Diane M. Stafford

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.

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

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 μg/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 μg/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 μg/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.

Diamide-induced cytotoxicity and thermotolerance in CHO cells

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide‐denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0°C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0°C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0°C. Thus, protein denaturation that occurred at 37.0°C, after proteins were chemically destabilized by diamide at 24.0°C [Freeman et al., J. Cell. Physiol., 164:356–366 (1995) Senisterra et al., Biochemistry 36: 11002–11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression.

Protocol for freezing thermotolerant cells and maintaining thermotolerance following thawing

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.