nochangein the sequenceof the polypeptide required. Totest the hypothesisthat deregulatedc-mycexpressionpredisposesto neoplasia, several strains of transgenic mice have been developedthat carry c-myelinked to different regulators (55, 56). Micebearinga normal mycgenetogether with several kilobases of 5’ and 3’ flanking DNA failed to developtumors(56). In contrast, those in whichmycexpressionhad been subjugated to a strong heterologouscontrol region exhibited a dramatic predisposition to tumors (55-57). Diverse tumor types were provoked, dependingon whichtissues expressedthe deregulatedgene. Beforereviewing these malignanciesin detail, however,it is worthnoting that a c-rnyc oncogenemaynot provoketransformation of all cell types. Thus, tumors did not developin lung, pancreas,and salivary gland expressingrelatively high levels of a deregulatedmy¢gene(42, 57). MMa’V/r,~YC Several strains of mice were developedthat bear a humancmyegene linked to the LTRof the mousemammary tumor virus (MMTV), whichprovides a hormonallyinducible promoter (55). The tissue distribution of expression varied betweenthe lines, presumablydue to the

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influence of the chromosomal insertion site. In mostlines, expressionwas restricted to the breast, salivary gland, and occasionallythe testis. Two multiparous founder animals developed malignant adenocarcinomasof the breast. The transgene was not amplified in the tumors but was expressed at higher levels than the endogenousc-mycgenes. In the strain studied in detail, all femaletransgenic offspring developedbreast tumors during their second or third pregnancy. Thus, mycexpression stimulated lactogenically appears to predisposethe mice to the onset of mammary adenocarcinomas. Not all the mammary glands in an individual becamemalignant;this strongly suggests that additional genetic changeis required(55). One exceptional MTV-myc lineage proved to express the transgene in diverse tissues, includingtestis, pancreas,lung, spleen, liver, and(in low amounts)kidney. This strain allowedan assessmentof the range of tissues susceptible to transformation involving c-mye (57). Some50%of the animals developedtumors, with a meanlatency of 14 months. Thevariety wasstriking: testicular tumors, B and T lymphomas, and mastcell tumors, as well as breast adenocarcinomas.Mice carrying a murine c-myc gene coupledto the SV40promoter/enhanceralso developeda range of tumors: a lymphosarcoma,a renal carcinoma, and a fibrosarcoma (56). Clearly deregulatedexpressionpredisposesdiverse cell types to becomemalignant. IG ENHANCER/MYCThe strong

association between c-myc deregulation and lymphoidneoplasia (54) provided a compellingcase for using transgenic mice to test the consequencesof constitutive mycexpressionwithin the lymphoidlineage. Twotransgenes were utilized, one involving the immunoglobulinheavy chain enhancer (E/~) and the other the kappa enhancer (Ex) (56). E#-myc geneused had been isola ted from an unusual plasmacytomain which 2.3 kb of the E# enhancer region had inserted just upstreamfrom the c-myc promoters (58). The Ex-mycgene wasa synthetic construct in whichmycexpression wascontrolled by the x light chain enhancerligated to the SV40promoter.TheE#-myctransgene proved remarkably potent: At least 13 of 15 primary mice developed lymphomas within a few monthsof birth. Thus, the heavy chain enhancer apparently dominatesexpression in manychromosomal contexts. The Exmyctransgene also induced lymphomas, albeit with lower frequency(6 of 17 primarymice) and longer latency. The lymphoidspecificity wasabsolute; no other tumor types were found either in the original study or subsequently. The high predisposition to tumor developmentin E#-mycmice has proven highly heritable. The mice typically succumbto a disseminated lymphoma affecting most lymphnodes, the spleen, and often the thymus;

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the disease is accompaniedby leukemia. All tumors so far analyzed (> 50) have proven to be of B lymphoid origin, even those primarily involving the thymus. All exhibit B lineage surface markers and immunoglobulin gene rearrangements; none carry Thy-1; and only one or two have undergone rearrangement of the a or fl T cell receptor loci. The absence of T cell tumors was initially surprising, since a complete immunoglobulin# transgene is expressed in T as well as B cells (59). But no expression of the Et~-mycgene has been detected in T cells (see below). A minority of the E#-rnyc tumors are comprised of mature B cells expressing sIg, while the rest can be assigned to different pre-B stages, exhibiting either D-J or V-D-J heavy chain rearrangement. None so far studied represents the very early maturation stage that lacks any heavy chain rearrangement. Most are relatively easy to establish as permanent lines in culture, and manyof the pre-B cell lines maturein vitro to sIg + B cell lines (A. Harris, W. Langdon, S. Cory, unpublished). The rearrangement patterns in certain mice suggested that maturation also sometimes occurs in vivo. Thus, myc-induced tumorigenesis need not prevent some subsequent differentiation. Compared to B lineage tumors generated by retroviruses bearing a variety of other oncogenes,including v-abl, a significant difference in the phenotype of El~-myc tumors is the absence of the 6C3 surface marker (W. Langdon, unpublished). This antigen (60) is also expressed at levels on stromal cells that support pre-Bcell proliferation and is speculated to be a growth factor or receptor (61). Tumors produced by myc retrovirus also lack this marker (62). Thus, it seems likely that myc-induced tumorigenesis involves a pathway different from that imposed by other oncogenes. Another distinctive feature of most E#-myctumors is the lack of the Lyl marker (W. Langdon, unpublished). This antigen is present on the surface of most conventional B lymphoid tumors and also on nontumorigenic B cell lines derived from normal mice (63). Most conventional lines therefore apparently derive from the minor, seeminglydistinct, Ly 1 + subset of B cells (64), whereas El~-mye-mediatedtransformation may garner a morerepresentative sample of all B lineage cells. To date, no plasmacytomas have arisen spontaneously in E#-mye mice, despite the fact that conventional plasmacytomas all carry chromosome translocations produced by recombination of the c-myc locus with an Ig heavy chain locus (54). This paradoxical observation maysimply reflect the different genetic background; the E#-myc gene is on a C57BL/6× SJL/J background, whereas plasmacytomas usually arise in BALB/eor NZB mice. Nevertheless, it maybe possible to induce plasmacytomasin E#-rnyc mice by providing the appropriate milieu. Experiments are currently in

