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The Creation of the AKR Strain, Whose DNA Contains the Genome of a Leukemia Virus 1

Jacob Furth

Institute of Cancer Research and Department of Pathology
Columbia University
New York, New York

This unique symposium is dedicated to the uses of inbred strains of mice. Among the developers of such strains, I am the only one who is not a mammalian geneticist. For me, the creation of the AKR strain was merely a means to get leukemic mice, and since their creation they have been widely used for understanding the development and control of the leukemias. 2

In 1927, an anonymous donor made a large donation to the University of Pennsylvania for the study of this then mysterious disease, leukemia, with a free hand to spend it all. Dr. E.L. Opie surveyed the literature ( 1) and arrived at the conclusion that such research was long overdue. In chickens, Ellerman found a virus that caused all types of leukemias ( 2). The classical geneticists, Tyzzer (cf. 3) and his disciple, C.C. Little (cf. 4), found that mice were a storehouse of solid tumors. Two mouse geneticists had leukemia strains.

Dr. Opie then chose me, his disciple, to undertake research in this field, and at the start he was my invaluable consultant. My prior experience was limited to microbial genetics and immunology (under Otto Weil and Karl Landsteiner).

The beginning was tough. A letter to Ellerman requesting his virus was returned "Deceased; virus lost." In my laboratory, however, I was soon able to isolate several strains of avian "leukosis" viruses, some producing Ellerman's "erythromyelosis" ( 5), some lymphosarcoma ( 6), some the so-called "Marek's disease" [alias fowl paralysis, neurolymphomatosis ( 7, 8)].

Efforts to get a murine leukemia also met with unexpected difficulties. The two geneticists who happened to have leukemia strains failed to give us breeding pairs, while they benefited by reading the excellent survey of Dr. Opie ( 1) pointing to possible avenues of leukemia research. This created tough competition by possessors of leukemia strains.

We began inbreeding three stocks of mice; two obtained from a commercial breeder were named "A" and "S." "A" mice were purchased from a dealer in Pennsylvania who was the supplier of mice to the long extinct cancer research laboratory (supported by the DuPont family) where I learned that leukemia did occur in the "A" stock, albeit infrequently. Stock "S" was obtained from a dealer in New Jersey (with no knowledge of leukemia in the stock); "R" mice, a gift of the Rockefeller Institute, were known to be relatively husky, long-lived, and rarely develop neoplasia. I began inbreeding the first two stocks for leukemias; "A" for the lymphoid type of leukemia, "S" for the myeloid type, and "R" to be leukemia free. "A" ultimately yielded the AKR strain, the topic of this report. "S" yielded a good myeloid leukemia strain which was lost, and "R" yielded a strain known as Rf.

Leukemia-oriented Research During Genetic Inbreeding of Stock A Mice (1928-1937)

Mammalian genetics began in mice with inbreeding for such genetic trains as coat color and readily detectable abnormalities. The pioneering work of Tyzzer and his students, foremost among them C.C. Little (cf. 4), also included studies of spontaneous solid tumors, which happened to occur in their strains. Little was especially interested in them and, to enable expansion of work in mouse cancer genetics, he founded The Jackson Memorial Laboratory in 1929.

Nobody, to our knowledge, ever attempted to inbreed de novo a leukemia strain. The difficulties connected with the problem of inbreeding for leukemia were learned by us the hard way, after we began doing it in 1928, in spite of the advice of expert geneticists.

Leukemia is a diffuse neoplasm that originates in internal leukopoietic tissues and remains usually undetected until post-mortem examination at about 8 to 16 months of age. At this age, living female siblings are usually infertile. Thus, to get a sufficient number of spontaneous leukemias, living male-female siblings have to be bred in numerous parallel lines. Further, a puzzling phenomenon was encountered in that numerous mice failed to express the leukemia genome, dying at 8 to 16 months of age, or even older, without any sign of leukemia.

Thus I would have been the victim of the dictum, "Publish or perish," were it not for the parallel approach to induce leukemia in mice with ionizing radiation, isolation of viruses producing avian leukemias, and analysis of the diverse types of leukemias and leukemoid reactions, as they were encountered.

