Normal human cells have a limited lifespan in culture and undergo replicative senescence whereas tumor cells commonly grow continuously and are immortal. Hence cellular senescence also functions in part as growth/tumor suppressor in a particular aspect of carcinogenesis. They have been exploiting SV40-transformed human fibroblasts (SV/HF) as a model to understand the molecular mechanism of immortalization. Utilizing matched sets of non-immortal (preimmortal) and immortal SV/HF, they eliminated changes in viral gene functions as being responsible and, therefore, sought to identify relevant cellular genes including an essential primary effector termed SEN6 (by molecular genetic approaches) and secondary effectors (by differential screening of a cDNA library) in the period of the last grant award. Each approach has resulted in the identification of a gene which had not previously been extensively studied or considered associated with senescence or immortalization. They propose to investigate their effects on normal cells and SV/HF. They have determined that a locus, which is inactivated upon immortalization, maps to 6q27 and have identified a gene in this region which has growth suppressor activity and has been mutated in immortal SV/HF, consistent with being SEN6. In addition, they have isolated a cDNA for a gene whose expression is reduced in immortal SV/HF (as compared to preimmortal SV/HF and normal cells) which has sequences indicative of a protein serine/threonine phosphatase (PSP). Proposed experiments will determine whether each is effective as a growth suppressor in SV/HF and other tumor-derived immortal cell lines, and assess the mechanism involved. Both SEN6 and PSP have properties which indicate that they might act at different stages of a common pathway involving MAP kinases so they will initially focus their attention on changes in those pathways in immortal SV/HF and upon overexpression (under the control of a regulatable promoter) of introduced sequences for the candidate for SEN6 and/or the protein phosphatase. They anticipate that the results will generate new insights into cellular senescence and its role in carcinogenesis.
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