The overall objective of this proposal is to investigate the basic cellular and molecular mechanisms of radiation responsiveness of quiescent cells. Quiescent cells exist in plateau phase monolayer cultures as well as multicellular spheroids and solid tumors and they have been implicated to be preferentially capable of repairing potentially lethal radiation damage (PLD). However, these cells have until recently been extremely difficult to identify and quantify, and the inherent radiation sensitivity of quiescent cells still remains unresolved especially for human tumor cells. Using the centrifugal elutriation and flow cytometry techniques recently developed in our laboratory to significantly enrich the quiescent cells, we propose to assess from plateau phase cultures and spheroids for: a) the radiation sensitivity of isolated quiescent tumor cells; b) radiation-induced alterations in the dynamics of recruitment from quiescent to proliferating compartments; c) the possible differential capacity of quiescent tumor cells for the repair of radiation-induced PLD; and d) the role of specific DNA- related ezymes (including one newly discovered enzyme) in the molecular mechanism(s) of repair. Relationships between cellular and molecular repair in quiescent cells will be studied by comparing the repair kinetics of DNA (strand breaks, DNA- protein crosslinks, DNA-DNA crosslinks) and chromosome damage to cell recovery at the same radiobiologically relevant doses. These studies will be performed with alkaline elution, DNA unwinding and premature chromosome condensation techniques. The results using these assays will be interrelated to determine which types of damage can be measured and related to radiation sensitivity of quiescent cells. Subsequent studies using inhibitors of DNA replication and repair will be performed on quiescent and proliferating cells and the effect of these inhibitors on the kinetics of the repair of radiation-induced PLD will be evaluated. Inhibitors are chosen to specifically inhibit DNA polymerases alpha, beta, delta I, delta II, topoisomerase I and II. A greater understanding of the mechanisms of quiescent cell radiation responsiveness, the kinetics of recruitment of quiescent to proliferating status, and the role of quiescent cells in the recovery of PLD, may provide basic information relevant to both radiation therapy and chemotherapy.
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