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progress (A. Harris, unpublished results) to determine whether pristane injection, which induces granulomas, will promote plasmacytoma formation in El~-myc mice as it does in BALB/cmice. E/z-myc induced tumorigenesis involves additional genetic change Despite the almost invariant onset of tumors in E#-mycmice, several observations argue that deregulated myc expression is almost certainly not the only critical factor. First, the lymphoidtissues of young E#-mycmice lacking any overt sign of disease express the transgene at the samelevel as do E#myc tumor cells (65). Nevertheless, in contrast to the tumor cells, they do not provoke malignancy on transplantation (66). Second, while the transgene disturbs B cell development in all E#-myc mice, even before birth (66), lymphomasarise sporadically--typically between one and six months of age (56, 67). Third, the pattern of immunoglobulin gene rearrangement indicates that nearly all the lymphomasare monoclonal (56). Finally, and most definitively, DNAfrom several of the El~-myc tumors transforms NIH3T3fibroblasts (S. Cory, W. Alexander, J. Adams, unpublished results). Since the EIz-myc gene does not transform NIH3T3 cells, these tumors must harbor an additional activated oncogene, presumably one that can collaborate with c-myc. The nature of the transforming gene(s) is presently under study. While all E#-myc mice eventually develop tumors, a minority succumb faster than most, and another group lag conspicuously. It seems possible that the variation in rate of onset reflects the variable genetic background of the mice. Because transgenic mice are produced more efficiently from hybrid eggs (2), each founder was an F2 derived from the C57BL/6and SJL/J strains, and the major colony has been maintained as an F2 pool. Preliminary backcross experiments (A. Harris, unpublished results) have revealed a major influence of strain: E#-myc mice of a largely C57BL/6 backgroundall still develop tumors, but the rate of onset is significantly lower. It appears relevant that old SJL mice can develop a lymphoid malignancy,perhaps as a result of a single recessive trait (68). The pre-neoplastic state in E#-mycmice To investigate howconstitutive c-myc expression establishes a high predisposition to malignancy, we analyzed B cell lymphoid differentiation in prelymphomagenic EIz-myc mice (66). The most conspicuous change was a remarkable polyclonal expansion of pre-B cells, somewhatat the expense of mature B cells. By 18 days of gestation, the proportion of pre-B cells in the fetal liver was already twice that in normal littermates. The number of pre-B cells increased dramatically after birth, reaching a plateau of about 65%of the bone marrowcells at 3 to 4 weeks of age. Pre-B cells were also found in the spleen; this is not the case in normal mice. The cells mayhave been

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generatedin situ as a result of increased splenic hemopoiesis.It is significant, however, that pre-B cells were not detected in the lymph nodes or thymus, even though these are the most commonsites for tumors. Thus, acquisition of the ability to invade the lymph nodes and thymus may be a critical step in progression towards malignancy. No significant changes were detected within the thymus of prelymphomagenicEl~-myc mice: The proportions of the four major T cell populations were unaltered. Because of reduced numbers of sIg + B cells, the proportion of T cells in the lymph node was greater than normal. The size of these cells was normal, however. Both the pre-B and B cells in the El~-mycmice were notably larger than most equivalent cells in normal mice. Late pre-B cells are thought to enter a quiescent nonproliferative phase as they mature to B cells. In El~-myc mice, however,no small pre-B or B cells were detectable. In addition, more than twice as manyof the pre-B and B cells were actively cycling in E#myc than in conventional mice. Since the analysis did not distinguish betweennoncycling cells and those traversing G1, it is conceivable that as a result of constitutive c-myc expression, none of the cells ceased cycling prior to their death. A large proportion of the cycling cells must in fact die to account for the steady state level found in adult El~-mye mice. The factors regulating the plateau level are not known,but one obvious possibility is that the supply of the requisite growthfactor(s) is limited. Overall, the total numberof pre-B cells in El~-myc mice was elevated over 4-fold, while the numberof mature sIg + B cells was depressed about 30% (66). Moreover, fewer splenic B cells had matured to surface IgD expression. These observations raised intriguing questions regarding the immunologicalcompetenceof B cells continually subjected to constitutive myc expression. Et~-mycB cell function was therefore investigated both in vitro and in vivo (68). While in vitro stimulation with either mitogens antigens led to proliferation and antibody production, the frequency of responsive B cells in E~-myccultures was only 30%that of B cells from control cultures. In vivo responses were sometimesdelayed but eventually reached normal levels, and switching from IgM to IgG production occurred. Thus, deregulated myc expression appears to retard but not prevent B cell differentiation; consequently, immunologicalfunction is not grossly impaired. To account both for the increased numberof cells early in the B lineage in Et~-mycmice and for the maintenance of at least a high proportion of the cells in cycle, it was proposed that c-myc normally plays an important role in control of differentiation as well as mitogenesis (66). The level c-myc expression was envisaged to set the balance between self-renewal and maturation within a cell lineage, the maturation requiring decreased

