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.
Similar content being viewed by others
References
Gartel A. L.; Serfas M. S.; Tyner A. L. p21—negative regulator of the cell cycle. Proc. Soc. Exp. Biol. Med. 213: 138–149; 1996.
Gopalakrishnan S.; Van Emburgh B. O.; Shan J.; Su Z.; Fields C. R.; Vieweg J.; Hamazaki T.; Schwartz P. H.; Terada N.; Robertson K. D. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding. Mol. Cancer Res. 7: 1622–1634; 2009.
Jiemjit A.; Fandy T. E.; Carraway H.; Bailey K. A.; Baylin S.; Herman J. G.; Gore S. D. p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage. Oncogene 27: 3615–3623; 2008.
Kanai Y.; Saito Y.; Ushijima S.; Hirohashi S. Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis. J. Cancer Res. Clin. Oncol. 130: 636–644; 2004.
Kim E.; Goren A.; Ast G. Insights into the connection between cancer and alternative splicing. Trends Genet. 24: 7–10; 2008.
Kumar S.; Cheng X.; Klimasauskas S.; Mi S.; Posfai J.; Roberts R. J.; Wilson G. G. The DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 22: 1–10; 1994.
Milutinovic S.; Knox J. D.; Szyf M. DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21(WAF1/CIP1/sdi1). J. Biol. Chem. 275: 6353–6359; 2000.
Ogino S.; Kawasaki T.; Kirkner G. J.; Ogawa A.; Dorfman I.; Loda M.; Fuchs C. S. Down-regulation of p21 (CDKN1A/CIP1) is inversely associated with microsatellite instability and CpG island methylator phenotype (CIMP) in colorectal cancer. J. Pathol. 210: 147–154; 2006.
Okano M.; Xie S.; Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19: 219–220; 1998.
Oridate N.; Lotan R. Suppression of DNA methyltransferase 1 levels in head and neck squamous carcinoma cells using small interfering RNA results in growth inhibition and increase in Cdk inhibitor p21. Int. J. Oncol. 26: 757–761; 2005.
Ostler K. R.; Davis E. M.; Payne S. L.; Gosalia B. B.; Exposito-Cespedes J.; Le Beau M. M.; Godley L. A. Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins. Oncogene 26: 5553–5563; 2007.
Ostler, K. R.; Yang, Q.; Looney, T. J.; Zhang, L.; Vasanthakumar, A.; Tian, Y.; Kocherginsky, M.; Raimondi, S. L.; Demaio, J. G.; Salwen, H. R.; Gu, S.; Chlenski, A.; Naranjo, A.; Gill, A.; Peddinti, R.; Lahn, B. T.; Cohn, S. L.; Godley, L. A. Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation and alters DNA methylation in human neuroblastoma. Cancer Res. 72: 4714–4723; 2012.
Robertson K. D.; Uzvolgyi E.; Liang G.; Talmadge C.; Sumegi J.; Gonzales F. A.; Jones P. A. The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res. 27: 2291–2298; 1999.
Saito Y.; Kanai Y.; Sakamoto M.; Saito H.; Ishii H.; Hirohashi S. Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 99: 10060–10065; 2002.
Shah M. Y.; Vasanthakumar A.; Barnes N. Y.; Figueroa M. E.; Kamp A.; Hendrick C.; Ostler K. R.; Davis E. M.; Lin S.; Anastasi J.; Le Beau M. M.; Moskowitz I. P.; Melnick A.; Pytel P.; Godley L. A. DNMT3B7, a truncated DNMT3B isoform expressed in human tumors, disrupts embryonic development and accelerates lymphomagenesis. Cancer Res. 70: 5840–5850; 2010.
Shen L.; Gao G.; Zhang Y.; Zhang H.; Ye Z.; Huang S.; Huang J.; Kang J. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA. Nucleic Acids Res. 38: 6054–6064; 2010.
Sperka T.; Song Z.; Morita Y.; Nalapareddy K.; Guachalla L. M.; Lechel A.; Begus-Nahrmann Y.; Burkhalter M. D.; Mach M.; Schlaudraff F.; Liss B.; Ju Z.; Speicher M. R.; Rudolph K. L. Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction. Nat. Cell Biol. 14: 73–79; 2011.
Staalesen V.; Leirvaag B.; Lillehaug J. R.; Lonning P. E. Genetic and epigenetic changes in p21 and p21B do not correlate with resistance to doxorubicin or mitomycin and 5-fluorouracil in locally advanced breast cancer. Clin. Cancer Res. 10: 3438–3443; 2004.
Su X.; Lv C.; Qiao F.; Qiu X.; Huang W.; Wu Q.; Zhao Z.; Fan H. Expression pattern and clinical significance of DNA methyltransferase 3B variants in gastric carcinoma. Oncol. Rep. 23: 819–826; 2010.
Tan H. H.; Porter A. G. p21(WAF1) negatively regulates DNMT1 expression in mammalian cells. Biochem. Biophys. Res. Commun. 382: 171–176; 2009.
Velicescu M.; Weisenberger D. J.; Gonzales F. A.; Tsai Y. C.; Nguyen C. T.; Jones P. A. Cell division is required for de novo methylation of CpG islands in bladder cancer cells. Cancer Res. 62: 2378–2384; 2002.
Wang J.; Bhutani M.; Pathak A. K.; Lang W.; Ren H.; Jelinek J.; He R.; Shen L.; Issa J. P.; Mao L. Delta DNMT3B variants regulate DNA methylation in a promoter-specific manner. Cancer Res. 67: 10647–10652; 2007.
Wang J.; Walsh G.; Liu D. D.; Lee J. J.; Mao L. Expression of Delta DNMT3B variants and its association with promoter methylation of p16 and RASSF1A in primary non-small cell lung cancer. Cancer Res. 66: 8361–8366; 2006a.
Wang L.; Wang J.; Sun S.; Rodriguez M.; Yue P.; Jang S. J.; Mao L. A novel DNMT3B subfamily, DeltaDNMT3B, is the predominant form of DNMT3B in non-small cell lung cancer. Int. J. Oncol. 29: 201–207; 2006b.
Young J. I.; Smith J. R. DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal. J. Biol. Chem. 276: 19610–19616; 2001.
Zhang K.; Zhang R.; Li X.; Yin G.; Niu X. Promoter methylation status of p15 and p21 genes in HPP-CFCs of bone marrow of patients with psoriasis. Eur. J. Dermatol. 19: 141–146; 2009.
Zheng Q. H.; Ma L. W.; Zhu W. G.; Zhang Z. Y.; Tong T. J. p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation. J. Cell. Biochem. 98: 1230–1248; 2006.
Acknowledgments
This project was supported by the National Natural Science Foundation of China (nos. 81060212, 81160244), China Postdoctoral Science Foundation (no. 20080430851), Inner Mongolia Science Foundation (no. 2010BS1104), and Inner Mongolia Educational Research Foundation (nos. NJ06011, NJZY12221, and NJZY12225). We thank Mrs. Jean Danforth and Dr. Edward Meister for their language-editing assistance.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Editor: T. Okamoto
An erratum to this article is available at http://dx.doi.org/10.1007/s11626-014-9864-9.
Rights and permissions
About this article
Cite this article
Shao, G., Zhang, R., Zhang, S. et al. Splice variants DNMT3B4 and DNMT3B7 overexpression inhibit cell proliferation in 293A cell line. In Vitro Cell.Dev.Biol.-Animal 49, 386–394 (2013). https://doi.org/10.1007/s11626-013-9619-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-013-9619-z