Normal human diploid fibroblasts (HF) undergo replicative senescence at the end of their limited lifespan and acute senescence in response to over expression of oncogenes, suggesting senescence is a tumor suppressor mechanism. We have demonstrated that transformation of HF by replication-defective SV40 sequences results in extension of lifespan (which terminates in crisis as apoptotic cell death) and promotes continuous cell proliferation, i.e. immortalization. We have developed an experimental system involving matched sets of SV40-transformed non-immortal HF (SV/HF) and their immortal derivatives to identify cellular genes involved in these processes. We have localized a gene (termed SEN6) with features of a growth suppressor to a region at 6q27, whose inactivation is involved in the immortalization of SV/HF. A series of complementary strategies are proposed to identify SEN6. We will test which of three mechanisms shown to result in inactivation of tumor suppressor genes in humans is (are) responsible; namely, loss of heterozygosity and mutation, haploinsufficiency, and/or gene silencing. Candidate genes for SEN6 identified using the Human Genome Database and the expression pattern of encoded mRNAs and ORF will be tested first by DNA-mediated gene transfer into non-immortal SV/HF for promotion of immortalization by small interfering RNA(s). We will initially assess the role of DLL-1, a ligand for the Notch receptor-signaling pathway, as a candidate based on recent information on the role of the Notch-1 in transformed cells and favorable experimental aspects of DLL-1 (moderate mRNA levels in SV/HF and availability of antibody to DLL-1). We have furthermore identified an SV40-transformed human cell line, which has a single copy of chromosome 6 (mono6) for direct test of the role of haploinsufficiency or as a target for mutagenesis of haploid SEN6. Alternatively, we will exploit YAC and/or BAC transfer to further localize the region encoding SEN6. Toward that goal, we have demonstrated that manipulation of the yeast host can markedly increase DNA transfer to human cells and propose to develop the approach further. Altogether these studies are expected to result in isolation of sequences encoding SEN6 and enhance our understanding of mechanisms of senescence and carcinogenesis as well as transformation/immortalization of SV/HF.
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