Thermotolerance expression in mitotic CHO cells without increased translation of heat shock proteins

Abstract

The objective of this study was to unequivocally demonstrate thermotolerance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected as an experimental system since their genome was in the form of condensed chromosomes and ostensibly incapable of being transcribed; thus, obviating stress-induced HSP gene expression. Asynchronous Chinese hamster ovary (CHO) cells were treated with 0.2 microgram/ml nocodazole to accumulate cells in mitosis for harvest by mitotic shakeoff. Cells were maintained in mitosis with nocodazole during thermotolerance induction, thermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the thermotolerance inducing heat shock, as indicated by gel mobility shift assay, no increase in steady-state HSP mRNA levels was detected, as expected. 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 determined by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 micrograms/ml cycloheximide during thermotolerance development confirming that new protein synthesis was not requisite. Parallel experiments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 micrograms/ml cycloheximide. As with mitotic cells, high levels of thermotolerance were attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this study demonstrated that high levels of thermotolerance could be expressed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermotolerance expressed in nonmitotic cells also occurs independently of induced HSP synthesis.