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Protection of chinese hamster ovary cells from heat killing by treatment with cycloheximide of puromycin involvement of hsps



Protection of chinese hamster ovary cells from heat killing by treatment with cycloheximide of puromycin involvement of hsps



Radiation Research 111(2): 237-253



Cyclohexamide (CHM) or puromycin (PUR) added for 2 h before heating at 43.degree. C followed by either PUR or CHM during heat greatly protected cells from heat killing. This protection increased with inhibition of protein synthesis. Since treatment with a drug both before and during heating was required for heat protection, and since one drug could be exchanged for the other after the 2-h pretreatment without affecting the heat protection, a common mode of action involving inhibition of protein synthesis is suggested for the two drugs. Drug treatment reduced the synthesis of heat-shock proteins (HSPs) as studied by one-dimensional gel electrophoresis by 80-98% relative to 37.degree. C untreated controls. Synthesis of large molecules (>30 kDa) was preferentially inhibited by PUR but not by CHM. Also for CHM, but not for PUR treatment, a 42 kDa band appeared along with a great reduction in the 43 kDa actin band during CHM treatment at both 37 and 43.degree. C. Furthermore, during CHM or PUR treatment, incorporation of [35S]methionine into HSP families 70, 87, or 110 was not increased relative to incorportion into total protein. However, synthesis of the 70 kDa HSP family was selectively suppressed when cells were incubated at 37.degree. C after CHM treatment, but when cells were incubated at 37.degree. C after treatment at 43.degree. C with CHM, synthesis of the 70 kDa HSP family resumed. When cells were labeled for 3 days, there was no preferential accumulation or turnover of HSP families during heating with or without CHM. Therefore, heat protection caused by treatment with CHM or PUR apparently involves a common mode of action not associated with changes in either total levels or synthesis of HSP families during drug treatment before and during heating. The significance of the changes observed in the synthesis of the HSP 70 family after heat is unknown. As thermotolerance developed during 5 h at 42.degree. C without drugs, synthesis of HSP families 70, 87, and 110, as studied with one-dimensional gels, increased 1.4-fold relative to synthesis of total protein, but compared to HSP families in cells labeled for 5 h at 37.degree. C incorporation was reduced by 40%. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. A significant observation was that these cells developing chronic thermotolerance during heating at 42.degree. C were much more thermally tolerant than cells that developed heat resistance to 43.degree. C by treatment with CHM. This thermotolerance was measured by a subsequent heat challenge treatment at 45.degree. C. This was particularly interesting since the survival curve for CHM-treated cells heated at 43.degree. C was biphasic and the same as untreated cells heated at 43.degree. C. Therefore, the greater thermotolerance observed at 42.degree. C compared to that at 44.degree. C with CHM may be due to enhanced synthesis of heat-inducible HSPs.

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Accession: 006209318

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PMID: 3628714


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