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levels. Constitutive expressionwouldthus favor self-renewal. Additional evidencesupportingthis modelis that deregulated mycexpressionwithin mouseerythroleukemiacells blocks the terminal differentiation induced by dimethylsulfoxide (69, 70). The model maynot hold for all lineages, however, because differentiated keratinocytes maintain high myc expression(71). All changesnoted in the E#-mycmice reflect alterations within the B lymphoidcompartment,consistent with evidence that the transgene is apparentlynot expressedin other lineages (65). It is significant that the level of transgene expression was found to be comparableto that in activated normallymphocytes.Thus, constitutive expression at physiological levels of a normalcellular genecan elicit all the profoundbiological effects cited above. This conclusion dramatically underlines the importance of understanding the molecular mechanismthat normally regulates c-myc expression. Astriking feature of all E#-mycB lymphoidcells, whetheror not they have becometumorigenic,is that the endogenousmycalleles are transcriptionally silent (56, 65), as is the untranslocatedmycallele in plasmacytomasand Burkitt lymphomas (72, 73). These results strongly support a negative feedbackmodelfor control of mycexpression(74, 75). A myc polypeptide concentration above a threshold level (which may vary betweenlineages or differentiation stages) is hypothesizedto repress further myctranscription, either directly or indirectly. TheE#-myctransgene, like a translocated mycallele, presumablyis recalcitrant to this repression. In viewof the immortalizingrole ascribed to the c-rnyc oncogene,it was surprising to find that pre-neoplasticE#-mycB lineage cells wereno easier to growin culture than their normalcounterparts. Whencultured under conventional conditions, with no source of growth factors other than serum, E#-myepre-B marrowcells in fact died more rapidly than their normal counterparts (W. Langdon, unpublished results). Moreover, splenic B cells from E#-mycmice did not display unlimited proliferative capacity, even in the presence of mitogens and rich sources of growth factors (68). Thus, the description of my¢as an immortalizinggene may be an oversimplification. At present, E#-mycpre-B cells can only be maintainedin vitro within Whitlock-Wittecultures (W. Langdon,unpublished results), in which unidentified growth factors are provided by an adherent stromal layer grownfrom normal bone marrowcells. In such cultures, the transgenic cells were again found to be larger than normalpre-B cells in parallel cultures. At least twiceas manywerein cycle, but theygrewto cell densities only slightly higher than those of their normalcounterparts, presumably

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because growth factor levels were limiting. Whenisolated from bone marrow,manyEla-myc pre-B cells display Ia (66), a marker normally confined to more mature cells within the B lineage. After one weekin culture, however,Ia expressionwaslost, perhapsbecausethe cultures lack factor(s) essential for Ia expression.Thus, all evidenceto date suggests that B lymphoidcells constitutively expressing c-myc maintain their requirement for growth factors, although perhaps at somewhatreduced levels (see also 76). Theseresults contrast markedlywith the report that infection with a v-myccontaining retrovirus abrogates the factor dependenceof certain hemopoietic cell lines (77). It is of great interest to see whetherthe entire E~-myctumorigenic processcan be reconstructedin vitro. Thecells in several long-termcultures of transgenic marrowcells markedlychangedtheir growthcharacteristics after several monthsof growth(W.Langdon,unpublished), reaching 10fold higher saturation densities but still requiting the stromallayer. One such culture finally becamefactor independentand tumorigenic.Thus, the events required in vivo for onset of tumors maywell include mutations subvertingfactor requirements. In summary,analysis of prelymphomagenic E#-mycmice has provided someinsight into whydysregulated mycexpression creates tumor-prone mice. Whenimposed on the B lymphoid lineage, constitutive myc expressioncauses a 4-fold elevation in cell numbers,and morethan twice as manycells are replicating. Since the total numberof divisions has been increased by an order of magnitude,the risk of genetic accident is also significantly increased. Similar conclusions presumablyhold for other lineages expressing a deregulated myc gene. The immunoglobulin rearrangement mechanismcould be an added risk factor for lymphoid cells if, as in E#-mycmice, the expandedpopulationis primarily composed of cells actively undergoingDNA rearrangement.These factors maynot, however,fully account for the very high predisposition to tumoronset, and it maywell provethat mycacts moredirectly to increase the frequency of mutation. ONCOGENE

