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. 2003 Mar;111(6):887-95.
doi: 10.1172/JCI15475.

Inactivation of hMLH1 and hMSH2 by promoter methylation in primary non-small cell lung tumors and matched sputum samples

Yi-Ching Wang  1 , Yung-Pin Lu, Ruo-Chia Tseng, Ruo-Kai Lin, Jer-Wei Chang, Jung-Ta Chen, Chuen-Ming Shih, Chih-Yi Chen
Affiliations

Inactivation of hMLH1 and hMSH2 by promoter methylation in primary non-small cell lung tumors and matched sputum samples

Yi-Ching Wang et al. J Clin Invest. 2003 Mar.

Abstract

We performed a genetic and epigenetic study of the hMLH1 and hMSH2 mismatch repair genes in resected primary tumors from 77 non-small cell lung cancer (NSCLC) patients. The molecular alterations examined included the loss of mRNA and protein expression as well as promoter methylation, and the allelic imbalance of the chromosomal regions that harbor the genes. We found that 78% and 26% of patients showed at least one type of molecular alteration within the hMLH1 and hMSH2 genes, respectively. Promoter methylation of the hMLH1 gene was present in 55.8% of tumors, and was significantly associated with the reduction in mRNA and protein expression (P = 0.001). A 72% concordance of aberrant methylation in sputum samples with matched resected tumors was found. In addition, a 93% consistency between the promoter methylation and the mRNA expression of the hMSH2 gene was found in 14 female NSCLC patients. However, no correlation was found between the expression of hMLH1 and hMSH2 proteins and the allelic imbalance of five microsatellite markers closely linked to the genes. Our results suggest that hMLH1 is the major altered mismatch repair gene involved in NSCLC tumorigenesis, and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMLH1 gene may be identified in sputum samples to serve as a potential diagnostic marker of NSCLC.

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Figures

Figure 1
Figure 1
Immunohistochemical staining of hMLH1 protein expression (a–c) and hMSH2 protein expression (d–f) in paraffin-embedded sections of lung tumor specimens. hMLH1 nuclear immunoreactivity was found in a and b. hMSH2 nuclear immunoreactivity was found in d and e. Tumors negative for hMLH1 and hMSH2 are shown in c and f, respectively. Original magnification ×100.
Figure 2
Figure 2
Representative examples of multiplex RT-PCR analysis of the hMLH1 and hMSH2 genes in lung cancer patients. Amplification of the β-actin gene indicated the initial RNA template used for each reaction. Patient numbers are the sample numbers. N and T represent the paired normal and tumor lung cells from the same patient. M, molecular marker.
Figure 3
Figure 3
Representative examples of promoter methylation analysis of the hMLH1 (a–d) and hMSH2 (e) genes. (a–c) HpaII-based multiplex PCR assay for the hMLH1 promoter. (a) Normal lung tissues, (b) tumor lung samples, and (c) matched sputum samples, indicated by n, t, and s before subject identification number. +, HpaII digest; –, mock digest. Positions of the target hMLH1 gene and control IFN-β1 gene are indicated. Normal lung tissues showed no hMLH1 promoter methylation. MSP assay for the hMLH1 promoter (d) and (e) hMSH2 promoter. Primer sets used for amplification are designated U for unmethylated or M for methylated genes. The data for lymphocytes containing unmethylated hMSH2 are shown for the experimental control. + and – in the rows labeled methyl and mRNA represent the status of promoter methylation and mRNA expression.
Figure 4
Figure 4
Concordance analysis between protein expression, mRNA expression, and promoter methylation of hMLH1 (a) and hMSH2 (b). The percentage of cases is indicated on the y axis, whereas the type of comparison is shown on the x axis. + indicates positive protein expression (protein), positive mRNA expression (RNA), and hypermethylation (methyl) of the promoter. Numbers above bars indicate percentages of the total concordant group (gray sections) and nonconcordant group (white sections). P = 0.001 for association between protein expression, mRNA expression, and promoter methylation in the hMLH1 gene; P = 0.002 for association between protein expression and mRNA expression in the hMSH2 gene.
Figure 5
Figure 5
Effects of 5-aza-dC treatment on CL2 and CL1-5-F4 cells. (a) Demethylation analysis by MSP assay of hMLH1 and hMSH2 in lung cancer cell lines after 5-aza-dC treatment. The presence of the unmethylated product (U) after 5-aza-dC treatment indicates demethylation of the promoter in these cell lines. (b) RT-PCR analysis of mRNA expression of the hMLH1 and hMSH2 genes in cell lines. (c) Immunohistochemistry analysis of hMLH1 (original magnification ×100) and hMSH2 (original magnification ×200) protein expression in cell lines.
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References

    1. Department of Health, The Executive Yuan, Republic of China. 2000. General Health Statistics, 1999. In Health and vital statistics, Republic of China. Republic of China Press. Taipei, Taiwan, Republic of China. 89–112.
    1. Fishel R, et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell. 1993;75:1027–1038. - PubMed
    1. Bronner CE, et al. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature. 1994;368:258–261. - PubMed
    1. Arzimanoglou II, Gilbert F, Barber HR. Microsatellite instability in human solid tumors. Cancer. 1998;82:1808–1820. - PubMed
    1. Kane MF, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 1997;57:808–811. - PubMed

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