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[Establishment of HSP90 overexpressing cell line and effects on cell proliferation]
1Lab of Thermal Environment Medicine, First Military Medical University, Guangzhou 510515, China.
Aim:
To establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response.
Methods:
The recombined plasmid pSmycHSP, which contained the full length DNA coding for human HSP90 B, was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion. After screened by G418, the positive clones were selected and identified by immunocytochemistry and Western-blotting. Contrasted with NIH-3T3 cells transfected with empty plasmid, the effect of high-level HSP90 on cell proliferation and cell cycle was analyzed by MTT method and flow cytometry.
Results:
The rising level of HSP90 was shown by immunocytochemistry and Western-blotting. Compared with control, the growth of HSP90 highly expressing cell line slowed down and the DNA content of S phase was lower.
Conclusion:
The NIH-3T3 derived cell line, which stably expressed high level of HSP90 was established. The effect of high-level HSP90 on cell proliferation was to retard cell growth by affecting cell cycle.