COOPERATIVITY

Theconclusion frommost of the transgenic oncogenestudies discussed is that expressionof one oncogeneis insufficient for malignancyto develop. (Ras oncogeneexpressionin pancreatic acinar cells maybe an exception-see 42.) Thetransgenic miceoffer uniqueopportunitiesto investigate the consequenceof exposingthe relevant cells to the action of additional oncogenes.Oneobvious wayis via retroviral infection. Neonatal mice carrying one oncogeneas a transgene can be infected with a virus bearing

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the secondoncogeneand monitoredfor tumoronset. Alternatively, cells isolated fromthe transgenic mice can be infected in vitro and their subsequent growthproperties monitoredin vitro and in vivo. Studies initiated along these lines with pre-B cells from E#-mycmice are proving most informative (W. Alexander, J. Adams,W. Langdon,S. Cory, unpublished results). Whenneonatal EIz-mycmice wereinfected with a helper virusfree virus carrying a mutated Ha-ras gene, lymphomainduction was greatly accelerated. Analternative strategy to investigate cooperativityis to cross lines of transgenic mice that express different oncogeneswithin the sametissue. Myc/rascooperativity has been demonstratedin breast epithelial cells by breeding a strain of MMTV-ras transgenic mice with an MMTV-myc line (78). The latency for onset of mammary carcinomas was considerably reducedbut their time of onset wasstill variable and the tumorsdid not involve the entire breast. Thusadditional somatic events appearedto be required for the onset of malignancyeven in the presence of both a deregulated mycgene and an activated ras gene. CONCLUSIONS

AND FUTURE

PROSPECTS

It is alreadyclear that transgenicmicewill substantially deepenour understanding of oncogeneaction. The efficacy of several genes as tumorpromotingagents in the living animalhas been demonstratedconclusively. In general, activity of morethan one cancer-promotinggene seemsto be required for the emergenceof a tumorigenic clone. The myc and ras oncogeneshave been shownto collaborate in the transformation of two cell types. Wecan look forwardto seeing the results of a great variety of such experiments, and these should establish the "rules" for oncogene cooperativityin diversecell lineages. Theclear demonstrationof an altered but preneoplastic phase in the transgenic animalsis particularly important. Duringthis stage, the biological effect of the primaryoncogenecan be analyzed.Moreover,attempts can be madeto alter the courseof the disease and perhapsevento prevent the onset of malignancy.Just as important,the effect of a rangeof chemical agents can be investigated for their tumor-promoting activity. Thecharacterization of additional tissue-specific promoter/enhancer sequenceswill extend the versatility of experimentstargeting oncogene expressionto specific cell lineages. Conversely,it will be of considerable interest to comparethe biological effects of different oncogeneswithin the same cell type. Already a comparisonhas been madeof the effects of mycand ras within mammary epithelial cells (55, 78). Moreover,several different oncogeneshave been coupledto the immunoglobulin heavychain

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enhancer (S. Cory, A. Harris, J. Adams,unpublished results), including N-myc, N-ras, v-myb, v-abl, and bcr-v-abl, a construct mimicking the hybrid oncogenethought to play a central role in chronic myeloid leukemia. Most of these genes have provoked lymphoid neoplasia in transgenic mice. The tumors observed to date include plasmacytomas and T lymphomas, as well as pre-B and B lymphomas. Transgenic mice harboring different growth factor genes will be an area for fruitful investigation, irrespective of whether their deregulated expression provokes tumors. In fact, certain oncogenes maywell prove to be growth factors expressed at very specific times of development: Int-2 has already been implicated as an embryonicgrowth factor (79). Finally, transgenic mice mayprove crucial to analysis of the recessive oncogenes, genes encoding products that normally prevent the emergence of tumors. High-level expression of an antisense copy of such genes in tumorigenic mice might mimic their loss and thereby induce malignancy. Since a strong candidate for the recessive retinoblastoma (Rb) susceptibility gene has been cloned recently (80), anti-Rb transgenic mice may well be the first of such analyses attempted.

