Molecular Studies on the Hyperthermic Inhibition of DNA Synthesis in Chinese Hamster Ovary Cells

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Abstract
The hyperthermic inhibition of cellular DNA synthesis was studied to elucidate the mechanism involved in killing S-phase cells. A time-temperature dependence for inhibition of DNA synthesis similar to that seen for cell survival was found in CHO cells. This inhibition was primarily an effect on the replication process although some inhibition of both [3H]TdR [thymidine] transport and endogenous synthesis of TTP was observed. Details of the inhibitory effect at the replicon level were studied for a 15-min-45.5.degree. C treatment, which reduced both DNA synthesis and survival of S-phase cells by 70-90%. Alkaline sucrose gradient analysis of the heat effect on cellular chain elongation of 3H-nascent DNA made immediately before treatment revealed a transient delay during heating and for about 5 min thereafter in the elongation and ligation of this nascent DNA into molecules of all sizes ranging from replicons to clusters. As incubation continued for up to 150 min after heating, a 2- to 3-fold reduction in the rate of chain elongation was also observed along with a decrease in the extent that nascent DNA was ligated into molecules > 120S. The initial delay in ligation mentioned above may cause the delay observed in the conversion of nascent DNA containing single-stranded regions into double-stranded DNA, as shown by BND[benzoylated naphthoylated DEAE]-cellulose chromatography, with no apparent heat effect on the number of single-stranded regions in 14C-bulk DNA. When DNA synthesized immediately or within 30 min after heating was analyzed from cells chased up to 120 min after labeling, a 3- to 5-fold reduction in the rate of chain elongation into molecules of all sizes was again observed, along with a 1.8-fold increase in the number of single-stranded regions present in the 3H-labeled nascent DNA synthesized after heat treatment. At later postheat incubation times (.apprx. 2-15 h), the DNA synthesized was predominantly around 120S in size, reflecting a greater reduction in the rate of new replicon initiation than the 3- to 5-fold reduction in the rate of elongation of replicons. By 15-18 h after heating, the rates of both replicon initiation and chain elongation returned to the control level. The retardation in DNA chain elongation caused by hyperthermia evidently leads to exchanges between DNA molecules, which in turn result in the chromosomal aberrations that are believed largely responsible for killing S-phase cells.