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Diamide-induced cytotoxicity and thermotolerance in CHO cells
M J Borrelli1, D M Stafford, C M Rausch
1Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073, USA. mborrell@ri-exp.beaumont.edu
Abstract:
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 degrees 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 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees 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. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.
Insights
Diamide treatment denatures cellular proteins, causing cell death. Thermotolerance protects cells by rapidly disaggregating these proteins, even without heat shock protein synthesis.
Area of Science:
- Cellular Biology
- Biochemistry
- Stress Response
Background:
- Prior studies implicated protein denaturation as oxidative damage activating heat shock transcription factor and inducing thermotolerance.
- Diamide, a sulfhydryl oxidant, denatures and aggregates cellular proteins.
Purpose of the Study:
- To characterize cellular response to diamide-denatured proteins.
- To investigate the involvement of diamide-denatured proteins in cytotoxicity.
- To explore the relationship between thermotolerance and diamide-induced protein aggregation.
Main Methods:
- Exposure of cells to diamide at different temperatures (37.0°C and 24.0°C).
- Assessment of protein denaturation and aggregation.
- Measurement of cell killing (cytotoxicity).
- Evaluation of thermotolerance induction and its effect on diamide toxicity and protein disaggregation.
Main Results:
- Diamide-induced protein denaturation at 37.0°C correlated with cell killing, but differently than heat shock.
- Diamide at 24.0°C was less cytotoxic, with minimal killing after return to 37.0°C.
- Thermotolerance protected against diamide cytotoxicity but did not reduce initial protein aggregation.
- Thermotolerant cells rapidly disaggregated proteins post-diamide, unlike non-tolerant cells.
- Nontoxic diamide induced thermotolerance without increased heat shock protein (HSP) synthesis.
Conclusions:
- Protein denaturation and aggregation are key cytotoxic consequences of stress and triggers for thermotolerance.
- Rapid protein disaggregation is a potential mechanism for thermotolerance against diamide toxicity.
- Diamide provides a method to induce thermotolerance independent of HSP synthesis, useful for studying stress responses.