Effect of Hyperthermia on CHO DNA Polymerases α and β

Abstract

The heat sensitivity of polymerase β (pol-β) has been studied in an attempt to assess the molecular lesions for heat killing and heat radiosensitization. Viable cells were heated, and then polymerase activity was determined in crude cell extracts by measuring the incorporation of $[{}^{3}{\rm H}]{\rm TMP}$ into an exogenous, activated calf thymus DNA. The thermal inactivation of pol-β activity was shown to correlate with hyperthermic cell killing and radiosensitization. Pol-β activity decreased exponentially when cells were heated at temperatures of 43°C and above, and a 10-min 45.5°C heat shock reduced pol-β activity by 80%. Also, pol-β activity showed a 3-fold greater heat sensitivity than polymerase α (pol-α) activity at 43.5 and 45.5°C. When cells were heated at temperatures of 42.2-42.5°C, pol-β activity decreased, but as for cell killing, tolerance to any further treatment was observed after 2 hr of heating. Thermal tolerance also was observed as a decrease in the rate of pol-β inactivation at 45.5°C if cells were given either a preconditioning heat treatment at 42.2°C for 6 hr, or 45.5°C for 10 min followed by 24 hr at 37°C. Furthermore, when cells were heated at 42.2°C in the presence of 7 mM procaine-HCl, both cell killing and loss of pol-β activity were greatly enhanced. Finally, the return of pol-β activity after thermal treatment correlated with the decrease in radiosensitization as evidenced by an increase in survival as a treatment (10 min, 45.5°C) and subsequent X-ray treatment were separated by increasing amounts of time at 37°C. For example, during incubation at 37°C after heat treatment, pol-β activity and the logarithm of survival increased over 4-fold, while pol-α activity increased only 1.4-fold to 75% of its control activity. Also, a decrease in X-ray-induced unscheduled DNA synthesis following heat treatment at 45.5°C correlated with the loss of pol-β activity at 45.5°C. These results support the hypothesis that the loss of pol-β activity may be a mechanism for heat killing and/or heat radiosensitization.