We had no difficulties in isolating diverse types of avian leukemia. Much of the work on avian neoplastic viruses was novel and highly productive, but since our theme is the history of the AKR strain, they are not detailed here. They were helpful in introducing us to techniques of identifying the virus of the AKR strain.

The radiation research was initiated after reading a single report on the high frequency of fatal leukemias among roentgenologists ( 9). It was already known that most pioneer roentgenologists and some of their patients developed cancers of the exposed parts of the body ( 10), but a connection between radiation exposure and leukemia had not previously been made. Following this lead, we exposed large numbers of mice to various types of whole-body radiation by single or fractional doses, thus hoping to minimize the development of localized, solid, non-leukemic neoplasms. Leukemias appeared in a small to moderate number of mice after several months of radiation. Following the advice of Dr. Opie, we did not sacrifice the surviving animals after our objective (leukemogenesis) was attained but kept them until natural death. The numerous neoplasms that appeared afterward led us to digress later into radiation biology.

These non-genetic studies on leukemia were impressive enough in 1932 (four years after their initiation) to honor them with a gold medal for original research at the annual meeting of the American Medical Association. The studies thus far were published in 24 papers (18 on viral leukemia in chickens, five on murine leukemias, and one on a membrane filter device) with nine associates ranging from students to expert scientists.

I recall three amusing episodes in this relation. A current weekly, guided by the appraisal of distinguished hematologists, expressed doubts about the conclusion of our prize-winning exhibit, stating, "What can he know about leukemia, he is an immunologist."

Shortly thereafter, after a key address at one of our annual conventions, the then leading oncologist, James Ewing ( 11), discussed my presentation. He accepted our work with mice, but he stated emphatically that from what he knew of human leukemia, it was not a neoplastic disease. Thereupon I wrote an article in the JAMA on the similarity of human and mouse leukemia ( 12). As I see now, this was good salesmanship, but not good statesmanship.

A similar episode occurred years later at an international meeting in St. Louis, after the explosion of the atomic bombs over Hiroshima and Nagasaki, but before the final "ballots" on their late effects were in. Here, too, leading clinical hematologists accepted our work on radiation leukemogenesis in mice but expressed strong doubts that ionizing radiations are leukemogenic in man. About two years later, biostatisticians of the ABCC (Atomic Bomb Casualty Commission) announced the development of leukemia in the exposed population in statistically significant numbers. Thus, I myself became a member of the ABCC and spent five years at Oak Ridge as a radiation pathobiologist. Among my young associates was Arthur Upton ( 13) who became the leading expert in radiation pathology. "Operation Greenhouse," a monumental study of an experimental atomic explosion, updates experimental work on the pathologic effects of ionizing radiation ( 14).

Spinoffs of these nine years of developing the AKR strain by inbreeding were the discovery of cell preservation in the living state by slow freezing ( 15) and proof that a single live lymphocyte but not crushed cells or fluid between them can transmit leukemia in very young adult mice ( 16).

Conventional Genetic Analysis of the AKR Strain After Homogeneity Was Attained (1937-1946)

The first up to date experimental studies on the genetics of spontaneous leukemia in AKR mice were made by Cole ( 17). In his studies the combined incidence of leukemia in AK mice (including the f, g, h, and i sublines) was 71.2% in females and 67.0% in males. The difference in values in the four sublines of Ak did not appear to be significant (64.0 to 76.3% in females and 42.1 to 70.0% in males). In our low-leukemia strain Rf, the corresponding values were 1.9% in females and 1.2% in males.

During the war years, Dr. Clara J. Lynch, the geneticist of the Rockefeller Institute, became the custodian of the AKR sublines ( 18). She made the most thorough genetic study of several lines originating in my laboratories and continued inbreeding them between 1940 and 1946. She published her protocols in a comprehensive monograph in 1954 ( 18). In the United States, most AKR mice in current use are derived from her inbred lines.