Literature Cited 1. Gordon, J. W., Ruddle, F. H. 1983. Gene transfer into mouseembryos: Production of transgenic mice by pronuclear injection. Methods Enzymol. 101: 41133 2. Brinster, R. L., Chen, H. Y., Trum¯ bauer, M. E., Yagle, M. K., Palmiter, R. D. 1985. Factors affecting the efficiency of introducing foreign DNAinto mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82:4438-42 3. Brinster, R. L., Palmiter, R. D. 1986. Introduction of genes into the germ line of animals. The Harvey Lectures 80:1-38 4. Palmiter, R. D., Brinster, R. L. 1986. Germline transformation of mice. Ann. Rev. Genet. 20:465-99 5. Hogan, B., Costantini, F., Lacey, E. 1986. Manipulatin9 the mouse embryo: A laboratory manual. Cold Spring Harbor Lab., Cold Spring Harbor, Me. 6. Woychik, R. P., Stewart, T. A., Davis, L., D’Eustachio, P., Leder, P. 1985. An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature 318:36-41 7. Elbrecht, A., DeMayo,F. J., Tsai, M. J., O’Malley, B. W. 1987. Episomal maintenance of a bovine papilloma virus vector in transgenic mice. Mol. Cell. Biol. 7:1276-79

8. Lacey, M., Alpert, S., Hanahan, D. 1986. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 322:609-12 9. Rassoulzadegan, M., L6opold, P., Vailly, J., Cuzin, F. 1986. Germline transmission of autonomous genetic elements in transgenic mousestrains. Cell46:513-19 10. Wagner,E. F., Covarrubias, L., Stewart, T. A., Mintz, B. 1983. Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line. Cell 35:647-55 11. Jaenisch, R. 1976. Germline integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. USA73:1260-64 12. Van der Putten, H., Botteri, F., Miller, A. D., Rosenfeld, M., Fan, H., Evans, R., Verma, I. 1985. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc. Natl. Acad. Sci. USA 82:6148-52 13. Jaenisch, R., J/ihner, D. 1984. Methylation, expression and chromosomal position of genes in mammals. Biochem. Biophys. Acta 782:1-9 14. Stewart, C. L., Stuhlmann, H., J/ihner, D., Jaenisch, R. 1982. De novo methylation, expressionand infectivity of retro-

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viral genomesintroduced into embryonal carcinomacells. Proc.Natl. Acad. Sci. USA79:4098-4102 15. Gautsch, J. W., Wilson, M. C. 1983. Delayedde novo methylationin teratocarcinomasuggests additional tissuespecific mechanisms for controlling gene expression. Nature301:32-37 16. Niwa,O., Yokota,Y., Ishida, H., Sugahara, T. 1983. Independentmechanisms involved in suppression of the Moloney leukemia virus genomeduring differentiation of murine teratocarcinoma cells. Cell 32:1105-13 17. Linney, E., Davis, B., Overhauser,J., Chao, E., Fan, H. 1984. Nonfunction of a Moloneymurine leukaemia virus regulatory sequence in F9 embryonal carcinomacells. Nature308:470-72 18. Gorman,C. M., Rigby, P. W.J., Lane, D. R. 1985. Negativeregulation of viral enhancersin undifferentiated embryonic stem cells. Cel142:519-26 19. Franz, T., Hilberg, F., Selinger, B., Stocking, C., Ostertag, W.1986. Retroviral mutantsefficiently expressedin embryonalcarcinomaceils. Proc. Natl. Acad. Sci. USA83:3292-96 20. Soriano, P., Cone, R., Mulligan, R., Jaenisch, R. 1987. Tissue-specific and ectopic expression of genes introduced into transgenicmiceby retroviruses.Science 234:1409-13 21. Rubenstein,J. L. R., Nicolas, J. F., Jacob, F. 1984.Constructionof a retrovirus capable of transducing and expressing genes in multipotential embryonic cells. Proc. Natl. Acad.Sci. USA 81:7137~,0 22. Stewart, L., Vanek,M., Wagner,E. F. 1985. Expressionof foreign genes from retroviral vectors in mouse teratocarcinomachimaeras. EMBO J. 4: 37019 23. Robertson,E., Bradley, A., Kuehn,M., Evans, M. 1986. Mousegermline transmission of gene sequences introduced into cultured pluripotential cells by a retroviral vector. Nature323:445~,8 24. Weinberg, R. A. 1985. The action of oncogenesin the cytoplasmand nucleus. Science 230:770-76 25. Rassoulzadegan,M., Cowie,A., Carr, A., Glaichenhaus, N., Kamen, R., Cuzin,F. 1982. Theroles of individual polyomavirus early proteins in oncogenic transformation. Nature300: 71318 26. Vander Elsen, P., de Pater, S., Houweling, A., vander Veer,J., vander Eb, A. 1982. The relationship betweenregion Ela and Elb of humanadenoviruses in cell transformation. Gene18:175-85

