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The Role of Cell Differentiation as a Determinant of Susceptibility to Virus Carcinogenesis* HENRY S. KAPLAN (Department of Radiology, Stanford University School of Medicine, Palo Alto, Calif.) Experimental analyses of the behavior of certain viruses in vitro, such as Dr. Dulbecco has presented to us on this memorable occasion, provide an exquisitely powerful method with which to dissect the intimate details of the cell-virus relationship. I t is to be hoped that such in vitro studies with the polyoma (10, 1~, ~9, 34), the Rous (s ~8), the myeloblastosis (7), and other carcinogenic viruses will soon yield new and fundamental information concerning the initial steps in the neoplastic transformation. I t has been one of the fond hopes of the proponents of the "virus theory" of carcinogenesis that the oncogenic viruses would provide a unifying conceptual framework for understanding the complex process of carcinogenesis. Yet, despite the admiration we may feel for the elegance of in vitro investigations such as have just been described, we are also increasingly aware that the advances of the past few years have provided us with new complexities and difficulties. We now know t h a t the oncogenic viruses are not a homogeneous family of viruses with similar special attributes. On the contrary, they exhibit as much diversity as other animal viruses. Some propagate in the cell nucleus (1), others in the cytoplasm (18, 18, $6); some contain D N A (11, 19, 31), others R N A (4, 6, 9), and those that have been clearly visualized by electron microscopy look disconcertingly like other animal viruses of comparable intracellular location and nucleic acid composition (5). Thus, we may begin to suspect that virus carcinogenesis will not prove to be a single process, but a diverse set of responses, perhaps differing characteristically for each group of oncogenic viruses. * This paper is based on an invited discussion of the first G. H. A. Clowes Memorial Lecture, entitled "Viral Carcinogenesis," presented by Dr. Renato Dulbecco at the annual meeting of the American Association for Cancer Research, Inc., Atlantic City, April 10, 1961. Previously unpublished work cited herein was supported by grant C-885~ from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. In the case of the family of viruses which elicit lymphomas a n d leukemias in the mouse (15, 83), we do not yet have the advantage of having an in vitro assay system, since these viruses do not produce cytopathogenic effects in any mammalian cells on which they have been inoculated to date, although they may be recovered from the culture fluids (14, 17). I t is therefore necessary to a t t e m p t to learn as much as possible about their mode of action from in vivo observations. Such investigations may yield valuable new insight concerning the factors which modify or determine cellular susceptibility to these viruses and thus contribute to the continuing search for quantitative in vitro methods. Thymic lymphoid neoplasms in irradiated strain C57BL mice have been shown to arise by a completely indirect process (~3). Cell-free filtrates and centrifugates prepared from such neoplasms elicited significant increases in lymphoma incidence when inoculated at birth into nonirradiated C57BL mice. These filtrate-induced tumors then yielded filtrates which exhibited enhanced potency on serial passage in newborn mice (~4). Similar observations with filtrates from lymphomas in irradiated strain C3H mice were independently reported by Gross (16). I t thus became apparent t h a t these supposedly radiation-induced lymphomas were in reality induced by a virus-like agent, and it seemed likely that irradiation set the induction sequence in motion by causing some change in the virus-host relationship. A few years ago (8), it was noted t h a t the earliest morphological changes associated with the induction of thymic implant lymphomas in irradiated C57BL mice bore a remarkable resemblance to arrested maturation in the lymphoid cell differentiation sequence. Accordingly, attention was focused on the process of differentiation in the mouse thymus and its possible relation to this tumor induction mechanism. Sainte-Marie and Leblond (~0), studying the rat thymus, traced a complex process of lymphoid 981 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1961 American Association for Cancer Research. 98s Cancer Research cell differentiation which proceeds in a series of steps from the undifferentiated reticulum or stem cell through the large lymphocyte (lymphoblast) and medium-sized lymphocyte stages to an end stage, the small lymphocyte, which is generally thought to be incapable of division. Although this sequence has not been quantitatively studied in the mouse, the same cell forms have been identified, and the great relative abundance of the small lymphocytes in the normal adult mouse thymic cortex makes it not unlikely that a similar pattern exists in that species. I t has long been known that bone marrow shielding or injection protects irradiated C57BL mice against the development of thymic lymphomas (ee). I t seemed desirable to compare the thymuses of shielded versus unshielded irradiated mice of this strain in a search for meaningful morphological antecedents of lymphoma induction. As noted elsewhere (20, ~1), these studies indicated rapid repair and maturation to the small lymphocyte stage in the radiation-damaged thymuses of the thigh-shielded animals. In contrast, there was an apparent arrest of maturation in the regenerating thymuses of the unshielded group, and their cortices remained filled with large and mediumsized lymphoid ceils, with a sustained high level of mitotic activity. A very similar