Deregulation Of Histone Modifications In Chemical Carcinogen MNNG And MNU Induced Gastric Epithelial Cell Malignant Transformation

Monofunctional alkylating agents N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and N-methyl-M-nitrosourea (MNU) are model chemical mutagens for studying of environmental N-nitrosamines compounds (NOC) initiated carcinogenesis. We have successfully induced malignant transformation of human gastric epithelial cell GES-1 by low-dose MNU or MNNG. To investigate epigenetic modifications involved in malignant transformation, we analyzed the alterations of major modifications on histone H3 and H4. Our results show that H3S10p and H3S28p are increased and H4K16ac is decreased after carcinogen exposure. Further studies reveal that kinase MSK1 is mainly responsible for H3S10p upregulation through transcriptional activation. ERK/MAPK pathway, the upstream signaling pathway of MSK1, however, only show little contribution to this regulation. In addition, we found that target genes downstream MSK1/H3S10p pathway such as c-fos, c-jun and jund are upregulated, suggesting that histone phosphorylation may participate in the regulation of important genes in malignant transformation. Furthermore, we found that the expression and activity of histone deacetylase HDAC1 are activated after carcinogen exposure, then it mainly induces the decrease of H4 acetylation. Inhibition of HDAC1 activity by inhibitors can abrogate this effect. Interestingly, another HDAC member, HDAC6, is also upregulated after carcinogen treatment. As the substrate of HDAC6, a-tubulin is deacetylated which effect can be reversed by HDAC6 knockdown. To understand the role of H4 hypoacetylation in gene regulation, we screened the altered tumor suppressor genes and found that FHIT is dramatically downregulated as an early response to carcinogen exposure. Further study indicates that there is no homozygous deletion of FHIT gene, and H4 hypoacetylation and DNA hypermethylation on FHIT promoter mainly contribute to the gene silencing. The results demonstrate that epigenetic regulations including histone modification and DNA methylation play an important role in controlling FHIT expression during cell malignant transformation. Finally, to elucidate the influence of histone modification dysregulation on NOC induced carcinogenesis, we treated transformed cells with inhibitors of MSK1, HDACs and DNA methyltransferase, alone or in combination. The reversion of epigenetic modifications inhibits malignant phenotype obviously.In conclusion, we investigated the regulatory mechanisms and functions of histone modifications deregulated in MNNG and MNU-induced cell transformation. This study will help us on the understanding of the important role of epigenetic mechanisms in NOC-induced cell malignant transformation and cancer initiation, and has prospective significance in the early diagnosis and therapy of gastric cancer.