Infrequent hypermethylation of the PTEN gene in Korean non-small-cell lung cancers

Abstract

CpG islands (CGIs) hypermethylation is implicated in the pathogenesis of many cancers, including lung cancer. The phosphate and tension homolog (PTEN) is a tumor suppressor that controls a variety of biological processes including cell proliferation, growth, migration, and death. The defects in PTEN regulation have a profound impact on carcinogenesis. Herein, we have examined the methylation status of the human PTEN gene in 137 primary non-small-cell lung cancers (NSCLCs) by using a methylation-specific PCR and correlated the results with clinicopathological features. Promoter methylation of the PTEN gene was observed in 5.1%, 2.9%, and 0.0% of three different CpG regions, which were localized at -1460 to -1263, -984 to -848, and -300 to -128 nucleotides upstream of the translation start site, respectively. Reverse transcription-PCR and immunohistochemical analysis showed the methylation of the CGI region at -984 to -848 correlated more accurately with PTEN expression. In addition, no significant correlation was found between PTEN methylation and clinicopathological factors, including the survival rates. These findings suggest that promoter methylation is not an important mechanism for PTEN deregulation in NSCLCs from Koreans.

Figure 1

Representative results of methylation‐specific PCR (MSP) and RT‐PCR analysis of the phosphate and tension homolog (PTEN) gene in non‐small‐cell lung cancer (NSCLC) patients. (A) Map of the PTEN promoter area. Open and closed boxes indicate 5′‐untranslated regions and protein coding domain of the first exon PTEN gene, respectively. +1 indicates the translation start site and the location of the three primer sets is shown. (B) The methylation status of PTEN gene was analyzed by MSP. CpGenome Universal methylated or unmethylated DNA (Chemicon, Temecula, CA, USA) was used as a positive control for the methylated or unmethylated products, respectively. Water was used as a negative control. N, non‐malignant tissue; T, tumor tissues; U and M, amplified product with primers that recognize the unmethylated or methylated sequences. (C) Expression of PTEN mRNA was performed on primary tissues by RT‐PCR. Amplified products were run on 2% agarose gel and appeared at positions corresponding to base pair lengths. GAPDH was used as an internal loading control.

Figure 2

Methylation‐specific PCR (MSP) and RT‐PCR analysis of the phosphate and tension homolog (PTEN) gene in 12 human lung cancer cell lines. The methylation status (A) and mRNA expression (B) of the PTEN gene was analyzed for 12 cell lines by MSP and RT‐PCR, respectively. (C) Methylation status of region 1 or 3 CGIs and expression of PTEN mRNA after demethylating agent 5‐aza‐2′‐deoxycytidine (5‐AzadC) treatment were examined by MSP and RT‐PCR analysis. (−) indicates vehicle alone treatment; (+) the 20 μm 5‐AzadC treatment for 3 days. The legend is the same as for Fig. 1.

Figure 3

Phosphate and tension homolog (PTEN) expression by immunohistochemistry in tumor and matched nonmalignant lung tissues. Representative photomicrographs of sections of lung tissue stained with a 1:100 dilution of polyclonal anti‐PTEN antibody. Tumor and matching nonmalignant lung tissues with unmethylated region 2 (sample 182 [A,B], sample 328 [E,F]) exhibited strong PTEN staining in the cytoplasm, but PTEN protein was undetectable in tumor tissue with methylation at the region 2 CGI (sample 291 [D]). PTEN expression was also detected in tumor tissue with methylation at the region 1 CGI (sample 182 [B]). Magnification, ×400.