process was noted during the course of regeneration and tumor development in subcutaneous thymic implants in thymectomized irradiated C57BL or hybrid hosts. This state of maturation arrest merged imperceptibly with the development of frank lymphomas, which remained confined to the thymic cortex for sonic weeks before invading the medulla, the capsule, and adjacent structures. I t was then noted that the normal thymus of 1-day-old C57BL mice differs strikingly from the adult pattern, by virtue of having almost a "pure culture" of large and medium lymphocytes, with a very high mitotic index, occupying the outer zone of the cortex and comprising from one-third to one-half of its entire width (Fig. 1). During the first days of life, this zone of immature cells differentiates rapidly and becomes indistinguishable from the inner cortical zone, composed predominantly of mature small lymphocytes. By 10 days Vol. ~1, S e p t e m b e r 1961 of age, the immature cell zone is only about one cell layer thick, and by s days it is gone (Fig. 2; 32). Since the only two hosts known to be susceptible to the strain C57BL lymphoma agent were (a) newborn mice and (b) irradiated (unshielded) mice, and since both of these revealed a similar and striking prevalence of immature lymphoid cell forms, it was postulated (~0) t h a t the viral agent may have a particular affinity or requirement for these undifferentiated cells as hosts and that it is not able to proliferate at an equilibrium-sustaining level leading to tumor development except in the presence of an adequate reservoir of such immature cells. If this view is correct, then the role of x-radiation in the induction of these tumors in strain C57BL mice may be plausibly attributed to its twofold action in (a) injuring the adult thymus, thus exciting a stimulus for regeneration which recapitulates the neonatal thymic lymphoid cell differentiation sequence and (b) injuring the bone marrow, thus interfering with the capacity of marrow cells to promote rapid regeneration (22) and maturation in the radiationinjured thymus. Further support for the view that the lymphoma viruses may have a particular requirement for immature lymphoid cells may now be found in the observation by Arnesen (~) and Metcalf (~5) t h a t the high-leukemia AK strain, in which irradiation is unnecessary for lymphoma development, is constitutionally adrenal-insufficient, with a concomitantly long-sustained thymic hyperplasia, in which immature cells are continuously abundant. Finally-, the low-leukemia C3H strain, which is susceptible to the freshly extracted AK lymphoma virus only during the postnatal period, has now been shown by the ingenious quantitative studies of Axelrad and Van der Gaag (3) to exhibit a transient zone of immature cells in the outer cortex, similar to t h a t in newborn C57BL mice, during a brief postnatal period which correlates well with the time of susceptibility to virus inoculation. Although such evidence is based on correlation, rather than direct experimental proof, these observations remain consistent under such diverse conditions as to make their chance occurrence highly unlikely. I t may therefore be concluded that the 1~o. 1.--Normal thymus of 1-day-old C57BL mouse. Note the zone of large, pale-stainingimmature lymphoid cells in the subeapsular region of the cortex. Darker-staining, mature small l~nphocytes are abundant in the inner cortex. X 1~5. FIo. ~.~Normal thymus of ~0-day-old C57BL mouse. The zone of large, pale-staining cells has disappeared. X 125. Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1961 American Association for Cancer Research. KAPLAN--Cell Differentiation and Virus Carcinogenesis state of differentiation of the lymphoid cells of the host is a significant determinant of susceptibility to this family of viruses. It seems likely that differentiation and/or physiological activity of target tissues will also prove to be important in the case of other oncogenic viruses, and that systematic investigations along these lines will be rewarding. REFERENCES 1. ALLmON,A. C., and ARMSTRONG,J. A. Abnormal Distribution of Nucleic Acid in Tissue Culture Cells Infected with Polyoma Virus. Brit. $. Cancer, 14: 313-18, 1960. 2. ARNESEN,K. The Adrenothymic Constitution and Susceptibility to Leukemia in Mice; a Study of the AKR/O and WLO Strains and Their Hybrids. Acta Path. et Microbiol. Scandinav. Suppl., 109:5-95, 1956. 3. AXELRAD,A. A., and VAN DER GAAG,H. C. Effect of Age on Thymus Cell Number, Histology, and Susceptibility to Induction of Lymphoma by Gross Passage A Virus in CaH,/Bi Mice. Proc. Am. Assoc. Cancer Research, 3:207, 1961. 4. BATHER,R. The Nucleic Acid Content of Partially Purified Rous No. 1 Sarcoma Virus. Brit. J. Cancer, 11:611-19, 1957. 5. BERNHARD,W. The Detection and Study of Tumor Viruses with the Electron Microscope. Cancer Research, 20:71227, 1960. 6. BONAR, R. A., and BEARD, ,1. W. Virus of Avian Myeloblastosis: XII. Chemical Constitution. J. Nat. Cancer Inst., 23:183-97, 1959. 7. BONAR,R. A. ; WEINSTEIN,D.; SOMMER,~'. R.; BEARD,D. ; and BEARD, J. W. Virus of Avian Myeloblastosis. XIII. Morphology of Progressive Virus-Myeloblast Interactions in Vitro. Nat. Cancer Inst. Monograph 4, pp. 251-90, 1960. 8. CARNES,W. H., and KAPLAN,H. S. Relation of Lymphoma Induction to Cortical Differentiation in Thymic Grafts in Irradiated C57BL Mice. Fed. Proc., 15:510, 1956. 9. CRAWFORD,L. 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The Role of Cell Differentiation as a Determinant of Susceptibility to Virus Carcinogenesis Henry S. Kaplan Cancer Res 1961;21:981-983. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/21/8/981.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1961 American Association for Cancer Research.