Differences in heat-induced cell killing as determined in three mammalian cell lines do not correspond with the extent of heat radiosensitization.

Three different cell lines, Ehrlich ascites tumour (EAT) cells, HeLa S3 cells and LM mouse fibroblasts, were used to investigate whether or not the extent of heat killing (44 degrees C) and heat radio-sensitization (44 degrees C before 0-6 Gy X-irradiation) are related. Although HeLa cells were the most heat-resistant cell line and showed the least heat radiosensitization, we found that the most heat-sensitive EAT cells (D0, EAT = 8.0 min; D0, LM = 10.0 min; D0, HeLa = 12.5 min) showed less radiosensitization than the more heat-resistant LM fibroblasts (TERHeLa less than TEREAT less than TERLM). Therefore, it is concluded that the routes leading to heat-induced cell death are not identical to those determining heat radiosensitization. Furthermore the inactivation of DNA polymerase alpha and beta activities by heat seemed not to correlate with heat survival alone but showed a positive relationship to heat radiosensitization. The possibility of these enzymes being a determinant in heat radiosensitization is discussed.

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol.

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.

DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments.

Possible relations between hyperthermic inactivation of alpha and beta DNA polymerase activity and hyperthermic cell killing or hyperthermic radiosensitization were investigated. Ehrlich Ascites Tumor (EAT) cells and HeLa S3 cells were treated with fractionated doses of hyperthermia. The heating schedules were chosen such that the initial heat treatment resulted in either thermotolerance or thermosensitization (step-down heating) for the second heat treatment. The results show that for DNA polymerase activity and heat radiosensitization (cell survival) no thermotolerance or thermosensitization is observed. Thus hyperthermic cell killing and DNA polymerase activity are not correlated. The correlation of hyperthermic radiosensitization and DNA polymerase activity was substantially less than observed in previous experiments with normotolerant and thermotolerant HeLa S3 cells. We conclude that alpha and beta DNA polymerase inactivation is not always the critical cellular process responsible for hyperthermic cell killing or hyperthermic radiosensitization. Other possible cellular systems that might determine these processes are discussed.

Strand break repair, DNA polymerase activity and heat radiosensitization in thermotolerant cells.

HeLa S3 cells were made thermotolerant by 'chronic' (5 h at 42 degrees C) or 'acute' (15 min at 44 degrees C followed by 5 h at 37 degrees C) heat treatments. Cell survival, repair of radiation-induced DNA strand breaks, alpha and beta DNA polymerase activity and radiation sensitivity following hyperthermia were all measured in both control and thermotolerant cells. The ability to repair DNA strand breaks correlated well with cell survival following hyperthermia. Hyperthermic inhibition of strand break repair was reduced in thermotolerant relative to control cells, although the thermal tolerance ratios for repair were less than for hyperthermic cell killing. Both radiosensitization and DNA polymerase inactivation by hyperthermia were only slightly reduced in thermotolerant relative to control cells. Hence a poor correlation was found between these two parameters and hyperthermic cell survival. For all heat treatments applied, alpha and beta DNA polymerase activity correlated well with the extent of hyperthermic radiosensitization.

Heat-induced alterations in DNA polymerase activity of HeLa cells and of isolated nuclei. Relation to cell survival.

The activity of DNA polymerase alpha and beta was assayed in heated HeLa S3 cells as well as in nuclei isolated from these cells. The enzyme activity as measured in cells and in nuclei has been compared with the extent of cell survival after the different hyperthermic doses. It was found that although the activity of the cellular DNA polymerases was related to cell survival after single heat doses, no correlation was found when thermotolerant cells were heated. When the activity of the DNA polymerases was determined in nuclei isolated from non-heated and heated cells, more polymerase activity was found in the nuclei of the heated cells. However, the heat sensitivity of DNA polymerase activity was the same for nuclei isolated from control, pre-heated and thermotolerant cells. Heat protection of polymerase activity by erythritol and sensitization by procaine was found when cells, but not when nuclei, were heated in the presence of these modifiers. It is concluded that (the nuclear bound) DNA polymerases are not to be considered as key enzymes in cellular heat sensitivity of HeLa S3 cells.

The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation.

Strand breaks were detected in the DNA of Ehrlich ascites cells as well as in HeLa S3 cells directly after 1-5 hr at 43-45 degrees C by the use of the unwinding in high salt/hydroxylapatite method. The strand breaks found could not be attributed to the decay of incorporated tritiated thymidine. When the cells were incubated at 37 degrees C after the hyperthermic treatments, the amount of strand breaks formed remained at a constant level. Hyperthermia inhibited the repair of "radiation-induced" strand breaks. The repair curves obtained this way show a heat-dose-dependent decrease of the relative weight of the fast component of repair. Similar repair curves of "radiation-induced" strand breaks could be obtained by mixing heat inactivated and vital control cells prior to irradiation. In the latter case, however, the DNA repair was inhibited to a greater extent for identical levels of cell survival. The possible underlying molecular mechanisms are discussed.

Inhibition of repair of radiation-induced strand breaks by hyperthermia, and its relationship to cell survival after hyperthermia alone.

Ehrlich ascites cells growing in vitro have been used to investigate the influence of hyperthermia on the formation and repair of DNA strand breaks after X-irradiation. Different heat pretreatments were given immediately prior to a dose of 6 Gy of X-rays. When a temperature of 42 degrees C was used, up to 4 hours of pretreatment had only a slight inhibitory effect on the repair of DNA strand breaks induced by radiation. At a temperature of 43 degrees C progressively more inhibition was observed with longer treatment times. This was also the case for temperatures of 44 degrees C and 45 degrees C. When the treatment times at 43-45 degrees C were longer, strand breaks in DNA were induced by the hyperthermic treatment alone. Under these conditions almost no repair was found of strand breaks induced by a subsequent radiation dose. The data obtained strongly suggest a correlation between the effect of the hyperthermic treatment alone on cell survival and the kinetics of repair of strand breaks in DNA as induced by radiation.