Methylation Silencing and Mutations of the p14ARF and p16INK4a Genes in Colon Cancer

Abstract

SUMMARY:The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16 INK4a and p14 ARF , whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14 ARF and p16 INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14 ARF and p16 INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1␤ of p14 ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14 ARF , documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14 ARF and p16 INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14 ARF was inversely correlated with p53 overexpression (p ϭ 0.02). p14 ARF and p16 INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p ϭ 0.02). Methylation of the p14 ARF gene occurred more frequently in well-differentiated adenocarcinomas (p ϭ 0.005), whereas the p16 INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p ϭ 0.002). The present results underline the role of p14 ARF and p16 INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14 ARF and p16 INK4a , might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways. (Lab Invest 2001, 81:217-229).

Regulation of p53 Stability and Function in HCT116 Colon Cancer Cells

“…Phosphorylation of Thr-18, which is predicted on structural evidence to block MDM2 binding, is unlikely to explain the absence of MDM2 regulation in HCT116 cells, because Thr-18 is not phosphorylated following DNA damage in these cells (19). Another explanation could be that MDM2 function is inhibited by p14ARF in HCT116 cells, but this is unlikely because one allele of the p14ARF gene is methylated and the other allele contains a codon 33 frameshift mutation (51). Finally, it is important to note that our results only demonstrate a lack of regulation by endogenous MDM2.…”

Human p14ARF-mediated cell cycle arrest strictly depends on intact p53 signaling pathways

“…Their phenotype, mostly in the context of a DNA damage response, has been extensively documented. In HCT cells, the endogenous p14 ARF gene is silenced by mutation of one and methylation of the other allele, respectively (Burri et al, 2001). Therefore, the introduction of p14 ARF into these cells substitutes missing p14 ARF expression instead of increasing already existing p14 ARF expression.…”

Multiple interacting domains contribute to p14ARF mediated inhibition of MDM2

“…Point mutations are unlikely to have a signi®cant destabilizing e ect on the ARF-MDM2 interaction and all of the single amino acid substitutions that we have analysed to date have proved indistinguishable from wild-type, at least based on the assays currently at our disposal. We are, however, aware of two frameshift mutations in exon 1b, one a single base deletion in a colon carcinoma cell line (Burri et al, 2001) and the other a 16 bp insertion in familial melanoma (Rizos et al, 2001). These would e ectively truncate ARF sequences after residues 33 and 21, respectively.…”