Effect of ornithine decarboxylase antizyme inhibitor-1 overexpression on the proliferation of B16-F1 mouse melanoma cells / 肿瘤

ABSTRACT

Objective: To study the effect of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) overexpression on the proliferation of B16-F1 mouse melanoma cells. Methods: OAZI-1 gene was cloned out from the cDNAs derived from H22 mouse hepatocellular carcinoma cells, and then subcloned into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/OAZI-1 was transfected into B16-F1 cells with LipofectAMINE2000 reagents. The positive clone with OAZI-1 overexpression was identified by Western blotting and real-time fluorescent quantitative PCR. The effects of OAZI-1 overexpression on the cell proliferation and the cell cycle distribution were detected by MTT and flow cytometry. The level of polyamine was detected by reversed-phase high performance liquid chromatography. The chemiluminescence analysis was used to determine the activity of spermine oxidase (SMO). Results: The B16-F1 clone with OAZI-1 overexpression was obtained successfully and named as B16/over. The expression level of OAZI-1 mRNA was 3.6-fold higher in the B16/over cells than that in the control B16/3.1 cells transfected with pcDNA3.1(+). The level of ornithine decarboxylase antizyme (OAZ) mRNA was decreased, and the level of ornithine decarboxylase (ODC) mRNA was elevated in B16/ over cells. Overexpression of OAZI-1 in B16-F1 cells could promote the cell proliferation and influence the cell cycle distribution, including decreasing the cell number in G0/G1 phase and increasing the cell number in S and G2/M phases. In B16/over cells, the content of putrescine was increased, but the contents of spermidine and spermine were decreased, and the activity of SMO was elevated. Conclusion: Overexpression of OAZI-1 may promote the proliferation of B16-F1 cells by elevating the content of putrescine, which indicates that OAZI-1 may be a potential target for melanoma therapy. Copyright© 2011 by the Editorial Board of Tumor.