[Stress response changes of NIH-3T3 cells with HSP90alpha expression inhibition by RNA interference].
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.


CONCLUSIONS
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.
Chen
Xue-mei
XM
Department of Occupational Hygiene, Southern Medical University, Guangzhou 510515, China. cxmcsz@fimmu.com
Zou
Fei
F
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004249:DNA Damage; D005455:Fluorescent Antibody Technique; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D018841:HSP90 Heat-Shock Proteins; D034741:RNA, Small Interfering