Demethylation of the hTERT promoter in normal human gastric mucosal epithelial cells following N-methyl-N'-nitro-N-nitrosoguanidine exposure

Cheng,Yong‑Bo; Fang,Dian‑Chun; Yao,Ping; Guo,Li‑Ping; Ning,Xiao‑Yan; Wang,Lei

doi:10.3892/br.2014.398

Demethylation of the hTERT promoter in normal human gastric mucosal epithelial cells following N-methyl-N'-nitro-N-nitrosoguanidine exposure

Authors:
- Yong‑Bo Cheng
- Dian‑Chun Fang
- Ping Yao
- Li‑Ping Guo
- Xiao‑Yan Ning
- Lei Wang
View Affiliations

Affiliations: Department of Gastroenterology, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054, P.R. China, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China
Published online on: December 9, 2014 https://doi.org/10.3892/br.2014.398
Pages: 176-178

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

N‑methyl‑N'‑nitro‑N‑nitrosoguanidine (MNNG) is an alkylating agent that can induce gastric carcinoma. As a well‑known human carcinogen, MNNG has been universally recognized as a methylating agent and is believed to act through methylation mechanism. In the present study, the epigenetic status of the human telomerase reverse transcriptase (hTERT) promoter was investigated in MNNG‑treated normal human gastric mucosal epithelial cells. After 4 h exposure to MNNG at different concentrations, 6.8 and 68 µM, bisulfite sequencing polymerase chain reaction showed that five methylated cytosines outside the CpG dinucleotides in the 290‑bp fragment from the hTERT promoter were demethylated and all the methylated cytosines in CpG dinucleotides remained intact. Furthermore, the epigenetic status of the target region following MNNG exposure was extremely similar to those of the BGC‑823, SGC‑7901 and MKN‑28 lines; the three cell lines from human gastric adenocarcinoma. The result indicates that MNNG‑induced demethylation in cytosines outside the CpG dinucleotides may be an early molecular lesion with the potential for impacting malignant transformation and a possible underlying carcinogenic mechanism of MNNG. Thus, it may provide another insight into the mechanisms of MNNG carcinogenesis.

View Figures

View References

1	Gombar CT and Magee PN: DNA-methylation by nitrosocimetidine and N-methyl-N′-nitro-N-nitrosoguanidine in the intact rat. Chem Biol Interact. 40:149–157. 1982. View Article : Google Scholar : PubMed/NCBI
2	Manikandan P, Murugan RS, Priyadarsini RV, Vinothini G and Nagini S: Eugenol induces apoptosis and inhibits invasion and angiogenesis in a rat model of gastric carcinogenesis induced by MNNG. Life Sci. 86:936–941. 2010. View Article : Google Scholar : PubMed/NCBI
3	Haerlin R, Sussmuth R and Lingens F: Mechanism of mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) v. methylation of DNA by N-trideuteriomethyl-N′-nitro-N-nitrosoguanidine (D3-MNNG). FEBS Lett. 9:175–176. 1970. View Article : Google Scholar : PubMed/NCBI
4	Bhalla A, Sachdeva G and Bamezai R: T-cell receptor-gamma rearrangement and c-myb methylation in MNNG exposed Bloom syndrome B-lymphoblastoid cells. Cancer Lett. 126:1–6. 1998. View Article : Google Scholar : PubMed/NCBI
5	Schroering AG and Williams KJ: Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment. DNA Repair (Amst). 7:951–969. 2008. View Article : Google Scholar : PubMed/NCBI
6	Tomicic MT, Christmann M, Fabian K and Kaina B: Apaf-1 deficient mouse fibroblasts are resistant to MNNG and MMS-induced apoptotic death without attenuation of Bcl-2 decline. Toxicol Appl Pharmacol. 207 (Suppl 2):117–122. 2005. View Article : Google Scholar : PubMed/NCBI
7	Sabah M, Cummins R, Leader M and Kay E: Expression of human telomerase reverse transcriptase in gastrointestinal stromal tumors occurs preferentially in malignant neoplasms. Hum Pathol. 35:1231–1235. 2004. View Article : Google Scholar : PubMed/NCBI
8	Neumeister P, Albanese C, Balent B, Greally J and Pestell RG: Senescence and epigenetic dysregulation in cancer. Int J Biochem Cell Biol. 34:1475–1490. 2002. View Article : Google Scholar : PubMed/NCBI
9	Gulmann C, Lantuejoul S, Grace A, Leader M, Patchett S and Kay E: Telomerase activity in proximal and distal gastric neoplastic and preneoplastic lesions using immunohistochemical detection of hTERT. Dig Liver Dis. 37:439–445. 2005. View Article : Google Scholar : PubMed/NCBI
10	Cheng YB, Fang DC, Guo LP, Wang ZQ, Luo YH and Zhou JC: A convenient primary culture of human gastric epithelial cells. Chin J Gastroenterol. 12:31–35. 2007.
11	Issa JP: Aging, DNA methylation and cancer. Crit Rev Oncol Hematol. 32:31–43. 1999. View Article : Google Scholar : PubMed/NCBI
12	Suzuki T, Fujii M and Ayusawa D: Demethylation of classical satellite 2 and 3 DNA with chromosomal instability in senescent human fibroblasts. Exp Gerontol. 37:1005–1014. 2002. View Article : Google Scholar : PubMed/NCBI
13	Liu L, Lai S, Andrews LG and Tollefsbol TO: Genetic and epigenetic modulation of telomerase activity in development and disease. Gene. 340:1–10. 2004. View Article : Google Scholar : PubMed/NCBI
14	Devereux TR, Horikawa I, Anna CH, Annab LA, Afshari CA and Barrett JC: DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene. Cancer Res. 59:6087–6090. 1999.PubMed/NCBI
15	Santourlidis S, Wernet P, Ghanjati F, Graffmann N, Springer J, Kriegs C, Zhao X, Brands J, Arauzo-Bravo MJ, Neves R, et al: Unrestricted somatic stem cells (USSC) from human umbilical cord blood display uncommitted epigenetic signatures of the major stem cell pluripotency genes. Stem Cell Res. 6:60–69. 2011. View Article : Google Scholar : PubMed/NCBI
16	Poole JC, Andrews LG and Tollefsbol TO: Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene. 269:1–12. 2001. View Article : Google Scholar : PubMed/NCBI
17	Fujimoto K, Kyo S, Takakura M, Kanaya T, Kitagawa Y, Itoh H, Takahashi M and Inoue M: Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT. Nucleic Acids Res. 28:2557–2562. 2000. View Article : Google Scholar : PubMed/NCBI