Human diploid cells derived from normal tissue undergo a characteristic pattern of growth in cell culture, ending with a non-replicative phase. Models of cellular aging have been developed which suggest analogy between this pattern and behavior of the organism in vivo; hence, the term senescence. Human diploid fibroblasts (HF) which have been transformed in vitro by the DNA virus SV40 have been found to overcome this phenomenon (i.e., immortalize) at low frequency. The investigators have isolated a series of SV40- transformed HF using origin-defective viral genomes containing either a wild-type T antigen (SV/HF) or that of tsA58 (SVtsA/HF). In each system, they have subsequently isolated immortalized derivatives for comparative studies. The data are most consistent with a 2-step model for immortalization. SV40 large T antigen(LT) function is required in both stages, as demonstrated by temperature-dependent (ts) cell proliferation in concordance with ts LT function in preimmortal and immortal SVtsA/HF-A cells. At step 1, LT functions as a mitogenic agent, in large part through its interaction with cellular proteins as pRb and p53. Step 2, which is specific to immortalization, involves changes in functions not directly dependent on LT. A rearrangement on the long arm of chromosome 6 (6q21) consistent with inactivation of a suppressor gene has been found to be specifically associated with step 2. The applicant has identified changes in gene expression through differential hybridization with cDNA libraries of preimmortal and immortal SV40- transformants and propose to identify the genes involved. The investigator will focus initially on those genes whose expression is reduced in several matched immortal transformants as compared to their parental preimmortal transformants, most consistent with the recessive nature of the latter in cell hybrids. Strategies are also presented to identify the responsible gene on 6q. In both cases, the genes identified will be assessed for growth suppression of immortal cell lines, a phenomenon relevant to both senescence and carcinogenesis. The investigators have also found that the cellular proto-oncogene c-myb is induced in an immortal HF cell line transformed by an intact but not a truncated LT. They propose to assess the viral and cellular functions responsible because of the importance of c-myb to cell proliferation and as a prototype of genes overexpressed in immortal SV40-transformants.
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