In this monograph she adopted the appellation AKR, changing my system in which the original stock is designated by a capital and the sublines by small letters (e.g., Akr). In this review and in earlier ones she also used the appellation "R.I.L." Yielding to the now common usage "AKR," I accepted this term. However, in citing earlier publications the appellations are those given therein.

The abbreviated tabulations of Lynch's data (Tables 1 and 2) give the best evidence indicating that her five lines were well inbred, and that the year to year fluctuation in leukemias incidence was due to some hidden non-genetic factor. Cole ( 17) carried his five lines in five sublines which showed year to year fluctuations comparable to those of Lynch.

"Homozygosity" was judged to attained when the incidence of leukemia could no longer be increased and when transplantation in young animals with leukemic cell suspension usually yielded close to 100% takes in females and males. These are tenuous criteria, as we now know from the studies of Snell ( 19) 3 Further, neoplastic cells have an individuality of their own, different from that of the normal cell from which they arose.

The leukemias, induced or spontaneous, behave as neoplasms, lymphosarcoma and leukemia being extreme variants of the same disease. I suggested that localization of lymphosarcoma may be due to antigenicity of the lymphoma by lowering the host resistance. This reasoning followed the now forgotten pioneer work of James Murphy on the relation of normal lymphocytes in resistance to neoplasms ( 20). Murphy failed to bring convincing positive evidence to his thesis, namely, that induced lymphocytosis would confer resistance to tumorous growth. Later development of "homozygous" (isogenic) strains ( 19) placed the earlier work with transplanted tumors in proper perspective, and led to approaches on immunotherapy of spontaneous neoplasms, as will be discussed.

All Ak leukemias were lymphoid. They were found most frequently in animals dying between 34 and 45 weeks of age, the oldest when 84 weeks old.

Summarizing her findings between 1940 and 1949, Lynch concluded that the variability in leukemia incidence from year to year cannot be accounted for by selection among lines, by longevity, by the season of the year in which the mice were born, by the age of the mother at parturition, or by litter seriation. Impressed by our work on the role of the thymus in leukemogenesis (described below), she concluded that possible intercurrent disease affecting the thymus may influence the occurrence of leukemia.

Appraising work in her and in my laboratories in 1941-1942 on foster nursing ( 21), she concluded that the AKR strain was susceptible to mammary tumor agents, but itself lacks such an agent or carries one of low infectivity.

Of the extensive early hybridization studies done in our laboratories ( 22), only two sets of abbreviated data are listed in Table 3. In the first by Schweitzer et al. ( 22), crossing mice of high mammary tumor-low leukemia strain C3H with Ak mice did not much inhibit leukemia development in the F1 hybrids, while crosses between the low leukemia-MT-free Rf mice [Exp. of Cole( 17)] caused marked inhibition especially when the Rf parent was female ( Table 3).

Table 4 presents Lynch's ( 18) list of the principal sublines of AKR in the USA, derived from her AKR/M line. She discontinued breeding AKRs in 1946, leaving her AKR/M at the Memorial Hospital. Dr. Lloyd Old and other investigators at the Memorial Hospital, who will speak in coming sessions, will enlighten us about the fate of the AKR/M lines.

The first major genetic contribution subsequent to the work of Lynch was made by Snell ( 19) who discovered that lines homologous by transplantation tests can differ genetically among themselves. The genetic studies on isogenicity nomenclature and other major developments in genetics will be topics in a session of this conference. The two editions of The Biology of the Laboratory Mouse, written by the staff of The Jackson Memorial Laboratory ( 3, 4), are informative textbooks for all students of the laboratory mouse.

Discovery of the Hormonal Role of Thymus in Leukemogenesis (1942-1946)

After inbreeding of the AKR strain was attained, the question was raised: Why the long latency of spontaneous leukemia (5 to 12 months) and when and where does the leukemic transformation of normal lymphocytes occur? The answer to this was first announced in 1942 at the conference of Gibson Island. 4 Until about five months of age, the lymphocytes and all hematopoietic organs of the AKR mice appeared normal, after which they went neoplastic transformation, first detected in the thymus by formation of isolated thymic lymphomas. From there they spread to many lymphoid organs. Sometimes only one, but usually both thymic lobes are involved. To probe this anatomic observation, thymectomies were performed ( 22, 24), the results of which are shown in Figure 1. This figure indicates that thymectomy prevented the development of leukemia, and thereby prolonged the life of AKR mice ( 24).

