ANALYSIS BY PULSED-FIELD GEL-ELECTROPHORESIS OF DNA DOUBLE-STRAND BREAKS INDUCED BY HEAT AND OR X-IRRADIATION IN BULK AND REPLICATING DNA OF CHO CELLS/

For a given amount of cell killing, heat alone (10-80 min, 45.5 degree s C) induced very few double-strand breaks (dsbs) compared with X-rays . Furthermore, 10 min at 45.5 degrees C immediately prior to X-rays ca used only a 1.3-fold increase in the slope of the X-ray-induced dsb do se-response curve, i.e. 0.67+/-0.006(95% confidence) dsbs/100Mbp/Gy fo r heated cells compared with 0.53+/-0.005 for unheated control cells. However, this same heat treatment caused > 5-fold inhibition in the ra te of repair of dsbs induced by 60-Gy X-rays, with the degree of inhib ition being much less in thermotolerant (TT) cells than in non-toleran t (NT) cells. This reduced inhibition of repair in TT cells correlated with the more rapid removal of excess nuclear protein from nuclei iso lated from TT cells than from NT cells. These results plus a TT ratio of 2-3 for both heat-induced radiosensitization and heal-inhibition of repairing dsbs are consistent with the hypothesis that heat radiosens itization results primarily from heat aggregation of nuclear protein i nterfering with access of repair enzymes to DNA dsbs. The selective he at-radiosensitization of S-phase cells, however, may result from an in crease in radiation-induced dsbs in or near replicating regions. For e xample, a preferential increase in dsbs in replicating DNA compared wi th bulk DNA was found following either hyperthermia alone (10-30 min, 45.5 degrees C) or a combined treatment (10 min, 45.5 degrees C before 60 Gy). A 30-min treatment at 45.5 degrees C induced dsbs equivalent to similar to 10 Gy in replicating DNA compared with 3-5 Gy in bulk DN A When cells were heated immediately before irradiation, the increase in dsbs induced in the replicating DNA by 60 Gy was equivalent to 200 Gy. We hypothesize that the observed 2-fold increase in single-strande d regions in replicating DNA after heat resulted in radiation selectiv ely inducing dsbs at or near the replication fork where the heat-induc ed increase in single-stranded DNA should occur. Thus, this preferenti al increase in dsbs in the replicating DNA by heat alone and especiall y when heat was combined with radiation may explain at least in part, the high sensitivity of S-phase cells to heat killing and heat radiose nsitization.