Induction of cell senescence is a prominent response of human tumor tissues to chemotherapy and radiation. Although senescent cells do not divide, they secrete many bioactive proteins implicated in cancer and other chronic diseases. Some of these proteins stimulate while others inhibit the growth and survival of neighboring tumor cells. Expression of cyclin-dependent kinase inhibitor (CDKI) proteins (such as p21/Waf1), which are frequently upregulated in senescent cells, enhances the production of tumor-promoting factors. Different types of senescent normal and tumor cells were found to produce different tumor-promoting or tumor-inhibiting paracrine activities. Understanding the spectra and determinants of cancer-relevant activities of senescent cells should help in developing biomarkers and strategies for managing the impact of tumor senescence on the long-term treatment outcome. In the first specific aim, HCT116 colon carcinoma and HT1080 fibrosarcoma cell lines will be treated with three different anticancer agents. Senescent and proliferating fractions of surviving cells will be separated by flow sorting. These fractions will be assayed for the production and secretion of bioactive proteins encoded by genes that are induced at the RNA level in senescent cells. In the second specific aim, senescent tumor cells will be tested for paracrine activities that affect tumor cell growth, survival, drug resistance, invasion or angiogenesis. Paracrine activities increased in senescent cells will be correlated with the secretion of proteins capable of conferring such activities, and the role of such proteins will be tested using specific inhibitors. The third specific aim asks whether inhibition of the CDKI-activated transcriptional pathway alters the secretory patterns of senescent tumor cells in favor of tumor-suppressing activities. For this analysis, secreted proteins and paracrine activities will be compared between senescent populations of HCT116 cells and their derivatives where CDKI pathway is inhibited. The fourth specific aim investigates production of bioactive proteins by senescent HCT116 cells grown in nude mice as xenografts and rendered senescent by irradiation in vivo. In addition, xenograft coimplantation assays will be used to investigate the effects of senescent HCT116 cells with different CDKI pathway status on tumor growth in vivo. The proposed program should help in elucidating different effects of tumor senescence on the outcome of cancer treatment and on its long-term implications for the patient's health.
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