We noted frequently that when the incidence of pneumonia and other debilitating diseases was high in our colony, the leukemia incidence was low and the thymus was atrophic. The relation of "stress" and thymic atrophy was known to Selye ( 25). The relationship of adrenal glucocorticoid to atrophy of lymphoid organs was discovered by Dougherty ( 26), and this became related to stress. At this stage, two experiments were called for: the demonstration of thymic atrophy by some simple, noninfectious agent, and the exploration of whether thymic grafts will restore the leukemia incidence in thymectomized Ak mice.

As an uncomplicated stressful agent, underfeeding was chosen. This was motivated by well controlled systematic studies of A. Tannenbaum ( 27) who confirmed the statistical analysis of the Metropolitan Life Insurance Company indicating that undernourishment tends to prolong the life of people and reduces the likelihood of developing cancer. Probing this idea, Saxton duplicated the effect of thymectomy in careful, well controlled experiments by rigid, uninterrupted underfeeding ( 28). Underfed mice outlived even the matched controls which had been subjected to thymectomy and the leukemia incidence among them was very low ( Figure 2 ).

The findings on the relation of the thymus to leukemia ( 23, 24) were a veritable "breakthrough." (Until then the thymus was regarded as just another lymphoid organ.) They attracted numerous investigators, whose contributions soon outclassed those made in my lab. The state of knowledge in 1959 in leukemogenesis and neoplasia in general is well surveyed in The Physiopathology of Cancer ( 29). In Chapter 12 ( 30) on leukemia and thymus 581 articles are cited. H.S. Kaplan et al. ( 31, 32) reviewed the relation of ionizing radions to leukemia and the effects of thymectomy and thymus grafts. L.W. Law et al. ( 33, 34) reported on carcinogen-induced leukemia, and genetic and thymic factors, and D. Metcalf ( 35, 36) on the thymic origin of his LSF (lymphocytosis stimulating factor) and its relation to leukemia.

To put the discovery of the thymus in historical perspective, the following is worthy of note. In the third decade of this century, I witnessed a prestigious lecturer calling the lymphocyte a cell of mystery: most abundant in the body, seen everywhere, dies daily in large numbers in the intestinal epithelium, but it is unknown where it is born and what its function is. Lymphocytes are concentrated in certain organs, known as "lymphopoietic."

The recognition of the relation of the thymus in lymphogenesis preceded that of its role in immunologic competence. Both are prevented by thymectomy and restored by thymic grafts. We suggested earlier that the thymus is the common base for both. Other vital factors affect both, operating via the thymus. Foremost of these are "stress" ( 25) induced by adrenal glucocorticoids ( 26) which are related to ACTH (adrenocorticotropic hormone).

In 1964, a symposium was held on "The Thymus" ( 37), where it was recognized as a special functional organ, playing a significant role in immunologic responsiveness ( 37; page 99) in carcinogenesis as well as leukemogenesis ( 37; page 121). A landmark of knowledge on the structure, function, and role of the thymus was the subject of another symposium ( 38) held at about the same time with contributions of 72 authors.

Several Ciba Foundation Symposia were held on leukemia and allied disturbances as noted in AKR mice ( 39, 40, 41, and 42). They mirror the development of knowledge in areas of leukemia, thymus, leukemia virus, and immunology. The first, held in 1954, was titled "Leukemia Research" ( 39). The second, held in 1959, had the same title ( 40). The third in 1962 was titled "Tumor Viruses of Murine Origin" ( 41). The fourth, held in 1966, was titled "The Thymus: Experimental and Clinical Studies" ( 42).

In the first symposium Ludwik Gross reported on a filterable agent in Ak mice, allied to Bittner's mammary tumor virus. I attributed his success to the use of neonatal and C3H mice. These are leukemia-free but are sensitive to AKR leukemias as was reported earlier ( 22). His sustained work and that of others are well described in his book Oncogenic Viruses ( 43).

