BIOLOGY DIVISION

4.BIOLOGY DIVISION

    The primary project of the Biology Division is to elucidate the molecular basis of multistage human lung carcinogenesis. Positional and functional cloning of several cancer-related genes is under way to achieve this goal. Once a gene that is genetically or epigenetically altered in lung cancer cells has been isolated, functional analyses are performed to clarify the biological significance of those alterations in lung carcinogenesis. To understand lung cancer phenotypes at molecular levels, gene expression profiling of lung cancer cells has been added to this project. Associations between genetic polymorphisms and lung cancer susceptibility are also being investigated to evaluate the risk for lung cancer in each individual and to develop a novel way of lung cancer prevention.
A Tumor Suppressor Gene, MYO18B

    Lung cancer is now the leading cause of cancer-related death in the world. To understand the molecular process of lung cancer progression, genetic alterations in various progression stages have been analyzed in the Division ⁽⁵⁵⁾. In 2002, a novel myosin family gene, MYO18B, was isolated in the Division as a gene frequently deleted, mutated and hypermethylated in lung cancers. More frequent reduction or loss of MYO18B expression and restoration of MYO18B expression by trichostatin A (TSA) treatment suggested the contribution of histone deacetylation to the epigenetic inactivation of the MYO18B gene. Thus, histone modification of the MYO18B gene was examined in 8 lung cancer cell lines ⁽⁵⁶⁾. In 6 of 7 cell lines with reduced MYO18B expression, the levels of histone H3/H4 acetylation were lower than those in a cell line with MYO18B expression. Following treatment with TSA, the levels of histone H3/H4 acetylation increased in all 6 cell lines with restoration of MYO18B expression by TSA, whereas neither H3 nor H4 acetylation increased in cells without such a restoration. Significant correlations were observed between the levels of histone H3/H4 acetylation and MYO18B expression. Thus, it was suggested that histone deacetylation plays an important role in MYO18B silencing and is involved in lung carcinogenesis.
    Genetic and epigenetic alterations of the MYO18B gene were also examined in ovarian cancers ⁽⁵⁷⁾. Mutations were detected in 1/4 cell lines and 1/17 primaries. MYO18B expression was reduced in 4/4 cell lines and 12/17 primaries. Hypermethylation was observed in 2/3 cell lines and in 2/15 primaries. Thus, it was indicated that MYO18B alterations, including both epigenetic and genetic alterations, play an important role in ovarian carcinogenesis.
Molecular Markers for Lung Cancer

    To identify genes overexpressed in lung adenocarcinoma (AdC) cells, type II alveolar cells and bronchiolar epithelial cells, as well as AdC cells, were isolated by laser capture microdissection, and subjected to cDNA microarray analysis ⁽⁵⁸⁾. Four genes, TOP2A, MMP15, MX2 and KOC1, were commonly overexpressed in AdC cells in comparison with epithelial cells. Quantitative RT-PCR analysis further revealed that the TOP2A, MMP15, MX2 and KOC1 genes were overexpressed in 10/10 (100%), 8/10 (80%), 5/10 (50%) and 3/10 (30%) AdC cases, respectively. The TOP2A gene was commonly overexpressed in lung AdC cells, as previously reported. In addition, the MMP15 and MX2 genes were identified, for the first time, as being commonly overexpressed in lung AdC cells. The results strongly indicate that the MMP15 and MX2 genes can be novel markers for the molecular diagnosis of lung AdC.
DNA Repair Genes

