Effect of Promoter Methylation of the p16 Gene on Phosphorylation of Retinoblastoma Gene Product and Growth of Hepatocellular Carcinoma Cells

Abstract

The biological significance of hypermethylation of p16 gene promoter in human hepatocellular carcinoma (HCC) cells remains to resolved. In order to clarify the significance of methylation of p16 gene promoter, we examined the methylation status of p16 gene in association with phosphorylation of retinoblastoma gene product (pRb) and cell growth in human HCC cell lines. The presence of methylation was examined by methylation-specific PCR. Expression and phosphorylation of p16 and pRb were examined by Western blot analysis. Genetic changes were analyzed by multiplex PCR and DNA sequencing. The effect of demethylation of p16 was assessed by cell growth. p16 gene promoter was methylated in HuH7 and HLF cells. The demethylating agent, 5-aza-2-deoxycytidine (5-Aza-CdR), upregulated p16 mRNA in HuH6 and HuH7 cells. 5-Aza-CdR increased p16 protein expression in HuH6, HuH7, and HLF cells, and it clearly decreased the phosphorylation level of pRb in HuH6, HuH7 and PLC/PRF/5 cells. Treatment with 5-Aza-CdR inhibited the growth of HuH7 cells. Homozygous deletion and significant mutations were absent. Methylation in the p16 promoter region is biologically significant, being associated with phosphorylation of pRb and cell growth in human HCC cells.

Predictive biomarkers for cancer therapy with PARP inhibitors

“…136 Along similar lines, histone deacetylase inhibitors can affect the DDR by modifying the expression of proteins involved in HR. 137 Indeed, the histone deacetylase inhibitor suberoylanilide hydroxamic acid efficiently inhibits the growth of hepatocellular carcinoma cells when associated with olaparib. 138 These findings are encouraging, but need further confirmation.…”

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

“…However, the frequency of methylation for many classically known tumor suppressors varies widely, some being as low as 15% (55–58). Using p16 as an example, analysis of the tumor samples with matched normals did not uncover tumor suppresors such as p16 and since we demonstrate the ability to detect p16 methylation in the cell line HuH7 (43) with known methylation it is likely that none of the tumors had methylation of p16 . Therefore, although improvements are always possible, we feel that our technical and analytical methods are sound.…”

“…Any ratio difference above this level was found to be methylated in one of the samples. To determine if our method could accurately identify known tumor suppressor CpG island methylation, we then analyzed the hepatocellular cancer cell line HuH7 with known methylation of the p16 promoter (43). In Figure 2F we show the detection of methylation of the region of the p16 gene CpG island commonly methylated and correlated with decreased gene transcription.…”

“…We then legitimatized the method's ability to detect methylation by bisulfite sequence validating over 15 fragments. We went on to analyze a number of samples that have been analyzed by others either by bisulfite sequencing or by other genome-wide approaches for methylation detection (43,45). In the case of Maeta et al ., we have validated specific methylation events and in the case of Sato et al .…”

Clinical implications of DNA methylation in hepatocellular carcinoma

“…Maeta et al . observed that when HCC cells were treated with demethylating agents, there was increased expression of p16 INK4a and subsequent cell growth inhibition, suggesting that modulation of the epigenetic control of these genes may be an important target in HCC therapeutics 28 . Lee et al .…”