The fourth Ciba Symposium ( 41 on the thymus was masterfully organized by Sir Macfarlane Burnet at the Hall Institute. Members of the Hall Institute included Jacques Miller, Metcalf, Nossal, and other researchers on the thymus. Sir Macfarlane graciously invited me even though I was a renegade in thymus research (having been attracted in the early 1950s to derangements of homeostasis and neoplasia without carcinogens). The main topic of this conference was the structure of the thymus, its role in cellular and humoral immunity, the function of lymphocytes, and autoimmune disease. AKR mice were used or referred to in studies by several contributors. I presented here my last fragmentary work on the thymus. It was done with Ioachim and other associates in tissue cultures of the thymus. It aimed at detection of cells which secreted the leukemogenic factor. Cultural epithelial reticulum ( Figure 3 ) restored the lymphocyte levels and immunocompetence in thymectomized rats to which the AKR mice became adapted.

With great intensification of cell culture research resulting in the development of numerous isolated cell lines, it seems likely that the various cellular elements of the thymus will be grown in pure cultures. Their interrelationship, nutritional requirements, promoters, inhibitors, transforming principles, and hormonal secretions will be identified.

Inasmuch as in vitro studies will likely use inbred strains, attention is called here to the splendid volume Readings in Mammalian Cell Culture ( 44) and to the proceedings of conferences at the Alton Jones Cell Science Center of the Tissue Culture Association in Lake Placid, New York. Prominent in this area are Grodon Sato and his associates. One of their major discoveries is that replacement of serum by hormones permits the growth of cells in a medium defined by them ( 45). Most pure functional cell lines, now preserved and distributed in the frozen state, are derived from Sato's laboratory.

The first comprehensive book on thymic hormones, edited by Luckey and published in 1973 ( 46), describes the technique of thymectomy, disorders created by thymic deficiency in man, and isolation and characterization of several thymic extracts with hormonal activity. Most known thymic hormones were isolated by A.L. Goldstein in collaboration with A. White (published in 14 articles in 1966-1972), while G. Goldstein et al., who called attention to an endocrine function of the thymus manifested in myasthenia gravis and lupus erythematosus, published in eight articles (1966-1970) as cited in this book.

A more updated book, The Biological Activity of Thymic Hormones, was edited by Van Bekkum in 1975 ( 47). In the introductory chapter Abraham White sums up the available experimental data, concluding that thymosin isolated by him and other hormones isolated by A.L. Goldstein fulfill five of six postulates essential for designating the thymus as an endocrine gland. The sixth essential postulate, the chemical synthesis of any of these hormones, has yet to be achieved. White postulated that the thymus produces a family of several hormones, each of which may alter the extent of expression of one or more of the immunological roles of the thymus in regulation of host competence. This book deals with the differentiation of thymic elements, their interrelationship, the diverse specific function of T lymphocytes, impairment of thymic function, administration of thymic extracts and thymosin to patients, mitogen, and mixed lymphocyte reaction for thymosin.

Inasmuch as my knowledge in the development of the diverse areas of research in which AKR mice are utilized is second-hand after 1966, I shall be brief and sketchy, citing original articles by major contributors as well as excellent semipopular surveys by them which began to appear in increasing numbers in the Scientific American articles written by them. A cursory review of a MEDLARS (Medical Literature Analysis and Retrieval System) search indicates that in a year covering 1974-1975 about 180 articles appeared on thymus hormones. In about the same year a literature search by the librarian of The Jackson Laboratory (the main source of AKR mice in the USA) has shown that in about the same number of articles AKR mice were used. My attempt to break down these publications per area of research proved exceedingly time-consuming because the topics covered are overlapping and are limited to a segment of the area which the author investigated in depth.

Leukemia Virus Genetics: The Transcriptase of the RNA Virus of AKR (1970) 5

The year 1970 was a landmark in molecular genetics and cancer research with the announcement of the discovery of the reverse transcriptase (RT) by Temin ( 48) and Baltimore ( 49). It triggered rapidly expanding research in molecular biochemistry and created a firm base for molecular genetics. It led to the discovery of many other enzymes linking the DNA-integrated silent viral segment of the AKR cell to its infectious RNA virus.

