Treatment of normal and tumor cells with DNA-damaging agents induces not only cell death but also rapid and irreversible proliferation arrest accompanied by phenotypic markers of cell senescence. This accelerated senescence response is positively regulated by p53 and p21 but can also occur in p53- or p21-deficient cells. p21-overexpressing tumor cells and normal senescent fibroblasts show increased expression of proteins with paracrine tumor-promoting activities. HCT1 16 colon carcinoma cells that became senescent after doxorubicin treatment were separated from proliferating cells and analyzed by differential cDNA microarray hybridization. Accelerated senescence was associated with downregulation of multiple genes that act in mitosis, DNA replication, or RNA processing. Senescent cells also showed increased expression of many intracellular and secreted growth inhibitors, as well as secreted mitogenic and angiogenic factors, proteases, extracellular matrix components and receptors, and different transcription factors. In the proposed study, p53-I- and p21-/- derivatives of HCT1 16 cells will be used to determine which of the genes that show altered expression in senescent cells are regulated through p53 or p21. Genes that show similar changes in the senescent cells of different tumor types will be identified as potential markers of accelerated senescence. HCT1 16 lines will be selected for improved recovery from drug-induced growth arrest and analyzed for changes in gene expression. Growth-inhibiting and transcription factor genes that show altered expression in senescent cells will be tested for their effects on cell growth, drug response and expression of other senescence-associated genes. Promoter sequences of genes that are up- or downregulated in accelerated senescence will be analyzed, and transcription factor binding sites shared by co-regulated genes will be identified. The role of candidate regulatory sites will be tested by promoter mutagenesis and transient transfection assays. Stably transfected cell lines with a reporter gene transcribed from the promoters of different senescence-associated genes will be developed as the basis for high-throughput screening of compounds that affect gene expression in accelerated senescence. The ability to modulate growth arrest and paracrine effects in accelerated senescence of tumor cells may help to improve the efficacy and decrease the side effects of cancer treatment.
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