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Construction of p70 /p85 ribosomal protein S6 kinase 1 expression vectors and functional assessment in human breast cancer MCF-7 cells
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Abstract:
Objective: To optimize the construction of eukaryotic expression vectors encoding p70/p85 ribosomal protein S6 kinase 1 (S6K1)and to evaluate the function of the constructed vectors in human breast cancer MCF-7 cells. Methods: Fragments of p70 S6K1 and p85 S6K1 cDNAs with restriction endonucleases sites were amplified by PCR with pRK7-HA-S6K1 as a template and cloned into an eukaryotic expression vector with a flag tag, pcDNA3.1 (-)-flag. MCF-7 cells were transfected with the constructed vectors, pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1. At 24 h after transfection, protein contents of p70 S6K1 and p85 S6K1 were assessed by Western blotting using anti-flag and anti-p70/85 S6K1 antibodies and cell death following induction with 1 mmol/L hydrogen peroxide for another 36 h was analyzed by microscopy. Results: Eukaryotic expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were successfully constructed; the full-length open reading frames were confirmed by DNA sequencing. Overexpression of S6K1 and S6K1 was detected in MCF-7 cells transfected with pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 respectively. Overexpression of p85 S6K1 but not p70 S6K1 enhanced MCF-7 cell death induced by 1 mmol/L hydrogen peroxide. Conclusion: Expression vectors pcDNA3.1(-)-flag-p70 S6K1 and pcDNA3.1(-)-flag-p85 S6K1 were constructed successfully. Overexpression of p85 S6K1 may enhance H2O2-induced breast cancer cell death.
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Project Supported:
Project supported by the National Natural Science Foundation for Young Taleats of China(No.81302357), the National Nautral Foundation of Guangdong Province (No. 2013040016493), and the Foundation for Distinguished Young Talents in Higher Education of Guangdong Province (No.2013LYM0075)
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