Temin ( 48, 50), studying a broad spectrum of tumor viruses, suggested that RNA tumor viruses are repeatedly evolving by emergence or escape from the genome of normal cells (protovirus hypothesis). The protovirus is sensitive to deoxyribonuclease and resistant to ribonuclease. (The infectious AKR virus is sensitive to ribonuclease.) By using a labeled DNA copy in hybridization tests, he found that DNA contains some of the infectious virion sequences. On the basis of his phylogenetic and ontologic studies, he postulated that the primary event is the evolution of nucleotide sequences in the DNA. The emergence of infectious RNA virion is a secondary event.

In his Clowes lecture, ( 48), Temin discussed how the genome of a normal cell changed into the genome of a cancer cell. The viruses that cause cancer have genes for cancer (now named T genes). Carcinogenesis by strongly transforming viruses involves the formation in infected cells of genes for cancer copied from a viral genome. Temin's protovirus hypothesis states "that the genes for neoplastic transformation arise in an organism as a result of misevolution of a normal system of DNA information-transfer."

In his 1975 Harvey lecture Baltimore sketched his strategy of RNA viruses ( 51). He contrasted three types of viruses: the polio virus, the virus of vesicular stomatitis, and RNA tumor viruses. They use different strategies for infecting cells. The strategy of the RNA tumor viruses is the use of reverse transcriptase in the virion to make a DNA copy which integrates into cellular DNA. This is illustrated schematically in Figure 8 of reference 51.

Once the proviral DNA is integrated into cellular DNA ( 49), it spreads the virus by inheritance ( 51). In the AKR mouse viral information has been shown to reside in two stable genetic loci and to segregate in genetic crosses as simple Mendelian traits (cf. Rowe et al., Science 178: 860, 1972, and Chattopadhyay et al., Proc. Natl. Acad. Sci. 71: 167, 1974).

Leukemia Virus Immunology

Soon after the discovery of transplantability of leukemic cells, immunologic techniques were used to characterize both the leukemic cells and the agents producing them. In 1941 Elvin Kabat, who spent three highly productive years in my laboratories, disclosed that the plasma of leukemic chickens contained huge quantities of virus sedimentable at high speed ( 52). Avian leukosis, in contrast to murine and human leukemias, contains colossal quantities of infectious virus in the blood. Further, the serum of leukotic chickens contains virus neutralizing antibodies ( 53).

Leukemic cells, as neoplastic cells in general, differ from their ancestral normal cells by a genetic deviation, detectable by transplantation tests, one of the tools of geneticists. The most sensitive early immunologic techniques such as complement fixation and hemagglutination (if the virus adhered to blood cells) were not sensitive enough to detect small quantities of tumor viruses.

Medawar, in his Harvey lecture of 1957 ( 54), discussed known factors in the immunology of transplantation. As he predicted years earlier immunology offers negotiable pathways in the central regions of biology as genetics and embryology.

The discovery of the role of the thymus as an endocrine organ ( 23, 24) was soon followed by studies on the effect of thymic ablation at various periods of life. This led to research on immunology of the thymus ( 55, 56) summarized by J.F.A.P. Miller in 1966 ( 42, page 153). He has proven that the thymus directs the maturation of immunologic capabilities by means of a humoral mechanism.

Subsequently, numerous books and symposia on the role of the thymus in immunology have appeared. Some have already been cited; others are listed by Spiegelman et al. ( 57, 58). The great discovery by Karl Landsteiner and M.W. Chase ( 59, 60) on how to recognize small molecules (haptens), which themselves are not detectable by conventional immunologic techniques, tremendously advanced the usefulness of immunology in tumor-virus-related research, yet to be fully exploited. 6 (This is reviewed in Scientific American in an informative, well illustrated article, "The Development of the Immune System," by M.D. Cooper and A.R. Lawton in 1974).

Thus the prophetic vision of Medawar materialized and became widely popular in the 1970s.

