Harry Rubin, D.V.M. - US grants

Heterogeneity of cells in tumors appears to be a major obstacle in the successful treatment of cancer. It is also of importance in understanding the progression of cells to autonomous growth including metastasis. The highest estimates of the rate of variation responsible for heterogeneity have been on the order of one heritable alteration per 10[unreadable]5[unreadable] cell divisions. In the past year we were able to isolate five clones among the progeny of a Balb/3T3 cell which had just undergone spontaneous transformation and to characterize their properties. We found that each of the clones could be distinguished from the others by morphology, colony-forming efficiency in agar (CFEag), and rate of tumor formation in nude mice. The implication of this finding is that cultured cells are so unstable soon after transformation that essentially every cell may differ from the others in one or more characteristics, i.e., that there is a truly radical heterogeneity. We have also studied the evolution of further changes in these properties among the five clonal populations over a 6-month period and conclude that the type of variation which led to the initial differences among the clones could also be responsible for continuing change akin to tumor progression. Since the frequency of this type of variation is very high and involves several properties in a quantitative but uncoordinated way, it is unlikely to be caused by conventional genetic mutations. Rather, it is likely to be an epigenetic event. This type of variation is also likely to play a role in our second major finding, which is that some properties of transformed cells are altered during tumor formation in the nude mouse and again when the tumor cells are cultured. The former contradicts the generally accepted view that cells are not changed by passage through nude mice. We find our example, that a decrease in CFEag of more than 100-fold, is frequently observed during tumor formation. Cells from some tumors regain their original high CFEag after repeated passaging in culture but others do not. That is, there is a heterogeneity of response, some changes being stable and others unstable. The evidence to date indicates the alterations in cells during tumor formation and subsequent cultivation are basically epigenetic in nature and similar in type to those described above which occur after transformation. The results promise to provide new ground for understanding the origin and progression of cancer. (J)

Inoculation into nude mice of spontaneously transformed Balb 3T3 cells has revealed that tumor development is a problem of cellular sociobiology, ecology and heritable variation. This is manifest in the observations that a minimum of 1000 cells is required to initiate a tumor and many such "low dose" tumors are inordinately slow in development until a burst of rapid growth occurs. The sequence is pathognomonic of tumor progression. By contrast, the innoculation of 10/6 cells uniformly results in rapid and sustained development of tumors. A similar population dependent multiplication of these cells can be produced in culture by using a medium deficient in specific growth factors, e.g., by substituting chicken serum for the conventionally used calf serum. The similarity between the in vivo and in vitro situations has pointed up the likelihood that the interstitial fluid, which is a filtrate of plasma rather than serum, and which serves as the medium for cells inoculated in subcutaneous tissue of the mouse, is even more deficient in growth factors than is plasma. Tumor development, therefore, depends on the microenvironment including cellular interactions and on heritable variation in the cells. Our plan is to study all the factors which contribute to cell multiplication and tumor progression in the easily quantified situation of cell culture. Alternative strategies will be applied for simulating the growth factor deficient environment of interstitial fluid, including the use of plasma, lymphatic fluid and combinations of growth factors used in commercial serum- free defined medium. The nature of the specific deficiency in chicken serum will be elucidated, and the effect of unfavorable growth environments on the frequency of heritable cell variation will be measured. A study will be made of the interactions among cells which permit rapid multiplication of large numbers of cells in a medium deficient in specific growth factors. Any conditioning on autocrine growth factors released by the cells will be characterized. The work in culture will be coordinated with quantitative experiments on tumor development in nude mice. The aim is to sort out the various processes, factors and interactions which contribute to tumor progression and thereby develop a rationale for its eventual control.

DESCRIPTION (provided by applicant):The broad, long-term objective of the project is to provide a solid foundation of knowledge about the cellular biology of cancer and aging. The first part of the project involves a critical analysis of the relationship between the limited lifespan of cells in culture and the long term replication of cells in the organism, particularly of the stem cells. This analysis will encompass the role of telomerase and telomeres in limiting the lifespan of human cells in culture and the relevance of these components to cell survival, differentiation and cancer in the organism. It will include consideration of the safety of the proposed use of artificially immortalized cells for therapy in humans. The second part of the project will compare the genetic stability of cells in culture, in the organism and in cancer. The role of tissue architecture in maintaining homeostasis in the organism will be extensively reviewed. The weakening of this top-down control with age will be considered in the origin of various chronic diseases associated with the aging process. The method is to use close, intensive reading of both the primary historical and the contemporary scientific literature relevant to the subjects. Emphasis will be placed on integrating the biological foundation of each field, often assumed but only superficially understood, with recent molecular findings to develop a more comprehensive, theoretical foundation of cellular change in cancer and aging. Several extensive reviews will be published m leading scientific journals to be followed eventually by unification into a monograph. Amalgamation of these and related subjects in a comprehensive volume is contemplated.