Determination of O 6 -Methylguanine DNA Methyltransferase Promoter Methylation in Non-Small Cell Lung Cancer

Introduction

One of the most common causes of death throughout the world is lung cancer (Li et al., 2009). In Turkey in 2005, it was reported that lung cancer is the first and fourth most common cause of cancer in men and women, respectively. Non-small cell lung cancer (NSCLC) accounts for more than 85% of primary lung cancers and approximately two-thirds of NSCLC patients are diagnosed at an advanced stage (Ministry of Health, Republic of Turkey, 2009).

Genetic and epigenetic alterations are a hallmark of several human diseases. Epigenetic alterations such as DNA methylation in a cell can alter the expression profile of the cell, allowing it to behave differently as in cancer. The most recognized epigenetic alteration in the initiation and progression of cancer was the finding of the DNA methylation drift in tumor-related genes including tumor suppressor genes and DNA repair genes.

O⁶-Methylguanine DNA methyltransferase (MGMT) removes mutagenic and cytotoxic adducts from the O⁶ position of guanine. MGMT plays an important role in protecting cells because alkylation of the O⁶ position of guanine is one of the most important events in mutagenesis.

Approximately 20%-25% of human tumors lack detectable MGMT activity, and one of the most common reasons for the loss of MGMT expression is the promoter methylation of the gene (Silber, 1998, 1999). It has been reported that the frequency of promoter methylation of MGMT was increased in a variety of human cancers, including lung cancer (Pegg, 2000; Esteller, 2007; Jacinto and Esteller, 2007).

The objective of the present study was to determine the frequency of MGMT methylation in NSCLC. We also examined the relationship between methylation of MGMT and clinicopathologic parameters including sex, age, smoking history, and clinical stage.

Materials and Methods

Results

The clinicopathological characteristics of the 80 patients described in this study are summarized in Table 2.

Discussion

CpG promoter methylation is one of the most common mechanisms associated with loss of expression of a number of cancer-associated genes in a variety of human cancers, including NSCLC. It is known that the methylation of tumor suppressor genes (TSGs) and other tumor-related genes is observed as an early event in lung carcinogenesis.

Esteller et al. (1999b) have studied the methylation status of different TSGs, including p16, DAP kinase, and GSTP1 and the MGMT gene promoter in NSCLC, and have detected MGMT promoter methylation in 66% of the tumors. In another study (Esteller et al., 1999a), they reported that increased protein expression and decreased enzymatic activity of MGMT were related to hypermethylation of MGMT in NSCLC. It was also found that the loss of its expression was associated with increased frequency of K-ras mutation in 21% of NSCLC (Zöchbauer-Müller et al, 2001). Brabender et al. (2003) observed higher hypermethylation frequency in 38% of cancerous tissues compared with 18% of normal tissues for MGMT gene. A list of all loci reported to be methylated in NSCLC have been reviewed recently by Anglim and Alonzo (2008). They observed that the frequency of promoter methylation of MGMT gene ranged from 10% to 100% in the patients. In this study, methylation of the MGMT gene promoter was seen in 64% of tumors. There was a significant correlation between MGMT methylation and tumor stage, and the frequency of MGMT promoter methylation was 75%, 61.7%, and 58.8% for stages I, II, and III, respectively. The methylation status identified with the primers used in our study has been shown to be associated with loss of MGMT protein expression in a variety of human tumors, including NSCLC. It is well known that the promoter methylation of the DNA repair gene MGMT resulting in the inactivation of the gene product is a common event in primary human cancer (Esteller et al., 1999a). Thus, our results strongly suggest that loss of expression of the MGMT gene by promoter methylation is a frequent and early event in the carcinogenesis of NSCLC.

The differences in the frequency of the promoter methylation of MGMT may be due to diversity in the methodology of bisulfite treatment, methylation-specific real-time polymerase chain reaction, and primer sequences used. Bisulfite treatment is an important step for accurate methylation-specific real-time polymerase chain reaction, and depurination has been identified as the main cause of DNA fragmentation during bisulfite treatment (Raizis et al., 1995; Genereux et al., 2008). Research on epigenetic changes resulting from internal and external environmental factors has also shown that the factors can have a strong effect on epigenetic changes.

In the present study, we did not observe statistically age-related methylation for MGMT gene, but MGMT was more frequently methylated among older patients compared with younger patients, indicating age-specific methylation. The presence of association of MGMT methylation with age is consistent with previous studies showing age-related methylation in NSCLC (Belinsky et al., 2002; Pulling et al., 2003; Liu et al. 2006).

There have been some studies that focused on the association between smoking and MGMT gene promoter methylation in lung cancer, and the results showed some disagreements among studies (Liu et al., 2006): promoter methylation of the MGMT genes was detected in 39.5% smokers and 12.2% nonsmokers. Our results showing higher frequencies of promoter methylation for the MGMT gene—but not significantly different—in smokers are consistent with those of previous studies (Pulling et al., 2003; Toyooka et al., 2003; Liu et al., 2006) reporting that the frequency of MGMT methylation was higher in nonsmokers (66%) than in smokers (47%) in lung adenocarcinomas. They also observed that the MGMT methylation affecting somatic mutations such as K-ras mutation in cancer cells was seen in the early stage of carcinogenesis. The reason for the disagreement on the frequencies of promoter methylation for MGMT gene may be due to limited sample size and differences among definitions of smoking status.

In conclusion, the present study provides strong evidence for a higher frequency of promoter methylation for the MGMT gene in NSCLC, indicating that it is a common event during the carcinogenesis of NSCLC, although the limitation of this study may be the small number of each clinicopathological parameter available for our study. It is still a matter of controversy whether the MGMT methylation is a biomarker of aggressive tumor behavior and also of cancer progression. In addition to genetic biomarkers, epigenetic biomarkers such as MGMT methylation associated with loss of its protein expression may be useful for early detection of preneoplastic lesions and also the progression of responses and outcomes in NSCLC treatment.