Retrospect: The New Strategy of Tumor-Virus Research

The history of the AKR strain is a good example of the old adage that problems never cease to exist. Solution of one usually brings to light many new ones. Science is cascading, thereby leading to increasing specialization, often to such a degree that a team of different specialists is required to tackle each basic problem. Peyton Rous, when in retirement, listened to a lecture to which he was invited and whispered to me, "In what they say about Rous sarcoma I do not recognize the tumor I worked with." Presently I, too, find it difficult to comprehend many lectures and publications in which the AKR strain is a basic tool of research.

AKR is a uniquely stable strain, perhaps because of the nine years of successive brother and sister matings required to obtain homozygosity. It took several years to identify its several cardinal features: onco-(leukemo)-genicity based on inheritance of leukemia genes, the expression of which (releasing an infectious RNA virus) is under control by some thymic hormone while other thymic hormones are related to immunogenicity.

The following are illustrative examples of the great diversification research has taken in the 1970s.

It has long been known that the genes are associated with a large number of proteins (basic histones and acidophilic proteins), but not their specific relation to the genes. The discovery of reverse transcriptase opened new vistas. Enzymes are proteins. Unlike nucleic acids, protein can be identified immunologically with ease and great precision. Many enzymes are needed for DNA-RNA function. Some are positive control elements wanted for activation or inactivation of the host genetic information. Others are negative control elements. There is also a cascade regulation, where several enzymes operate sequentially. The new strategy of the regulation of the viral genome calls for identification of these controlling enzymes. The background knowledge obtained from thorough studies of bacterial viruses to mammalian cells in general had to be adapted to cells with tumor viruses. For research on oncornaviruses, AKR is an excellent tool for formulation of new tumor and viral genome strategies. The discovery of reverse transcriptase broke the ground.

The structure and function of chromatin (i.e., nucleic acid associated proteins) was the topic of a Ciba Symposium in 1975 ( 61). By 1976 Maclean could speak in more precise terms on gene expression in eukaryotes ( 62). The involvement of ribosomal (rs) RNA, transfer (t) RNA, and messenger (m) RNA in gene expression was recognized. His studies were done with pure cell lines grown in dissociated cell cultures. His Table 6 lists 14 hormones, whose actions are mediated by changes in levels of cyclic AMP active in multicellular organisms.

Analysis of virus-linked proteins (enzymes) led to the development of probes for identification of viruses in cells and information on gene function. First of these was linking leukemia viruses to a protein in the blood sedimentable at 60-70 g ( 58, page 11). He was one of the over 100 participants in an international symposium on the Molecular Approach to the Etiology of Human Cancer ( 58). In his article Spiegelman presented his molecular hybridization with radioactive probes, giving references to the historical research including the pioneer oncogenic theory of Huebner and Todaro ( 63, 64).

Research in immunology, both hormonal and cellular, vastly expanded. The book Lymphocyte Differentiation, Recognition and Regulation is a well organized, balanced review of some 2,317 references on this subject ( 65). "From investigation on the division of lymphocytes into the two great families, the regulatory role of T cells in antibody formation, the one cell one antibody rule, the genetics of immune responsiveness, the cellular mechanism whereby the immune system distinguishes 'self' from 'not self,' the physiology of lymphocyte surface receptors for antigens, major discoveries have resulted" (citing the excellent review by G.J.V. Nossal, who is a scholar on this subject).

The Thymus and Self ( 66) is a monograph by Rygaard on the immunobiology of the mouse mutant nude discovered in 1968. It is born without a thymus and, therefore, forms a baseline for study and isolation of the normal thymus functions. This gave an opportunity to the author to review the state of knowledge about the immune system structure and function with emphasis on the apparent dichotomy of the immune system, supportive and inhibitory.

These are but morsels of the rapidly growing and diversifying AKR-related literature in the current decade.

Figure 4.


I gratefully acknowledge the editorial assistance of Dr. Herbert C. Morse III and his assistants. Apologies are extended to many colleagues for omitting to cite their relevant work due to the limited scope of this article, and time and space limitations.


1The investigation was supported by Mr. Edward Mallinckrody, Sr. (anonymously) and by numerous donors named in the publications cited.

