Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive-representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome.

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The ING1 gene is involved in the regulation of the cell cycle, senescence, and apoptosis and is a novel candidate tumor suppressor gene. ING2, another gene in the ING family, was identified and cloned. The functions of ING1 and ING2 largely depend on the activity of p53. To determine whether an alteration in these genes plays a role in carcinogenesis and tumor progression in lung cancer, we screened 30 human lung cancer cell lines and 31 primary lung cancer tumors for mutations in these genes using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing. Our findings failed to uncover any mutations in these genes. We also examined the expression of ING1 and ING2 in lung cancer cell lines that either had or lacked a p53 mutation, and in a control bronchial epithelium cell line, using quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR). ING1 expression was up-regulated in all 7 lung cancer cell lines that had a p53 mutation, while the expression of ING2 was down-regulated in 6 of 7 lung cancer cell lines that had a p53 mutation. These results suggest that the ING1 and ING2 genes have different roles in lung carcinogenesis and progression, and the ING2 gene may be an independent tumor suppressor candidate on p53.

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Patients with lung cancer have a poor prognosis because of the high metastatic potential of the neoplasm. Therefore, identifying new molecular targets for anti-metastatic therapy is very important. To identify novel key factors of tumor metastasis in lung cancer, we established the gene expression profiles of two adenocarcinoma cell line variants, PC9/f9 and PC9/f14, by use of genome-wide human cDNA microarray analysis and comparing these profiles with that of the parental cell line, PC9. The PC9/f9 and PC9/f14 cell lines were selected for analysis because of their high metastatic potential. We identified five genes in the highly metastatic cell lines that showed a significantly enhanced or reduced expression and that had not been reported to be involved in metastasis of lung cancer. One of the overexpressed genes that was identified encoded the beta-galactoside-binding protein LGALS3 (Galectin 3). LGALS3 has been reported to be overexpressed in a variety of human cancers, but not in lung cancer, and to be involved in tumor metastasis. We examined the expression of LGALS3 by use of real-time quantitative reverse transcription-polymerase chain reaction in 38 lung cancer cell lines and in tumor tissue obtained by thoracoscopic biopsy. A population (10/30) of the non-small-cell lung cancers examined was found to overexpress the LGALS3 gene at levels three times higher than those of normal epithelial cells. In contrast, all small-cell lung cancers either failed to express the gene or expressed it at a very low level. The mean of the relative expression of the LGALS3 gene in non-small-cell lung cancer (3.065 +/- 3.976) was significantly higher than those of small-cell lung cancer (0.02 +/- 0.03) (P < 0.025). This is the first report of alterations of LGALS3 gene expression in lung cancer. These results, together with the previous reports on Galectin 3 function, suggest that Galectin 3 may play a role in the process of metastasis in non-small-cell lung cancer that overexpresses Galectin 3, but not in small-cell cancer. Accordingly, LGALS3 may be a phenotypic marker that excludes small-cell lung cancer and may represent a novel target molecule in non-small-cell lung cancer therapy.