Abstract
OBJECTIVE To investigate the mechanism of hMLHl deregulation in non-small cell lung cancer (NSCLC).
METHODS A genetic and epigenetic study of the hMLHl gene was performed using surgical primary tumors from 40 NSCLC patients and their corresponding noncancerous tissues. The molecular alterations examined included promoter méthylation by Hpa II/Msp I-based PCR analysis, loss of heterozygosity (LOH) by D3S1621 locus PCR-electrophore sis-silver staining, as well as the loss of protein expression by immunohistochemical analysis.
RESULTS The frequencies of hypermthylation, LOH and loss of protein expression of hMLHl were 67.5% (27/40), 65% (26/40) and 72.5% (29/ 40), respectively. Among 26 hMLHl gene LOH (+) cases, 21 (80.8%) showed hypermthylation, which was significantly higher than the group of LOH (-). The frequency of the double inactivation of the hMLHl gene by hypermthylation and LOH related to a loss of protein expression of 72.4% (21/29).
CONCLUSION Biallelic inactivation of the hMLHl gene by hypermthylation and LOH most likely will cause loss of hMLHl protein expression and play an Important role in the development of NSCLC. Therefore, controlling and monitoring for hypermthylation of the hMLHl gene may be partially useful for treatment and early diagnosis of NSCLC
keywords
Lung cancer is one of the most common malignancies in the world and is the leading cause of cancer mortality in China. Lung cancer is divided into two major histological categories, small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC).[1] In China, more than 80% of the lung cancers are NSCLC. Lung squamous cell carcinoma and adenocarcinoma are the two major types of NSCLC.[2]
Hypermethylation of cytosines in CpG-rich islands of the promoter regions of genes is one of the mechanisms of gene silencing. [3] In cancer, hypermethylation of the promoter regions is associated with transcriptional inactivation and loss of expression of tumor suppressor and other regulatory genes, constituting an alternative, epigenetic way for the loss of gene function. [4] The hMLHl gene, one of six human mismatch repair (MMR) genes, is a tumor suppressor gene that is located in the 3p21.3-22 region. To investigate the mechanism of hMLHl deregulation in NSCLC, in this research, we performed a genetic and epigenetic study of the hMLHl gene in resected primary tumors from 40 NSCLC patients and corresponding noncancerous tissues. The molecular alterations examined included promoter hypermethylation, loss of heterozygosity (LOH) as well as immunohisto chemical analysis.
Materials and Methods
Samples
The primary tumor samples and their corresponding noncancerous tissues were obtained from 40 Chinese patients who underwent surgery for NSCLC (31 male and 9 female; median age, 61 years, range, 42-70 years). Twenty eight of the tumors were squamous cell carcinomas and 12 were adenocarcinomas. Eighteen were Stages I+II and 22 were Stages III+IV, according to the Tumor Node Metastasis classification.
Genomic DNA extracted
Genomic DNA was extracted from frozen samples of the lung tumors and normal lung tissues using pro teinase-K digestion and phenol/chloroform purification followed by ethanol precipitation.
Aberrant methylation analysis of the hMLHl gene
The promoter methylation status of the hMLHl gene was investigated using Hpa II/Msp Lbased PCR analysis. Hpa II is a methylation sensitive enzyme and Msp I is a methylation insensitive enzyme. If the promoter region can be amplified after Hpa II digestion and cannot be amplified after Msp I digestion, it was defined as aberrant methylation. Primers of the hMLHl gene promoter region: 5'-CGC TGC TAG TAT TCG TGC-3' (sense), 5’-TCA GTG CCT CGT GCT CAC-3' (antisense), 603 bp. PCR was performed using the following conditions: 50 ng of DNA template, 10 pmol of each primer, 2.5 mM MgCl2, 1.5 mM dNTP mix, 1 x PCR buffer, and 1 unit of Ampli Taq in a 20-μl final volume. PCR cycles included one cycle of 95 °C for 10 min followed by 35 cycles at 95°C for 30 s, 55°C for 45 s, and 72 °C for 30 s, 72 °C 10 min, in a Perkin Elmer Gene Amp PCR system 9700.
LOH analysis of the hMLHl gene
The microsatellite polymorphic marker D3S1621 links together closely with the hMLHl locus. Cases were defined as LOH when an allele peak signal from tumor DNA was reduced by 50% compared with the normal counterpart. The methodology has been described by Tamotsu K et al.[5] Primers of D3SI621: 5'-TCT TTT AGT CAG CAG TTA TGT C-3' (sense), 5'-CCC ATA AGA AAT GTT ACT CTA C-3’ (antisense), 210 bp. PCR products were loaded on a 7% denaturing gel and then silver stained. PCR was performed on the genomic DNA samples using the following conditions: 200 ng of genomic DNA template, 10 pmol of each primer, 2.5 mM MgCl2, 1.5 mM dNTP mix, 1 xPCR buffer, and 1 unit of Ampli Taq in a 20-μl final volume. PCR cycles included one cycle of 95 °C for 10 min followed by 35 cycles at 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 30 s, 72°C 10 min, in a Perkin Elmer Gene Amp PCR system 9700.
Immunohistochemistry assay of hMLHl protein expression
The normal staining patterns for hMLHl are nuclear. Tumor cells that exhibited an absence of nuclear staining in the presence of non-neoplastic cells and infiltrating lymphocytes with nuclear staining were considered to have an abnormal pattern.
Statistical analysis
The statistical analysis was performed with the Student’s t-test and Chi-square test. P<0.05 was regarded as statistically significant.
