Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma

Abstract

One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14 ARF , p15 INK4B M ethylation of normally unmethylated CpG islands is an alternative mechanism for loss of function of tumor suppressor genes in human cancer. p16 INK4A is one of the most important tumor suppressor genes belonging to the family of cell cycle inhibitors, encoding a protein that binds competitively to cyclin-dependent-kinase-4 proteins. In hepatocellular carcinoma (HCC), methylation of p16 INK4A promoter has already been documented with variable frequency. [1][2][3][4] Wong et al. 5 recently reported p16 INK4A promoter methylation in 81% of sera samples of patients with methylated HCC. This result is particularly interesting for early diagnosis and possible therapeutic implications because promoter methylation is potentially reversible. It is known that cirrhosis is a favorable background for HCC development so that, not unexpectedly, p16 INK4A methylation was also recently reported by Kaneto et al. 6 in 30% of cirrhoses, suggesting its potential clinical use for selecting patients at high risk for HCC.The functional significance of promoter methylation was also established in a number of studies showing that this epigenetic abnormality usually corresponds the abrogation of messenger RNA (mRNA) synthesis and protein expression in both tumoral and nontumoral liver tissue. 3,4,6 Interestingly, concurrent inactivation of tumor suppressor genes of RB1 gene-related pathways such as p16 INK4A , p14 ARF , and p15 INK4B by promoter methylation has been shown not only in many types of human tumors, such as meningiomas, central nervous system lymphomas, and glial tumors, 7-9 but also in early preneoplastic Abbreviations: HCC, hepatocellular carcinoma; PCR, polymerase chain reaction. From the

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer

“…RASSF1A, E-Cadherin and p16INK4A methylation As previously reported (Roncalli et al, 2002), 1 mg of genomic DNA in a volume of 50 ml was denatured by NaOH for 10 min at 371C; 30 ml of 10 mM hydroquinone (Sigma, Saint Louis, MO, USA) and 520 ml of 3 M sodium bisulphite (Sigma, Saint Louis, MO, USA) at pH 5, both freshly prepared, were added and mixed, and samples were incubated at 501C for 16 h. Modified DNA was purified using the Wizard DNA purification resin according to the manufacturer's instruction (Promega, Milano, Italy) and eluted into 50 ml of water. Modification was completed by NaOH treatment (final concentration 0.3 M) for 5 min at room temperature, followed by ethanol precipitation.…”

“…One section from each tissue was stained with hematoxylin -eosin to distinguish neoplastic tissue from the nonmalignant counterpart. After manual dissection, DNA was extracted from the unstained section of the malignant tissue as described previously (Roncalli et al, 2002). Briefly, tissue sections were deparaffinised using 100% xylene (Sigma, Saint Louis, MO, USA) followed by 100% ethanol.…”

Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis

“…Kaneto et al, using methylation specific PCR and immunohistochemistry, had detected methylation of p16 promoter in HCC (72.6%, 16/22) and loss of expression in all methylation positive HCC [13] . Roncalli et al reported that methylation of p16 promoter with complete loss of immunoreactivity occurred in 27 of 33 HCC (82%) [15] . Thus, our result, which was consistent with other reports, suggested that association of p16 gene 5'CpG island methylation with transcription inactivation might play an important role in hepatocarcinogenesis, and 5'CpG island methylation was the major mechanism of p16 gene inactivation.…”

“…Wong et al reported a high frequency of p15 methylation (64%, 15/25) in HCC. Yang et al showed methylation of p15 in 49% of HCC [14][15][16] . Based on Northern blot analysis, our study found that a complete loss of p15 mRNA expression accounted for a small portion of the p15 methylation positive cases, while the majority had decreased transcription level.…”

“…It has been demonstrated that aberrant methylation of CpG islands, which are CpG dinucleotide rich region located mainly in the promoter regions of many gene, serves as an alternative mechanism for inactivation of the tumor suppressor gene in cancers [10] . Recently, aberrant methylation of the p16 and p15 promoters has been reported [11][12][13][14][15][16] . The discordant events in which low P16 protein expression was not companied with deletion or mutation could be resulted from the low mRNA expression level of p16 gene due to hypermethylation of its CpG islands.…”