The limited ability of cells to proliferate is a fundamental property of normal diploid human cells both in tissue culture and in vivo. Tumor cells in culture have become immortal, and there is increasing evidence that escape from the normal mechanisms of cellular senescence may constitute a critical step in the multistage process of tumorigenesis. Genes originally discovered as tumor suppressors (pRB and p53) may be involved in regulating cellular senescence, and it is possible that additional molecules discovered as part of the present application to play roles in cellular senescence will prove to be important in cancer. The overall objective of this project is to define the molecular basis of cellular senescence, with the long term goals of applying this knowledge both to the biology of cancer as well as to the determination of whether or not cellular senescence plays a contributing role in organismal aging. Human telomeres (the ends of chromosomes) are composed of multiple repeats of the sequence TTAGGG. In the absence of the enzyme telomerase, which adds these sequences to the 3' end of DNA molecules, the telomeres shorten every time a cell divides. One possibility to be tested is that the regulatory loci that control the M1 (Mortality Stage 1) mechanisms of cellular senescence are located adjacent to telomeres and that their expression is modified by telomere length. Induction of the M1 mechanism constitutively activates the tumor suppressor proteins pRB and p53 into their antiproliferative states. Blocking M1 with agents that bind pRB and p53 permit cells to continue to divide until most telomeric repeats are lost, which causes M2 (Mortality Stage 2). Escape from M2 may be due to the inactivation of factors in the pathway that represses telomerase, and the derepression of telomerase then results in an immortal cell line.
The specific aims of this application are directed towards establishing molecular proof for these hypotheses by: 1) Cloning and characterizing expressed genes in the subtelomeric DNA, showing that their expression is regulated by telomere length, and demonstrating their functional roles in the regulation of M1; 2) Using insertional mutagenesis to clone M2 genes, and investigating their functional role in M2 and the derepression of telomerase; and 3) Developing additional evidence that M1 represents a cellular program common to different lineages, which involves a senescence specific transcriptional regulation of a variety of genes in addition to those controlling proliferation. The identification of the specific molecular events controlling both M1 and M2 should permit detailed studies of their functional roles in oncogenesis and the degenerative pathologies of aging.
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