The role of cell proliferation status on radiobiological parameters will be studied using EMT6/Ro tumors in vitro and in vivo. We propose first to evaluate the proliferating (P) and quiescent (Q) tumor cell compartments under normal growth conditions with monolayer and multicell spheroid cultures to be utilized for the in vitro investigations. The P and Q cells will be identified and quantitated using several assays including: a) two-step acridine orange (AO) staining and flow cytometric analysis; b) "chromatin conformation" as assessed by acid AO staining; c) nucleolar morphology; and d) 3H-thymidine labelling and autoradiographic analysis. The results using these assays will be interrelated to probe for possible tumor cell heterogeneity, particularly in the cell compartment. To enrich for Q cells, these studies will be performed in conjunction with cell separation procedures including centrifugal elutriation, density qradient separation and selective spheriod dissociation. Biochemical measurements will be performed on the Q enriched spheroid and tumor cells, and the results will be compared with those from Q cells obtained by serum deprivation. Subsequent radiobiological investigations are proposed to assess for: a) the radiation sensitivity of isolated and in situ Q tumor cells; b) radiation-induced alterations in P and Q compartment dynamics; c) the possible differential capacity of Q tumor cells for the repair of radiation-induced potentially lethal damage (PLD) and d) the role of radiation-induced alterations in cell proliferation status on the cellular sensitivity of fractionated radiation treatment. These investigations should lead to a more precise understanding of the biology of Q cells, and of tumor heterogeneity in general. Also, the radiobiological studies proposed may provide basic information relevant to clinical radiation therapy.