Splice variants DNMT3B4 and DNMT3B7 overexpression inhibit cell proliferation in 293A cell line.

Abstract

DNA methyltransferase 3B (DNMT3B) is critical in abnormal DNA methylation patterns in cancer cells. Nearly 40 alternatively spliced variants of DNMT3B have been reported. DNMT3B4 and DNMT3B7 are two kinds of splice variants of DNMT3B lacking the conserved methyltransferase motif. In this study, the effect of inactivation of DNMT3B variants, DNMT3B4 and DNMT3B7, on cell proliferation was assessed. pCMV-DNMT3B4 and pCMV-DNMT3B7 recombinant plasmids were developed and stably transfected into 293A cells. 293A cells transfected with plasmid pCMV-DNMT3B4 or pCMV-2B were then treated with G418 to the stable cell lines. After that, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method was used for testing the proliferation level, and flow cytometry was used to test cell cycle distribution of the cell line. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 promoter was detected by methylation-specific PCR (MS-PCR). It was found that DNMT3B4 and DNMT3B7 overexpression could inhibit cell proliferation and increase the expression of p21. Cell cycle analysis demonstrated that inactivation of DNMT3B variants overexpression inhibited cell cycle progression. Inactivation of DNMT3B variants overexpression facilitated p21 expression to delay 293A cell proliferation. These findings indicate that inactivation of DNMT3B variants might play an important role in cell proliferation correlating with the change of p21.

• Authors: Guo Shao, Ran Zhang, Shu Zhang, Shuyuan Jiang, You Liu, Wei Zhang, Yanbo Zhang, Jinping Li, Kerui Gong, Keri Gong, Xin-Rong Hu, Shi-Wen Jiang
• Journal: In vitro cellular & developmental biology. Animal (In Vitro Cell Dev Biol Anim) Vol. 49 Issue 5 Pg. 386-94 (May 2013) ISSN: 1543-706X [Electronic] Germany
• PMID: 23636939 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• CDKN1A protein, human
• Cyclin-Dependent Kinase Inhibitor p21
• DNA Primers
• Gentamicins
• Protein Isoforms
• Tetrazolium Salts
• Thiazoles
• antibiotic G 418
• DNA (Cytosine-5-)-Methyltransferases
• DNA methyltransferase 3B
• thiazolyl blue

Topics

• Blotting, Western
• Cell Proliferation
• Cyclin-Dependent Kinase Inhibitor p21 (metabolism)
• DNA (Cytosine-5-)-Methyltransferases (genetics, metabolism)
• DNA Methylation (genetics)
• DNA Primers (genetics)
• Flow Cytometry
• Gene Expression Regulation (physiology)
• Gene Silencing
• Gentamicins
• HEK293 Cells
• Humans
• Plasmids (genetics)
• Protein Isoforms (genetics, metabolism)
• Real-Time Polymerase Chain Reaction (methods)
• Tetrazolium Salts
• Thiazoles