Fractionation of Combined Heat and Radiation in Asynchronous CHO Cells: II. The Role of Cell-Cycle Redistribution

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Abstract
Asynchronous Chinese hamster ovary (CHO) cells treated with conditioning hyperthermia (10 min at 45.degree. C) followed immediately by X-irradiation with 4 Gy [grays] at room temperature display a decreased radiation response when either irradiated, or heated and irradiated again 24 h later [a potential cancer therapy procedure]. In addition, conditioning by 4 Gy of X-radiation alone followed at 24 h by the combined heat and radiation treatment also resulted in a larger radiation Do [mean lethal dose]. Since the differential cell-cycle responses to heat- and X-ray-induced lethality are well established for CHO cells, the potential role of cell-cycle redistribution in the observed radiation desensitization was investigated by flow cytometry. At 24 h after the combined treatment, the fraction of the cells found in the G2 + M portion of the DNA histograms was about three times that of the controls and there was a concomitant reduction in both the G1 and S fractions. Similar observations were made after the conditioning heat treatment alone but cells irradiated with 4 Gy only did not display any marked redistribution at 24 h. A comparison of harsh acriflavin-Feulgen vs. gentle mithramycin DNA-staining techniques as well as Ficoll centrifugation to remove dead and dying cells indicated that the DNA histograms generated at 24 h after conditioning heat and/or radiation represented the population present at the time of second treatment. Analysis of unsorted and sorted cells for mitotic figures and doublets indicated that the accumulated cells in the G2 + M area of the histogram were in G2. Comparisons of these cell-cycle redistributions to the observed alterations in radiosensitivity (Do) lead to the conclusion that they are not directly related. Rather, the common parameters for alterations in sensitivity appear to be the initial radiation exposure combined with heat given simulataneously or 24 h later. Further experiments designed to monitor DNA damage and repair processes following irradiation may elucidate the role of hyperthermia in the alterations in radiosensitivity seen with these particular protocols.