Methylation of CpG islands of p16 associated with progression of primary gastric carcinomas

Abstract

Inactivation of p16 by methylation of CpG islands is a frequent early event in gastric carcinogenesis. The positive relationship between p16 methylation and the clinical characteristics of gastric carcinomas (GC) has not been reported to date. In the present study, a DHPLC assay to quantify p16 methylation was established (detection limit by fluorescence detector: 1:255 (Methlyated vs Unmethylated)). The proportion of methylated p16 in the representative samples was confirmed and standardized by clone sequencing. Then, the DHPLC and two regular methylation-specific PCR (MSP) assays were used to detect p16 methylation in 82 paired, resected GCs and their adjacent normal tissues. Results showed that the average proportion of methylated p16 in GCs was significantly higher than that in their adjacent samples (12.90 vs 0.63%; t-test P ¼ 0.005). A much higher proportion of methylated p16 was detected in GCs with metastases (local or distant) than without metastases (14.76 vs 2.61%; t-test P ¼ 0.014). A proportional relationship was observed between clinical stages and positive rates of p16 methylation in GCs and/or adjacent tissues: 27.3, 37.5, and 58.8% (by DHPLC) for stage-I, -II, -III-IV of GCs, respectively (two-sided Fisher's exact test P ¼ 0.016). To confirm the data obtained by DHPLC, two MSP primer sets (p16-M and p16-M2) were also used to analyze p16 methylation in the same set of samples simultaneously. Data of MSP assay using the primer set p16-M2, but not p16-M, correlated with that of DHPLC. These results imply that the primer set p16-M2 might be more suitable than p16-M to detect p16 methylation in gastric tissues. In conclusion, the present data indicates that p16 methylation correlates with progression of GCs significantly.

Methylation of p16 CpG Island Associated with Malignant Progression of Oral Epithelial Dysplasia: A Prospective Cohort Study

“…Although the quantitative MethyLight results in both initial biopsy and follow-up rebiopsy samples were consistent with those of the methylation-specific PCRdenatured high performance of liquid chromatography assay in the present study, we did not observe a significant increase of oral cancer risk in patients with oral epithelial dysplasia with the strong p16 methylation by MethyLight. Unlike in cultured cell lines, we reported that methylation status of CpG sites within the p16 CpG island in human tissue samples was not always homogeneous (17). Thus, different primer sets used in two kinds of assays might be the main reason accounting for detection difference of p16 methylation in these oral epithelial dysplasias.…”

“…S1; 150 bp for methylated copy and 151 bp for unmethylated copy) and analyzed using denatured high performance of liquid chromatography at Peking University School of Oncology. Paraffin blocks of xenografted p16-methylated RKO and p16-unmethylated MGC803 human cancer cell lines were used as positive and negative control for each experiment throughout the analysis procedure (including DNA extraction, bisulfite modification, methylation-specific PCR, and denatured high performance of liquid chromatography detection), respectively (17).…”

The p16-Specific Reactivation and Inhibition of Cell Migration Through Demethylation of CpG Islands by Engineered Transcription Factors

“…The methylation status of p16 CpG island was detected by the 150/151-bp methylation-specific PCR (MSP), 392-bp denatured high-performance liquid chromatography (DHPLC), or clone sequencing as described (Herman et al, 1996;Luo et al, 2006). In brief, the bisulfite-modified genomic DNA was amplified using the methylated/unmethylated p16-specific primer sets in the MSP assay or using the CpG-free universal primer set in the DHPLC and sequencing assays (Supplementary Table 4S).…”

Differentiation and adaptation epigenetic networks: Translational research in gastric carcinogenesis

“…Because the crucial CpG sites are not characterized for most CpG island-containing genes, we used the DHPLC assay to validate alterations of the methylation status of all 90 genes. DHPLC is a convenient assay for quantifying the proportion of methylated CpG islands we established previously [33,34]. Like bisulfite sequencing, PCR products (400-1000 bp) of methylated and unmethylated CpG islands amplified with CpG-free primers are analyzed with this assay.…”