BIOLOGY DIVISION

4.BIOLOGY DIVISION

    In the Biology Division, genetic and functional analyses of genes whose structure and/or expression are altered in cancer cells are performed to understand the molecular basis of human carcinogenesis. It is important to elucidate the molecular basis of metastasis, thus, isolation and characterization of metastasis-associated genes is a major project of the Division. The associations of genetic polymorphisms and germline mutations, in genes such as tumor suppressors or DNA repair genes, with cancer susceptibility are also being investigated. In particular, involvement of base excision repair genes is being investigated by analyzing the functional diversities among polymorphic and mutated genes.
Genetic Alterations in Human Cancers

    Several chromosomal regions have been identified as being deleted, amplified or translocated in human cancers,^(61-⁶⁵⁾ and a novel gene, MEL1, was cloned as a target of chromosomal translocation in t(1;3)(p36;q21)-positive acute leukemia.⁽⁶⁶⁾ Extensive mutational analyses of the candidate tumor suppressor genes, p51, PPP1R3, DCC, BAX and SNF5/INI1, have been performed in several types of human cancers.^(67-⁷²⁾ p51 was not mutated in hepatocellular carcinoma.⁽⁶⁷⁾ PPP1R3 was infrequently mutated in several types of cancers.^(71,⁷²⁾ DCC mutations were rare, but expression was reduced in small cell lung carcinoma.⁽⁶⁹⁾ SNF5/ INI1 was not mutated in lung cancer.⁽⁷⁰⁾ Microsatellite instability as well as BAX mutations were frequently detected in T cell acute lymphoblastic leukemia cell lines.⁽⁶⁸⁾
Functional Analysis of Cancer-related Genes

    Functional regulation of p53 protein was investigated by analyzing the phosphorylation status of the protein. The Chk1 and Chk2 checkpoint kinases were shown to mainly phosphorylate Ser20 of p53.⁽⁷³⁾ In contrast, ATM kinase phosphorylates Ser15 of p53. Thus, ATM kinase was purified,⁽⁷⁴⁾ and it was shown that ATM kinase was activated by ionizing radiation throughout the cell cycle.⁽⁷⁵⁾ It was also found that stress signals utilize multiple pathways to stabilize p53,^(76,⁷⁷⁾ and that cooperative phosphorylation at multiple sites is required to activate p53.⁽⁷⁸⁾ In cells immortalized by the Tax gene of HTLV-1, p53 is inactive but abundant. It was now found that Tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration.⁽⁷⁹⁾ Upon DNA damage, p53 protein accumulates rapidly through a post-transcriptional mechanism(s) and is also activated as a transcription factor, which then leads to growth arrest or apoptosis. It is an important issue how these two different pathways are selected by p53. It was shown that phosphorylation of Ser46 regulates the transcriptional activation of apoptosis-inducing genes by p53.⁽⁸⁰⁾
Isolation and Characterization of Metastasis-associated Genes

    Several metastasis-associated genes were isolated by the mRNA differential display method.^(81-⁸³⁾ Ehm2, which is a novel NF2/ERM/4.1 superfamily gene, was cloned as a gene specifically expressed in high-metastatic K1735 murine melanoma cells.⁽⁸¹⁾ The LAMB3 and LAMC2 genes, encoding the laminine-5 subunits, were differentially expressed between small cell and non-small cell lung carcinomas.⁽⁸³⁾
Functional Diversities among Polymorphic Base Excision Genes

    To develop novel methods for the evaluation of cancer risk in each individual, genetic polymorphisms in the base excision DNA repair genes were determined in association with their enzymatic activities.^(84,⁸⁵⁾ Various types of OGG1 mRNA transcripts were identified, and among them, type-1a (nuclear form) with the Ser326Cys polymorphism was dominantly expressed in both cancerous and non-cancerous human cells. Adenine excisional repair function of the MYH protein was specific to A:8-hydroxyguanine under physiological salt concentrations. No apparent functional difference was detected between the two types of polymorphic MYH proteins.
Genetic Factors for Familial Aggregation of Gastric Cancer

    Criteria and guidelines for management of familial gastric cancer were defined by an international collaboration.⁽⁸⁶⁾ Since germline CHK2 mutations were previously identified in families with Li-Fraumeni syndrome, possible involvement of the CHK2 gene in familial clustering of gastric cancer was investigated. However, no germ-line CHK2 mutations were detected in familial gastric cancer cases.⁽⁸⁷⁾




