Lipopolysaccharide Induces Aberrant Hypermethylation of Hic-1 in Mouse Embryonic Fibroblasts Lacking p53 Gene

Abstract

Background: The present study asked whether continuous administration with lipopolysaccharide (LPS), a potent inflammatory agent, induces aberrant methylation in the promoter region of tumor suppressor genes and p53 and/or inducible nitric oxide synthase (iNOS) genes involved in its aberrant methylation. Materials and Methods: Mouse embryonic fibroblasts (MEFs) were prepared from mice harboring four different genotypes (p53^+/+iNOS^+/+, p53^+/+iNOS^-/-, p53^-/-iNOS^+/+ and p53^-/-iNOS^-/-). The MEFs were immortalized by 3T3 procedure and continuously cultured under a medium containing LPS or LPS plus interferon (IFN)-γ during 40 passages. The methylation status in the CpG site of hypermethylated in cancer-1 (Hic-1) exon 1a and p16 promoter region was monitored using bisulfite-sequencing methods. Results: LPS and LPS plus IFN-γ induced de novo methylation in the CpG sites of the Hic-1 gene. This site was methylated only in p53^-/- MEFs, and the mRNA expression of Hic-1 decreased in p53^-/- MEFs compared to p53^+/+ MEFs. The methylation patterns of Hic-1, however, were not affected by iNOS gene status. The promoter region of p16 was methylated by increasing the passage, even under the control medium, with LPS administration promoting methylation, particularly in MEFs lacking the iNOS gene. However, the methylation pattern was not significantly different between the p53 genotypes. Conclusion: Our preliminary study suggests that LPS induces de novo methylation in the CpG site in MEFs. For the Hic-1 gene, but not p16, the p53 gene might protect against aberrant methylation. The iNOS gene might not be involved in methylation of the Hic-1 gene, whereas the promoter region of p16 could be prone to methylation in MEFs lacking the iNOS gene.