27. Land, H., Parada, L., Weinberg,R. A. 1983. Tumorigenicconversion of primaryembryofibroblasts requires at least two co-operating oncogenes. Nature 304:596~00 28. Ruley, E. 1983. Adenovirusearly region 1Aenables viral and cellular transforminggenesto transformprimarycells in culture. Nature304:602~5 29. Land,H., Chen,A. C., Morgenstern,J. P., Parada,L. F., Weinberg,R. A. 1986. Behaviourof mycand ras oncogenesin transformation of rat embryofibroblasts. Mol.Cell. Biol. 6:1917-25 30. Brinster, R. L., Chert, H. Y., Messing, A., Van Dyke, T., Levine, A. J., Palmiter, R. D. 1984. Transgenicmice harboring SV40T-antigengenes develop characteristic brain tumors. Cell 37: 367-79 31. Palmiter, R. D., Chen,H. Y., Messing, A., Brinster, R. L. 1985. SV40enhancer and large-T antigen are instrumentalin developmentof choroid plexus tumors in transgenic mice. Nature 316:45740 32. Faas, S., Pan, S., Pinkert, C., Brinster, R., Knowles, B. 1987. Simian virus 40 SV40-transgenic mice that develop tumoursare specifically tolerant to SV40 T antigen. J. Exp. Med.165:417-27 33. VanDyke,T., Finlay, C., Levine, A. J. 1985. A comparisonof several lines of transgenic mice containing the SV40 early genes. Cold Spring HarborSymp. Quant.Biol. 50:671-78 34. Messing,A., Chen, H. Y., Palmiter, R. D., Brinster, R. L. 1985. Peripheral neuropathies, hepatocellular carcinomasandislet cell adenomas in transgenic mice. Nature 316:461~3 35. Mahon, K. A., Chepelinsky, A. B., Khillian, J. S., Overbeek,P. A., Piatigorsky, J., Westphal, H. 1987. Oncogenesis of the lens in transgenic mice. Science 235:1622-28 36. Hanahan,D. 1985. Heritable formation of pancreatic r-cell tumoursin transgenic miceexpressing recombinantinsulin/simian virus 40 oncogenes. Nature 315:115-22 37. Adams,T. E., Alpert, S., Hanahan,D. 1987. Non-toleranceand autoantibodies to a transgenicself antigenexpressedin pancreaticfl cells. Nature325:223-28 38. Ornitz, D., Hammer,R., Messing,A., Palmiter,R., Brinster, R. 1987.Science. In press 39. Small, J. A., Khoury, G., Jay, G., Howley,P. M., Scangos, G. A. 1986. Early regions of JC virus and BK virus inducedistinct andtissue-specific tumorsin transgenic mice. Proc. Natl. Acad. Sci. USA83:8288-92

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TRANSGENICMICE AND ONCOGENESIS 40. Small, J. A., Scangos,G. A., Cork,L., Jay, G., Khoury, G. 1986. The early region of humanpapovavirus JC inducesdysmyelination in transgenicmice. Cell 46:13-18 41. Barbacid, M. 1986. Humanoncogenes. In ImportantAdvancesin Oncology,ed. V. DeVita, S. Hellman,S. Rosenberg, pp. 3-22. Philadelphia:Lippincott 42. Quaife,C. J., Pinkert, C. A., Ornitz, D. M.,Palmiter,R. D., Brinster, R. L. 1987. Pancreatic neoplasia induced by ras expressionin acinar cells of transgenic mice. Cell 48:1023-34 43. Andres, A. C., Sehonenberger,C. A., Groner, B., Henninghausen, L., LeMeur,M., Gerlinger, P. 1987. Ha-ras oncogeneexpression directed by a milk protein gene promoter: Tissue specificity, hormonalregulation, and tumor inductionin transgenicmice.Proc.Natl. Acad. Sci. USA84:1299-1303 44. Metcalf, D. 1985. The granulocytemacrophage colony stimulating factors. Science 229:16-22 45. Lang, R. A., Metcalf, D., Gough,N. H., Dunn, A. R., Gonda, T. J. 1985. Expressionof a hemopoieticgrowthfactor eDNA in a factor dependentcell line results in autonomous growth and tumorigenicity. Cell 43:531-42 46. Rosenthal,A., Lindquist, P. B., Bringman,T. S., Goeddel,D. V., Derynck,R. 1986.Expressionin rat fibroblasts of a humantransforming growth factor c~ cDNA results in transformation.Cell46: 301-9 47. Hapel, A. J., VandeWonde,G., Campbell, H. D., Young,I. G., Robbins, T. 1986. Generationof an autocrine leukemia using a retroviral expression vector carrying the interleukin-3 gene. Lymphokine Res. 5(4): 249~50 48. Lang, R. A., Metcalf, D., Cutherbertson, R. A., Lyons,I., Kelso, A., Kannourakis,G., Williamson,J., Klintworth, G., Gonda,T. J., Dunn,A. R. 1987. Transgenic mice expressing a haemopoieticgrowth factor gene (GMCSF)develop an accumulationof activated macrophages, blindness and a fatal syndromeof tissue damage.Submitted 49. M/iller, R. 1986.Cellular and viral fos genes: Structure, regulation of expression and biological properties of their encoded products. Biochem. Biophys. Acta 823:207-25 50. Jenuwein,T., M/iller, R. 1987. Structure-function analysis offos protein: A single aminoacid changeactivates the immortalising potential of v-fos. Cell 48: 647-57

