The synthesis and distribution of HSPs (HSP-70 in particular) will be correlated with thermotolerance for heat killing and heat radiosensitization. Chinese hamster ovary (CHO) cells in culture can be made equally resistant to heating at 43 degrees C in a variety of ways, e.g., incubation at 37 degrees C after a trigger with arsenite, intermediate concentrations of puromycin, or heat; or with a high concentration of cycloheximide or puromycin before and during heating at 43 degrees C. However, these different treatments result in large differences in thermotolerance to a subsequent challenge at 45 degrees C, and patterns of protein synthesis studied with one-dimensional polyacrylamide gel electrophoresis also differ. Therefore, two dimensional gels and antibodies to HSPs will be used to quantify the synthesis and distribution in the cell of constitutive and inducible HSPs. These findings will be related to the amount of thermotolerance for both heat killing and heat radiosensitization. Heat radiosensitization studied in synchronous CHO cells will be related to chromosomal aberrations; activities of DNA polymerases; and to the induction, repair, and residual amount of base damage and double-strand breaks in DNA. Studying DNA double-strand breaks by both neutral elution and new alternating field gradient electrophoresis (AFGE) techniques will be used to define the relationship between chromosomal aberrations and the initial and residual double-strand breaks. The use of restriction enzymes to produce DNA fragments of particular sizes separated by AFGE and identified by Southern blots with unique DNA probes may provide information concerning misrepair and degradation in particular domains in the DNA. The effects of heat during S-phase on DNA synthesis (initiation, elongation, and fork displacement) will be studied in detail to determine if alterations in any of these endpoints result in chromosomal aberrations and cell lethality. During S-phase, chromatid aberrations are induced by either heat or ionizing radiation, and for both modalities, have the same correlation with lethality. Therefore, chromatid aberrations and residual double- strand breaks will be studied in relation to replicating regions, after heat, radiation, and heat-radiation combined.
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