2The medal given me carries the inscription "Venienti Occurite Morbo." (Treat the disease as you find it.)

3Mapping the cell surface antigens is the topic of Session IV ( Amos, Graff, Old, Flaherty and Taylor).

4This was the site of the now popular summer conferences named after Dr. Gordon who initiated them. They are now held in New England.

5The genetic regulation of viruses is analyzed in Session III by Lilly, Rowe, Chused, Cardiff, Schlom, and Law.

6The relationships between worldwide and domestic mice (genetic, virus content, immunogenicity) are topics of later sessions.


1. Opie, E.L. (1928). Medicine 7: 31.

2. Ellerman, V. (1918). Die übertragbare Hühnerleukose. Springer Verlag, Berlin.

3. Snell, G. (1941). Biology of the Laboratory Mouse, 1st ed. The Blakiston Co., Philadelphia. (Gives refernce to E.E. Tyzzer, C.C. Little and other pioneer workers on the genetics of tumor formation.)

4. Green, E.L. (1966). Biology of the Laboratory Mouse, 2nd ed. McGraw-Hill Book Co., New York.

5. Ellerman, V. (1921). The Leucosis of Fowls and Leucemia Problems. Gyldendal, London.

6. Furth, J. (1933). J. Exp. Med. 58: 253.

7. Furth, J., and Stubbs, E.L. (1934). Proc. Soc. Exp. Biol. 32: 381.

8. Furth, J. (1935). Arch. Path. 20: 379.

9. Aubertin, C. (1931). Bull. Off. Soc. Franc. d'Electrol. Radiol. 40: 218.

10. Furth, J., and Furth, O.B. (1936). Am. J. Cancer 28: 54.

11. Ewing, J. (1940). Neoplastic Diseases, 4th ed. W.B. Saunders, Philadelphia.

12. Furth, J., Ferris, H.W., and Reznikoff, P. (1935). J. Am. Med. Assoc. 105: 1824.

13. Furth, J., Upton, A.C., and Kimball, A.W. (1959). Radiat. Res., Suppl. 1: 243.

14. Upton, A.C., Kimball, A.W., Furth, J., Christenberry, K.W., and Benedict, W.H. (1960). Cancer Res. 20; No. 8, Part 2.
See also PubMed.

15. Breedis, C., Barnes, W.A., and Furth, J. (1937). Proc. Soc. Exp. Biol. Med. 36: 220.

16. Furth, J., and Kahn, M.C. (1937). Am. J. Cancer 31: 276.

17. Cole, R.K., and Furth, J. (1941). Cancer Res. 1: 957.

18. Lynch, C.J. (1954). J. Natl. Cancer Inst. 15: 161.
See also MGI.

19. Snell, G. (1966). In Biology of the Laboratory Mouse, 2nd ed. (E.L. Green, ed.), p. 457. McGraw-Hill Book Co., New York.

20. Murphy, J.B. (1944). Cancer Res. 4: 622.

21. Barnes, W.A., and Cole, R.K. (1941). Cancer Res. 1: 99.

22. Schweitzer, M.D., and Furth, J. (1939). Am. J. Cancer 37: 224.

23. McEndy, D.P., Boon, M.C., and Furth, J. (1944). Cancer Res. 4: 377.

24. Furth, J. (1946). J. Geront. 1: 46.
See also MGI.

25. Selye, H. (1950). Stress. Acta, Inc., Montreal.

26. Dougherty, T.F. (1952). Physiol. Rev. 32: 339.

27. Tannenbaum, A. (1959). In The Pathophysiology of Cancer, 2nd ed. (F. Homburger, ed.), p. 517. Hoeber-Harper, New York.

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41. Wolstenholme, G.E.W. (1962). Ciba Foundation Symposium on Tumor Viruses of Murine Origin. J. & A. Churchill, Ltd., London.

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*Another comprehensive book, The Lymphocyte, Vol I, II, edited by J.J. Marchalonis, published by Marcel Dekker, New York, appeared in the same year (1977), which I had no opportunity to review.

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