O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

Abstract

Background: O⁶-methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the mispairing base O⁶-methyl-guanine induced by environmental and experimental carcinogens. It can transfer the alkyl group to a cysteine residue in its active site and became inactive. The chemical carcinogen N-nitroso compounds (NOCs) can directly bind to the DNA and induce the O⁶-methylguanine adducts, which is an important cause of gene mutation and tumorigenesis. However, the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis, especially in the initiation phase, remains largely unclear.

Aim: To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.

Methods: We established a gastric epithelial cell malignant transformation model induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitroso-urea (MNU) treatment. Cell proliferation, colony formation, soft agar, cell migration, and xenograft assays were used to verify the malignant phenotype. By using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis, we detected the MGMT expression in malignant transformed cells. We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry. MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR. The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.

Results: We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment. Moreover, we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells. Inhibition of the MGMT expression by O⁶-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes. Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU. Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues, but downregulated in the gastric cancer tissues.

Conclusion: Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation. The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis, which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

Keywords: DNA methylation; Epigenetic regulation; Gastric carcinogenesis; Malignant transformation; O6-methylguanine-DNA methyltransferase.

Figure 1

O⁶-methylguanine-DNA methyltransferase expression is enhanced in early stage gastric cancer. A: Representative images of immunohistochemistry staining for O⁶-methylguanine-DNA methyltransferase (MGMT) in early stage gastric tumor and normal tissues (n = 19); B: The mRNA level of MGMT in early stage gastric tumor and adjacent normal tissues (n = 19). The mRNA expression was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); C: MGMT mRNA and protein expression in normal gastric epithelial cells and cancer cells by quantitative real-time polymerase chain reaction and immunoblot assays. GAPDH was used to normalize MGMT expression. The analyses were repeated three times, and the results are expressed as the mean ± SD. ^aP < 0.05; ^bP < 0.01. MG-C: MNNG-induced malignant transformed cell; MU-C: MNU-induced malignant transformed cell; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

N-nitroso compound treatment induces gastric epithelial cell malignant transformation. A: Cell proliferation monitored by cell counting in N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)/N-methyl-N-nitroso-urea (MNU)-treated and control cells; B and C: Cell anchorage-independent growth on soft agar and cell colony formation. Top, representative images; bottom, quantitative results of cell colony per field; D: Wound healing assay. Top, representative images of wound healing assay; right, relative percentage of wound closure after treatment; E and F: Tumor growth curve and tumor weight in nude mice injected subcutaneously with the transformed cells induced by MNNG/MNU and control cells. The analyses were repeated three times, and the results are expressed as the mean ± SD. ^aP < 0.05; ^bP < 0.01.

Figure 3

O⁶-methylguanine-DNA methyltransferase is downregulated in N-nitroso compound-induced gastric epithelial cell malignant transformation. A and B: O⁶-methylguanine-DNA methyltransferase (MGMT) mRNA and protein expression in transformed gastric epithelial cells induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)/N-methyl-N-nitroso-urea (MNU) for 1, 4, and 8 wk; C: Cell anchorage-independent growth on soft agar for subcolones of MNNG/MNU-induced cells. C1-28: Different subcolones of MNNG/MNU-induced cells; MGMT(+): MGMT expression is upregulated in these subcolones; MGMT(-): MGMT expression is downregulated or no-changed in these subcolones; D: Apoptosis assay of MNNG/MNU-transformed subcolones after doxycycline treatment. The analyses were repeated three times, and the results are expressed as the mean ± SD. ^aP < 0.05; ^bP < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

DNA hypomethylation contributes to O⁶-methylguanine-DNA methyltransferase upregulation in cell malignant transformation. A: Luciferase reporter assay in control and N-nitroso compound-transformed cells using PGL3-O⁶-methylguanine-DNA methyltransferase (MGMT) promoter; B and C: Methylation specific polymerase chain reaction and bisulfite genomic sequence analysis of the DNA methylation level of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced transformed cells compared with control cells; D: Correlation of MGMT expression and DNA methylation level of MGMT promoter based on the CCLE database; E: ChIP assay with anti-DNMT1 and anti-H3K9Me3 and H3K4Me2 antibodies for analyzing the DNMT1 binding to the MGMT promoter and the H3K9Me3 and H3K4Me2 levels in the MGMT promoter. The analyses were repeated three times, and the results are expressed as the mean ± SD. ^aP < 0.05; ^bP < 0.01. M: Methylated; U: Unmethylated; IgG: Immunoglobulin G.

Figure 5

Inhibition of O⁶-methylguanine-DNA methyltransferase contributes to the N-nitroso compound-induced cell malignant phenotype. A and B: Cell anchorage-independent growth on soft agar and cell colony formation of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced cells after O⁶-BG treatment; C: Cell anchorage-independent growth on soft agar of cells with O⁶-methylguanine-DNA methyltransferase (MGMT) knock-down; D: Knock-down efficiency of MGMT detected by Western blot; E and F: Cell anchorage-independent growth on soft agar and cell colony formation of MGMT overexpressing cells; G: The mRNA expression of MGMT in gastric endoscopic biopsy samples. The analyses were repeated three times, and the results are expressed as the mean ± SD. ^a,cP < 0.05. ^cP < 0.05, precancerous lesion and early cancer vs advanced cancer. EV: Empty vector; MGMT: MGMT overexpression; MGMT: O⁶-methylguanine-DNA methyltransferase.