Results
Aberrant methylation frequency of hMLHl in NSCLC
Among the 40 cases of NSCLC, there were 27 cases (67.5%) which demonstrated aberrant methylation of the hMLHl gene promoter region (Fig. 1, Table 1). The difference compared to the control group was significant (P<0.01).
Aberrant methylation of the hMLHl gene promoter region. H: Hpa II digestion, M; Msp I digestion, C: control, Mar: Marker (ϕX 174-Hinc II). 1:hyper methylation, 2: un-methylation.
Aberrant methylation frequency of hMLHl in NSCLC
LOH frequency in the hMLHl gene in NSCLC
Of the 40 cases of NSCLC, there were 26 cases (65%) showing LOH in the hMLHl gene (2, Table 2). In Fig.2, the upper band stands for one allele and the lower band stands for the other allele. Cases were defined as LOH when an allele peak signal from tumor DNA was reduced by 50% compared with the normal counterpart. The difference compared to the control group was significant (P<0.01).
LOH in the hMLHl of the D3S1621 locus. N:normal, T:tumor.
LOH frequency of the hMLHl gene in normal tissue and NSCLC
The relationship between LOH and methylation of hMLHl in NSCLC
In 26 of the hMLHl gene LOH (+) cases, 21 cases (80.8%) had hypermethylation of promoter regions, which was significantly higher than the group of LOH (-)(P<0.05, Table 3). This result suggested that aberrant methylation was related to LOH of hMLHl.
Correlation between methylation of hMLHl and LOH in NSCLC
Protein expression of hMLHl in NSCLC
The hMLHl protein expression in the control group was 100% (40/40). Among the 40 cases of NSCLC, 29 (72.5%) demonstrated loss of hMLHl expression. The difference with the control group was significant (P<0.05, Table 4). Of these 29 cases, there were 20 cases (69%) of squamous cell carcinoma and 9 cases (31 %) of adenocarcinoma.
Loss of hMLHl expression in NSCLC
Upon statistical analysis, in 27 hMLHl gene aberrant methylation cases, 26 cases (96.3%) had loss of protein expression of hMLHl, which was significantly higher than the un-methylated group (P<0.05). This result indicated that aberrant methylation was related to loss of protein expression of hMLHl. In 26 hMLHl gene-LOH (+) cases, 23 cases (88.5%) had loss of protein expression of hMLHl, which was significantly higher than the group of LOH (-)(P<0.05). These results also suggested that LOH was relatedo loss of protein expression of the hMLHl gene.
Among the 29 cases showing loss of hMLHl protein expression, 21 cases (72.4%) displayed LOH and aberrant methylation. On the other hand, of the 11 cases showing positive hMLHl expression, 7 cases (63.6%) had neither LOH nor aberrant methylation. These results suggested that aberrant methylation combined with LOH was related to loss of hMLHlprotein expression.
DISCUSSION
The development of human cancer is generally thought to be a multiple process that involves multiple genetic and epigenetic changes. Recently, several studies have shown that methylation of CpG islands located within the promoter regions of tumor suppressor genes is a frequent event in the development of several human malignancies.[6] This epigenetic modification has been proposed to be an alternative way of inactivation of tumor suppressor genes in cancer.[7] Since gene promoter hypermethylation and chromatin structure alteration are comparable with genetic mutations or deletions of tumor suppressor genes in cancer, it is very urgent to find some target genes that are inactivated by hypermethylation. In fact, there are many examples of CpG-rich islands methylation-mediated transcriptional silencing of tumor suppressor genes, such as pl5, pl6, APC, Rb, hMLHl, E-cad etc. In our study, we have found that hMLHl is a target gene for hypermethylation in NSCLC. This aberrant methylation can cause cancer by inactivation of the hMLHl gene and lead to a series of molecular events.
The hMLHl gene, one of the six human mismatch repair (MMR) genes, is a tumor suppressor gene that is located in the 3p21.3-22 region. MMR can correct base mismatchs during the DNA replication progress, eliminating the unpaired base sequence.[8, 9] Inactivation of the hMLHl gene leading to loss of protein expression plays an important role in NSCLC carcinogenesis. Chromosome 3p allelic losses are frequent events in many types of cancers including lung cancer. LOH at certain chromosomal loci accumulates during tumor progression.[10] Previous studies have shown that chromosomal 3p21 regions were found to be hemizygously deleted in 55% of the patients [11] and that LOH of hMLHl plays an important role in NSCLC carcinogenesis. At the present time, the mechanism of hMLHl gene transcriptional inactivation in NSCLC is still unclear.
In the present study, the hypermethylation in the promoter region and loss of heterozygosity (LOH) of the hMLHl gene were frequent (65% and 67.5%, respectively) in NSCLC. We found that 72.4% (21/29) of the cases with negative hMLHl protein expression showed hypermethylation and LOH at the same time. Our results suggested that hMLHl may be inactivated in NSCLC in accordance with the Knudson’s two-hit inactivation model,[12] involving deletion of one allele and hypermethylation of the other. Previous work has shown that LOH at the hMLHl gene region was observed frequently in NSCLC. The present study demonstrated that hMLHl hypermethylation significantly correlated with LOH at the hMLHl region in NSNCLC. These results suggest that hMLHl gene alterations may play a role in the NSCLC carcinogenesis via a two-hit mechanism, including epigenetic changes for tumor suppressor-gene inactivation. Biallelic inactivation of the hMLHl gene by hypermethylation and LOH most likely will cause loss of hMLHl expression and play an important role in the development of NSCLC. Therefore, controlling and monitoring for hypermethylation of the hMLHl gene may be useful for treatment and early diagnosis of NSCLC.
- Received April 23, 2006.
- Accepted May 26, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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