Normal human cells in vitro exhibit a stringent limitation of division capacity in contrast to tumor-derived and virus-, carcinogen- or irradiation- transformed cells that can divide indefinitely (immortal). We do not yet understand the mechanisms that limit the division potential of normal human cells or the changes occurring to yield immortal cells. However, from cell hybrid studies we have found that the immortal phenotype results from recessive changes in normal cell growth control. We have exploited this fact to separate twenty-eight different immortal cell lines into four complementation groups for indefinite division, indicating that there are at least four paths to immortality. A fortuitous result of this study was the assignment of seven of eight immortal SV40 transformed cell lines of different origin to the same complementation group. This indicated that SV40 immortalizes different human cells via the same mechanisms. Since many studies had indicated that the presence and expression of the SV40 genome was not sufficient to maintain immortalization in human cells, we questioned whether any viral genes were required to maintain the immortal state. We have found that loss of active T antigen (T ag) results in loss of proliferation, indicating that some function(s) of T ag is required. In this proposal we plan to identify this function(s) of T ag by the use of mutants of T ag and genes that share sequence and functional homology with T ag. Existing data indicates that immortalization of human cells by SV40 also involves an event that occurs at very low frequencies (<1 in 10-7) when the cells enter a stage called crisis. To remain consistent with our observation that immortality is recessive, we hypothesize that this event involves inactivation of a cellular gene that suppresses cell growth, either directly or by suppressing other genes. We propose to identify this gene by two approaches: retroviral insertional mutagenesis, restriction fragment length polymorphism (RFLP) analysis of matched pairs of normal and immortal SV40 transformed cell. Results from these studies should greatly increase our understanding of the genes involved in growth regulation as well as the mechanisms by which DNA tumor viruses such as SV40 affect these genes.
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