47

51. Riither, U., Garber, C., Komitowski, D., Muller, R., Wagner,E. F. 1987. Deregulatedc-fos expressioninterferes with normalbone developmentin transgenie mice. Nature325:412-16 52. Kelly, K., Siebenlist, U. 1985. Therole of c-mycin the proliferation of normal andneoplasticcells. J. Clin. Immunol. 5: 65-77 53. Studzinski,G. P., Brelvi,Z. S., Feldman, S. C., Watt, R. A. 1986. Participation of c-myc protein in DNAsynthesis of humancells. Science 234:467-70 54. Cory, S. 1986. Activation of cellular oncogenesin haemopoietic cells by chromosome translocation. Adv. CancerRes. 47:189-234 55. Stewart,T. A., Pattengale,P. K., Leder, P. 1984. Spontaneousmammary adenocarcinomas in transgenic mice that carry and express MTV/mycfusion genes. Cell 38:627-37 56. Adams,J. R., Harris, A. W., Pinkert, C. A., Corcoran,L. M., Alexander,W.A., Cory, S., Palmiter, R. D., Brinster, R. L. 1985. The c-myc oncogenedriven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318:533-38 57. Leder,A., Pattengale,K., Kuo,A., Stewart, T. A., Leder, P. 1986.Consequence of widespreadderegulationof the c-myc gene in transgenic mice: Multiple neoplasms and normal development. Cell 45:485-95 58. Corcoran,L. M., Cory, S., Adams,J. A. 1985. Transposition of the immunoglobulin heavychain enhancerto the myc oncogene in a murine plasmacytoma. Cell 40:71-79 59. Grosschedl,R., Weaver,D., Baltimore, D., Costantini, F. 1984. Introductionof a # immunoglobulin gene into the mouse germline: Specific expressionin lymphoid cells andsynthesisof functionalantibody. Cell 38:647-58 60. Tidmarsh,G., Dailey, M., Whitlock,C., Pillemer, E., Weissman,I. L. 1985. Transformed lymphocytes from Abelson-derivedmice express levels of a B lineage transformationassociated antigen elevated from that foundon normal lymphocytes. J. Exp. Med. 162: 142130 61. Whitlock,C. A., Tidmarsh,G. F., MUller-Sieburg, C., Weissman,I. L. 1987. Bonemarrowstromalcell lines with lymphopoieticactivity expresshighlevels of a pre-B neoplasia-associatedmolecule. Cell 48:1009-21 62. Morse,H. C., Tidmarsh,G. F., Holmes, K. L,, Fredrickson, T., Hartley, J., Pierce, J., Langdon,W.Y., Dailey, M.,

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Weissman, I. L. 1987. Expressionof the line. Nature322:748-50 6C3 antigen on murine hematopoietic 71. Dotto, G. P., Gilman, M. Z., Maruneoplasms. J. Exp. Med.165:920-25 yama,M., Weinberg,R. A. 1986. c-myc and c-fos expressionin differentiating 63. Braun, J., Forouzanpour,F., King, L., Teheranizadeh,T., Bray, M., Kliewer, mouseprimary kerotinocytes. EMBO J. ÷ S. 1986. B-Lyl cells: ImmortalLy-1 5:2853-57 B lymphocytecell lines spontaneously 72. Bernard, O., Cory, S., Gerondakis,S., arising in maturesplenic cultures. ImmuWebb,E., Adams,J. M. 1983. Sequence nol. Rev. 93:5-21 of the murine and humancellular myc 64. Hayakawa,K., Hardy, R., Herzenberg, oncogenesand two modesof mye transcription resulting from chromosome L. A., Herzenberg, L. A. 1985. Progenitorsfor Ly-1Bcells are distinct from translocation in B lymphoidtumors. progenitors for other B cells. J. Exp. EMBOJ. 2:2375-83 Med. 161:1554~58 73. Nishikura,K., Ar-Rushidi,A., Erikson, 65. Alexander,W., Schrader, J. W., Adams, J., Watt, R., Rovera, G., Croce, C. M. J. M. 1987. Expression of the c-myc 1983.Differential expressionof the normal and of the translocated humanconcogene under control of an immunoglobulin enhancer in E#-myctransmyc oncogenesin B cells. Proe. Natl. genic mice. Mol. Cell. Biol. 7: 1436Acad. Sci. USA80:4822-26 74. Leder,P., Battey,J., Lenoir, G., Moulding, L., Murphey,W., Potter, H., Stew66. Langdon,W. Y., Harris, A. W., Cory, S., Adams,J. A. 1986. Thec-myc oncoart, T., Taub, R. 1983. Translocation gene perturbs B lymphocyte develamong antibody genes in human opmentin E#-myctransgenic mice. Cell cancer. Science 222:765-71 47:11-18 75. Rabbits, T., Forster, A., Hamlyn,P., 67. Harris, A. W.,Pinkert, C. A., Crawford, Baer, X. 1984. Effect of somaticmutaM. C., Brinster, R. L., Adams,J. M. tion within translocated c-mycgenes in 1987. The E#-myctransgenic mouse:A Burkitt’s lymphoma.Nature 309: 592modelsystem for high incidence spon97 taneous lymphoblastic lymphomaand 76. Cory, S., Bernard,O., Bowtell,D., Schleukemiaof early pre-Bcell origin. Subrader, J. W.1987. Murinec-myc retromitted viruses alter the growthrequirementsof 68. Vaux,D. L., Adams,J. M., Alexander, myeloidcell lines. Oncogene1:61-76 W.S., Pike, B. L. 1987. Immunological 77. Rapp,U., Cleveland,J., Brightman,K., competence of B cells subjected to Scott, A., Ihle, J. 1985. Abrogationof IL-3 and IL-2 dependence by recomconstitutive c-rnyc oncogeneexpression in E#-myctransgenic mice. J. Immunol. binant murineretroviruses expressingvIn press myc oncogenes. Nature 317:434-38 68a. Bubbers,J. E. 1984. Identification and 78. Sinn, E., Muller, W., Pattengale, P., linkage analysis of a gene, Rcs-I supTepler, I., Wallace,R., Leder, P. 1987. pressing spontaneous SJL/J lymphoma Coexpression of MMTV Iv-Ha vas and expression.J. Natl. CancerInst. 72: 441MMTV I c-myc genes in transgenic mice: 46 synergistic action of oncogenesin vivo. 69. Coppola, J. A., Cole, M. D. 1986. Cell 49:465-75 Constitutive c-myconcogeneexpression 79. Dickson,C., Peters, G. 1987. Potential oncogeneproduct related to growthfacblocks mouse erythroleukemia cell differentiation but not commitment. tors. Nature326:833 Nature 320:760-63 80. Lee, W.-H., Bookstein, R., Hong, F., Young,L.-J., Shew,J.-Y., Lee, E. Y.-H. 70. Dmitrovsky,E., Kuehl, W.M., Hollis, G.F., Kirsch,I. R., Bender,T. P., Segal, P. 1987. Humanretinoblastoma susS. 1986. Expression of a transfected ceptibility gene: Cloning,identification humanc-myconcogeneinhibits differand sequence. Science 235:1394-99 entiation of a mouseerythroleukemia



