THERMOTOLERANCE EXPRESSION IN MITOTIC CHO CELLS WITHOUT INCREASED TRANSLATION OF HEAT-SHOCK PROTEINS

The objective of this study was to unequivocally demonstrate thermotol erance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected a s an experimental system since their genome was in the form of condens ed chromosomes and ostensibly incapable of being transcribed; thus, ob viating stress-induced HSP gene expression. Asynchronous Chinese hamst er ovary (CHO) cells were treated with 0.2 mu g/ml nocodazole to accum ulate cells in mitosis for harvest by mitotic shakeoff. Cells were mai ntained in mitosis with nocodazole during thermotolerance induction, t hermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the ther motolerance inducing heat shock, as indicated by gel mobility shift as say, no increase in steady-state HSP mRNA levels was detected, as expe cted. Preferential synthesis of HSPs from extant mRNA was not detected during thermotolerance development and cellular levels of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins remained constant, as determin ed by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 mu g/ml cycloheximide during thermotolerance development co nfirming that new protein synthesis was not requisite. Parallel experi ments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 mu g/ml cy cloheximide. As with mitotic cells, high levels of thermotolerance wer e attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this s tudy demonstrated that high levels of thermotolerance could be express ed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermo tolerance expressed in nonmitotic cells also occurs independently of i nduced HSP synthesis. (C) 1996 Wiley-Liss, Inc.