4.BIOLOGY DIVISION



	In the Biology Division, genetic and functional analyses of genes whose structure and/or expression are altered in cancer cells are performed to understand the molecular basis of human carcinogenesis. It is important to elucidate the molecular basis of metastasis, thus, isolation and characterization of metastasis-associated genes is a major project of the Division. The associations of genetic polymorphisms and germline mutations, in genes such as tumor suppressors or DNA repair genes, with cancer susceptibility are also being investigated. In particular, involvement of base excision repair genes is being investigated by analyzing the functional diversities among polymorphic and mutated genes. Genetic Alterations in Human Cancers Several chromosomal regions have been identified as being deleted, amplified or translocated in human cancers,^(61-⁶⁵⁾ and a novel gene, MEL1, was cloned as a target of chromosomal translocation in t(1;3)(p36;q21)-positive acute leukemia.⁽⁶⁶⁾ Extensive mutational analyses of the candidate tumor suppressor genes, p51, PPP1R3, DCC, BAX and SNF5/INI1, have been performed in several types of human cancers.^(67-⁷²⁾ p51 was not mutated in hepatocellular carcinoma.⁽⁶⁷⁾ PPP1R3 was infrequently mutated in several types of cancers.^(71,⁷²⁾ DCC mutations were rare, but expression was reduced in small cell lung carcinoma.⁽⁶⁹⁾ SNF5/ INI1 was not mutated in lung cancer.⁽⁷⁰⁾ Microsatellite instability as well as BAX mutations were frequently detected in T cell acute lymphoblastic leukemia cell lines.⁽⁶⁸⁾ Functional Analysis of Cancer-related Genes Functional regulation of p53 protein was investigated by analyzing the phosphorylation status of the protein. The Chk1 and Chk2 checkpoint kinases were shown to mainly phosphorylate Ser20 of p53.⁽⁷³⁾ In contrast, ATM kinase phosphorylates Ser15 of p53. Thus, ATM kinase was purified,⁽⁷⁴⁾ and it was shown that ATM kinase was activated by ionizing radiation throughout the cell cycle.⁽⁷⁵⁾ It was also found that stress signals utilize multiple pathways to stabilize p53,^(76,⁷⁷⁾ and that cooperative phosphorylation at multiple sites is required to activate p53.⁽⁷⁸⁾ In cells immortalized by the Tax gene of HTLV-1, p53 is inactive but abundant. It was now found that Tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration.⁽⁷⁹⁾ Upon DNA damage, p53 protein accumulates rapidly through a post-transcriptional mechanism(s) and is also activated as a transcription factor, which then leads to growth arrest or apoptosis. It is an important issue how these two different pathways are selected by p53. It was shown that phosphorylation of Ser46 regulates the transcriptional activation of apoptosis-inducing genes by p53.⁽⁸⁰⁾ Isolation and Characterization of Metastasis-associated Genes Several metastasis-associated genes were isolated by the mRNA differential display method.^(81-⁸³⁾ Ehm2, which is a novel NF2/ERM/4.1 superfamily gene, was cloned as a gene specifically expressed in high-metastatic K1735 murine melanoma cells.⁽⁸¹⁾ The LAMB3 and LAMC2 genes, encoding the laminine-5 subunits, were differentially expressed between small cell and non-small cell lung carcinomas.⁽⁸³⁾ Functional Diversities among Polymorphic Base Excision Genes To develop novel methods for the evaluation of cancer risk in each individual, genetic polymorphisms in the base excision DNA repair genes were determined in association with their enzymatic activities.^(84,⁸⁵⁾ Various types of OGG1 mRNA transcripts were identified, and among them, type-1a (nuclear form) with the Ser326Cys polymorphism was dominantly expressed in both cancerous and non-cancerous human cells. Adenine excisional repair function of the MYH protein was specific to A:8-hydroxyguanine under physiological salt concentrations. No apparent functional difference was detected between the two types of polymorphic MYH proteins. Genetic Factors for Familial Aggregation of Gastric Cancer Criteria and guidelines for management of familial gastric cancer were defined by an international collaboration.⁽⁸⁶⁾ Since germline CHK2 mutations were previously identified in families with Li-Fraumeni syndrome, possible involvement of the CHK2 gene in familial clustering of gastric cancer was investigated. However, no germ-line CHK2 mutations were detected in familial gastric cancer cases.⁽⁸⁷⁾