Annual Reviews www.annualreviews.org/aronline


Ann. Rev. lmmunol. 1988. 6 : 4943 Copyright © 1988 by Annual Reviews Inc. All rights reserved

KININ FORMATION: MECHANISMS AND ROLE IN INFLAMMATORY DISORDERS David Proud Division of Clinical Immunology,Johns Hopkins University School of Medicine at The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Maryland 21239

Allen P. Kaplan Division of Allergy, Rheumatologyand Clinical Immunology, State University of NewYork, Stony Brook, NewYork 11794-8161

INTRODUCTION Bradykinin and lysylbradykinin are potent vasoactive peptides liberated from ~2 globulins, called kininogens, by the actions of various proteases, known collectively as kininogenases. The pharmacologic properties of kinins--including their abilities to increase vascular permeability, to cause vasodilatation and pain, to contract most smooth muscle preparations, and to stimulate arachidonic acid metabolism--have led several investigators to suggest that these peptides may be important inflammatory mediators in humans(1-3). Only in recent years, however, with improved assay technologies and an increased awareness of the mechanismsregulating kinin levels, has meaningfuldirect evidence to support a role for the kinin system in humaninflammatory disorders begun to accumulate. In the present chapter we review our current knowledgeregarding the three different pathwaysthat maylead to kinin formation in inflammatory events: (a) Generation of bradykinin as a result of the activation of the Hagemanfactor-dependent pathways and production of plasma kallikrein, (b) the generation of lysylbradykinin by tissue kallikreins, and (c) the potential role of cellular proteases in kinin formation. Wealso critically 49 0732-0582/88/0410-0049502.00

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PROUD & KAPLAN

evaluate the evidence of activation of one or more pathways and the involvcment of kinins during inflammatorydisordcrs in humans.


MECHANISMS

OF KININ

FORMATION

Hageman Factor Dependent Pathway (4) The formation of bradykinin in humanplasmais dependentuponthe interactionof certain negativelychargedsurfaces withthree plasmaproteins; namely,FactorXII (Hageman factor) (5), prekallikrein(6-8), high molecularweight (HMW) kininogen(9-11) (Figure 1). These proteinsare also the initiating factors requiredfor the intrinsic blood coagulationandfibrinolytic pathways.In plasma,prekallikreinandHMW kininogencirculate as a complex with1 : 1 molarstoichiometry (12), while Hageman factor is not complexed.These proteins bind to initiating surfaces, the Hageman factor becomes activated, prekallikreinis converted to kallikrein, andHMW kininogenis digested to release the vasoactive peptidebradykinin(Figure1). A secondmajorplasmasubstrateof activated Hageman factor, factor XI, also circulates boundto HMW kininogen(13); it is convertedto factorXIa(14), whichcontinuesthe intrinsic coagulationcascade. Hageman factor (HF)(coagulationfactor XII) is a single-chainglobulin of molecularweight80,000(15). It is activateduponcontactwithcertain

surfoce

FACTOR Xl HMWKininogen ~ [

"PRO"-KALLIKREIN Intr~cellulorEnzymes ~ Plosmin PIosmoKollik~’~in

~

TISSUEKALLIKREIN(SECRETED)

FACTOR ~IA

LMWKININOGEN1:~] PREKALLIKREIN - ~ KALLIKRF’IN

i COAGULATION

~

No title

No title

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