    The OGG1 gene, isolated in the Division, encodes a protein to suppress mutagenesis induced by 8-hydroxyguanine (8OHG), an oxidatively damaged pro-mutagenic base. The mutation suppressive ability was compared between two common polymorphic proteins, OGG1-Ser326 and OGG1-Cys326, using a supF forward mutation assay ⁽⁵⁹⁾. Mutations by 8OHG were more efficiently suppressed in Ser326 transduced cells than Cys326 transduced cells, indicating therefore that Cys326 has a lower ability to prevent mutagenesis by 8OHG than Ser326 in human cells in vivo, supporting the results of association studies that Cys326 is a risk allele for several types of human cancers. In the human OGG1 gene, a genetic polymorphism is also present at codon 46 (Arg46Gln). Therefore, the abilities of three forms (Ser326, Cys326 and Gln46) to suppress oxidative mutagenesis were examined ⁽⁶⁰⁾. All three forms efficiently suppressed chemically induced and spontaneously occurring oxidative mutagenesis, and Gln46 had a weaker ability to suppress mutations. An electronic microarray technique for analysis of nine single-nucleotide polymorphisms in the OGG1 gene was developed ⁽⁶¹⁾.
    The DLD-1 colon cancer cell line is known to display a high mutation rate, and to carry mutations in the MSH6 and POLD1 gene. Therefore, the effect of wild-type MSH6 and POLD1 expression on the mutation rate at the HPRT locus was examined ⁽⁶²⁾. The mutation rates decreased more than 4-fold in MSH6-transfected cells, while they did not significantly decrease in POLD1-transfected cells, thus indicating that mutations in the MSH6 gene, and not in the POLD1 gene, are primarily responsible for the high mutation rates in DLD-1 cells.
Lung Cancer Susceptibility Genes

    To clarify whether alterations of smoking behavior and risk for lung cancer can be found in subjects with CYP2A6 variants, an epidemiological study was performed in male Japanese smokers ⁽⁶³⁾. The result indicated that CYP2A6 is a principal determinant not only of smoking behavior but also susceptibility to tobacco-related lung cancer.
HrK-Mediated Apoptosis

    Bcl-2 homology domain (BH) 3-only proteins of the Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. Hrk/DP5 is a BH3-only protein, however, the molecular mechanism of Hrk-mediated apoptosis remains unclear. By a yeast two-hybrid screening, p32, a mitochondrial protein, was identified as an Hrk-interacting protein. Hrk-induced apoptosis was suppressed by the expression of p32 mutants, and small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis, thus suggesting that p32 is a key molecule that links Hrk to mitochondria and is involved in the regulation of Hrk-mediated apoptosis ⁽⁶⁴⁾




