The goal of this project is to determine radiobiological and molecular mechanisms that regulate the entry of quiescent cells (G-0) into proliferative phases of the cell cycle. Quiescent cells often constitute a large portion of the total tumor cell population and remain clonogenic after radiation treatment. Thus, measurement of growth fraction and the study of recruitment of quiescent cells into proliferation compartment is essential for understanding the responses of tumors to radiation therapy. Using monoclonal antibodies made to proliferation associated nuclear antigens, PCNA and Ki-67, we propose to determine 1) the kinetics of radiation induced redistribution of quiescent cells, and 2) the changes in PLD and SLD repair following the recruitment of cells from quiescent to proliferating compartments. The role of specific antisense RNA that binds to the PCNA mRNA and inactivates the protein synthesis will be evaluated to determine if PCNA is required for the progression of quiescent cells into proliferating cycle. These studies will be performed by introducing antisense RNA to the cells, and then measure its effects on level of PCNA protein and mRNA, and on progression of cells from quiescent to proliferating compartments. Subsequent measurement of PCNA protein and mRNA from irradiated quiescent cells will determine if PCNA is involved in the radiation induced recruitment of quiescent cells. Suppression of PCNA synthesis using an antisense oligonucleotide will be performed to determine whether PCNA expression is necessary for efficient repair of radiation induced potentially lethal damage. We also plan to clone cDNA and express the mRNA for a human tumor specific, proliferation associated nuclear antigen, Ki-67. Northern hybridization and Western blot analysis using the probe prepared from the full length cloned cDNA of Ki-67 will be performed to evaluate the transcriptional and translational regulation on the radiation induced recruitment of quiescent cells in human tumors.
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