Objective: To establish a heat shock protein 90alpha (HSP90alpha) expression-inhibited cell line and study the effect of lowered HSP90alpha level on cell stress response.
Methods: The recombinant plasimid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90alpha was subcloned, purified and identified by DNA sequence analysis before introduced into mouse fibroblast cell line NIH-3T3 by electroporation. After G418 selection, the positive clones were identified by immunofluorescence and Western blotting. NIH-3T3 cells were subjected to hyperthermia at 44 degrees C for 40 min to simulate oxidative stress, and flow cytometry was performed to analyze the effect of low-level HSP90 on DNA damage under stress condition.
Results: Immunofluorescence and Westen blotting showed lowered HSP90 levels in the transfected cells. Compared with the control cells, cells subjected to hyperthermia displayed intensified DNA damage.
Conclusion: Low-level HSP90alpha causes the cells to be more vulnerable to oxidative stress condition, and HSP90 content can be associated with cell protection against such condition.