4.BIOLOGY DIVISION



	The primary project of the Biology Division is to elucidate the molecular basis of multistage human lung carcinogenesis. Positional and functional cloning of several cancer-related genes is under way to achieve this goal. Once a gene that is genetically or epigenetically altered in lung cancer cells has been isolated, functional analyses are performed to clarify the biological significance of those alterations in lung carcinogenesis. To understand lung cancer phenotypes at molecular levels, gene expression profiling of lung cancer cells has been added to this project. Associations between genetic polymorphisms and lung cancer susceptibility are also being investigated to evaluate the risk for lung cancer in each individual and to develop a novel way of lung cancer prevention. A Tumor Suppressor Gene, MYO18B Lung cancer is now the leading cause of cancer-related death in the world. To understand the molecular process of lung cancer progression, genetic alterations in various progression stages have been analyzed in the Division ⁽⁵⁵⁾. In 2002, a novel myosin family gene, MYO18B, was isolated in the Division as a gene frequently deleted, mutated and hypermethylated in lung cancers. More frequent reduction or loss of MYO18B expression and restoration of MYO18B expression by trichostatin A (TSA) treatment suggested the contribution of histone deacetylation to the epigenetic inactivation of the MYO18B gene. Thus, histone modification of the MYO18B gene was examined in 8 lung cancer cell lines ⁽⁵⁶⁾. In 6 of 7 cell lines with reduced MYO18B expression, the levels of histone H3/H4 acetylation were lower than those in a cell line with MYO18B expression. Following treatment with TSA, the levels of histone H3/H4 acetylation increased in all 6 cell lines with restoration of MYO18B expression by TSA, whereas neither H3 nor H4 acetylation increased in cells without such a restoration. Significant correlations were observed between the levels of histone H3/H4 acetylation and MYO18B expression. Thus, it was suggested that histone deacetylation plays an important role in MYO18B silencing and is involved in lung carcinogenesis. Genetic and epigenetic alterations of the MYO18B gene were also examined in ovarian cancers ⁽⁵⁷⁾. Mutations were detected in 1/4 cell lines and 1/17 primaries. MYO18B expression was reduced in 4/4 cell lines and 12/17 primaries. Hypermethylation was observed in 2/3 cell lines and in 2/15 primaries. Thus, it was indicated that MYO18B alterations, including both epigenetic and genetic alterations, play an important role in ovarian carcinogenesis. Molecular Markers for Lung Cancer To identify genes overexpressed in lung adenocarcinoma (AdC) cells, type II alveolar cells and bronchiolar epithelial cells, as well as AdC cells, were isolated by laser capture microdissection, and subjected to cDNA microarray analysis ⁽⁵⁸⁾. Four genes, TOP2A, MMP15, MX2 and KOC1, were commonly overexpressed in AdC cells in comparison with epithelial cells. Quantitative RT-PCR analysis further revealed that the TOP2A, MMP15, MX2 and KOC1 genes were overexpressed in 10/10 (100%), 8/10 (80%), 5/10 (50%) and 3/10 (30%) AdC cases, respectively. The TOP2A gene was commonly overexpressed in lung AdC cells, as previously reported. In addition, the MMP15 and MX2 genes were identified, for the first time, as being commonly overexpressed in lung AdC cells. The results strongly indicate that the MMP15 and MX2 genes can be novel markers for the molecular diagnosis of lung AdC. DNA Repair Genes The OGG1 gene, isolated in the Division, encodes a protein to suppress mutagenesis induced by 8-hydroxyguanine (8OHG), an oxidatively damaged pro-mutagenic base. The mutation suppressive ability was compared between two common polymorphic proteins, OGG1-Ser326 and OGG1-Cys326, using a supF forward mutation assay ⁽⁵⁹⁾. Mutations by 8OHG were more efficiently suppressed in Ser326 transduced cells than Cys326 transduced cells, indicating therefore that Cys326 has a lower ability to prevent mutagenesis by 8OHG than Ser326 in human cells in vivo, supporting the results of association studies that Cys326 is a risk allele for several types of human cancers. In the human OGG1 gene, a genetic polymorphism is also present at codon 46 (Arg46Gln). Therefore, the abilities of three forms (Ser326, Cys326 and Gln46) to suppress oxidative mutagenesis were examined ⁽⁶⁰⁾. All three forms efficiently suppressed chemically induced and spontaneously occurring oxidative mutagenesis, and Gln46 had a weaker ability to suppress mutations. An electronic microarray technique for analysis of nine single-nucleotide polymorphisms in the OGG1 gene was developed ⁽⁶¹⁾. The DLD-1 colon cancer cell line is known to display a high mutation rate, and to carry mutations in the MSH6 and POLD1 gene. Therefore, the effect of wild-type MSH6 and POLD1 expression on the mutation rate at the HPRT locus was examined ⁽⁶²⁾. The mutation rates decreased more than 4-fold in MSH6-transfected cells, while they did not significantly decrease in POLD1-transfected cells, thus indicating that mutations in the MSH6 gene, and not in the POLD1 gene, are primarily responsible for the high mutation rates in DLD-1 cells. Lung Cancer Susceptibility Genes To clarify whether alterations of smoking behavior and risk for lung cancer can be found in subjects with CYP2A6 variants, an epidemiological study was performed in male Japanese smokers ⁽⁶³⁾. The result indicated that CYP2A6 is a principal determinant not only of smoking behavior but also susceptibility to tobacco-related lung cancer. HrK-Mediated Apoptosis Bcl-2 homology domain (BH) 3-only proteins of the Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. Hrk/DP5 is a BH3-only protein, however, the molecular mechanism of Hrk-mediated apoptosis remains unclear. By a yeast two-hybrid screening, p32, a mitochondrial protein, was identified as an Hrk-interacting protein. Hrk-induced apoptosis was suppressed by the expression of p32 mutants, and small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis, thus suggesting that p32 is a key molecule that links Hrk to mitochondria and is involved in the regulation of Hrk-mediated apoptosis ⁽⁶⁴⁾