Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity

The mechanism by which a cell protects itself from the lethal effects of he at shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA re plication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells thro ugh S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 muM aphidicolin th e measured clonogenic survival was similar to that for exponentially growin g cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with th e nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, corr elates with lethality of S-phase cells following heat shock under four diff erent experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis t hat heat-induced changes in the binding of proteins associated with DNA rep lication factories are the potentially lethal lesions, which become fixed t o lethal lesions by S-phase progression but are repairable if S-phase progr ession is delayed. (C) 2001 Wiley-Liss, Inc.