Biochemistry Division

2.BIOCHEMISTRY DIVISION

    Genetic background, genomic stability and various environmental factors play important roles in human carcinogenesis. The Biochemistry Division seeks to clarify various genetic alterations and the interactions between genetic background and environmental factors in multistage carcinogenesis using animal models. The biological roles of poly(ADP-ribose) polymerase and glycohydrolase with respect to DNA-damage responses and carcinogenesis are being elucidated, and the molecular mechanisms involved in maintaining genomic stability are also under investigation.
Multistep Genetic Alterations in Colon Carcinogenesis of Rats Induced by PhIP

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant carcinogenic heterocyclic amines produced while cooking meat and fish.^(40,⁴¹⁾ PhIP induces aberrant crypt foci (ACF) and colon cancers in rats.^(42-⁴⁴⁾ ACFs induced by PhIP could be categorized histologically into three types;namely, ACF of hyperplasia, ACF of mild dysplasia and ACF of severe dysplasia. Accumulation of b-catenin and/or decreased Apc protein were detected in a subset of ACFs, possibly putative precancerous lesions of PhIP-induced colon carcinogenesis.
Exploration of Candidate Susceptibility Genes for Colon Carcinogenesis by PhIP

    Using ACF as a surrogate biomarker, the colon cancer susceptibility gene (RCS) was mapped to chromosome 16 between D16Rat40 and D16Rat60. N8 generation was used to create congenic rat lines, and were subsequently intercrossed to produce progeny with a homozygous sensitive allele of F344 (F/F) and progeny with a homozygous resistant allele of ACI (A/A). Genes with 2-fold or greater changes in mRNA expression between F/F and A/A were screened. Thus far, of the 8740 genes on the Rat Genome U34A array, 5 were differentially expressed between F/F and A/A without PhIP-treatment and 25 after 2-week administration of PhIP. These genes are possible candidates for the gene governing susceptibility to colon carcinogenesis by PhIP.
Molecular Mechanisms of the Induction of Minisatellite Mutations

    Minisatellite DNA sequences (MNs) are frequently mutated in various types of tumors. The minisatellite repeat (5'-GGGCA-3')n in the mouse MN Pc-1 was found, by NMR and CD spectrum analyses, to form an intrastrand anti-parallel quadruplex (G4') structure. Since DNA polymerase progression in vitro is blocked at d(GGG)sites, the higher structure of these repeats could be partially responsible for the hypermutability of MNs in vivo. Several MN Pc-1 binding proteins have been isolated from a cultured mouse cell line. MNBP-B was demonstrated to bind to a (5'-GGCAG- 3')n sequence and to partly unfold the G4'-structure. MNBP-F was localized in the cytoplasm and transient expression of MNBP-F in HeLa cells induced nuclear shape deformities, indicating its possible involvement in the mitotic phase. Reports related to this work can be found in the attached list of publications.⁽⁴⁵⁾
Role of Poly(ADP-ribose) Polymerase-1 in Carcinogenesis

    Poly(ADP-ribose) polymerase-1 (Parp-1) is activated in nuclei and catalyzes poly(ADP-ribosyl)ation of proteins using NAD as a substrate, ⁽⁴⁶⁾ and is involved in recovery from various types of DNA damage.⁽⁴⁷⁾ Synthesized poly(ADP-ribose) is mainly degraded by poly(ADP-ribose) glycohydrolase (Parg).⁽⁴⁸⁾ To investigate the role of Parp-1 in carcinogenesis, Parp-1 knockout mice were subjected to carcinogenesis experiments, using N-nitrosobis(2-hydroxypropyl) amine (BHP) and azoxymethane (AOM). The incidence and numbers of liver hemangiosarcomas and lung adenomas induced by BHP and of colon cancers by AOM were significantly higher in Parp-1^-/- mice than in Parp-1^+/+ mice. These findings indicate Parp-deficiency to possibly be a risk factor for carcinogenesis. The relationship of Parg to carcinogenesis is now under investigation.
Lack of Parp-1 Confers Tumor Phenotype and Trophoblast Differentiation

    Parp-1^-/- ES cells developed teratocarcinoma-like tumors with trophoblastic giant cells (TGCs) which possess single or multiple megalo-nuclei, when injected subcutaneously into nude mice. The TGCs were present within large intratumoral hemorrhages. In situ hybridization analysis revealed that the tumors from Parp-1^-/- ES cell clones expressed placental lactogen I and 4311, indicating that they consisted of heterogeneous trophoblast cell subtypes. TGCs also expressed an angiogenic factor, proliferin, as well as the vasodilatory factors, adrenomedullin and eNOS. These findings indicated that a full spectrum of trophoblast differentiation may occur during tumor development specifically in the Parp-1 deficient state and that lack of Parp might promote differentiation of ES cells into spongiotrophoblast.




2.BIOCHEMISTRY DIVISION



	Genetic background, genomic stability and various environmental factors play important roles in human carcinogenesis. The Biochemistry Division seeks to clarify various genetic alterations and the interactions between genetic background and environmental factors in multistage carcinogenesis using animal models. The biological roles of poly(ADP-ribose) polymerase and glycohydrolase with respect to DNA-damage responses and carcinogenesis are being elucidated, and the molecular mechanisms involved in maintaining genomic stability are also under investigation. Multistep Genetic Alterations in Colon Carcinogenesis of Rats Induced by PhIP 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant carcinogenic heterocyclic amines produced while cooking meat and fish.^(40,⁴¹⁾ PhIP induces aberrant crypt foci (ACF) and colon cancers in rats.^(42-⁴⁴⁾ ACFs induced by PhIP could be categorized histologically into three types;namely, ACF of hyperplasia, ACF of mild dysplasia and ACF of severe dysplasia. Accumulation of b-catenin and/or decreased Apc protein were detected in a subset of ACFs, possibly putative precancerous lesions of PhIP-induced colon carcinogenesis. Exploration of Candidate Susceptibility Genes for Colon Carcinogenesis by PhIP Using ACF as a surrogate biomarker, the colon cancer susceptibility gene (RCS) was mapped to chromosome 16 between D16Rat40 and D16Rat60. N8 generation was used to create congenic rat lines, and were subsequently intercrossed to produce progeny with a homozygous sensitive allele of F344 (F/F) and progeny with a homozygous resistant allele of ACI (A/A). Genes with 2-fold or greater changes in mRNA expression between F/F and A/A were screened. Thus far, of the 8740 genes on the Rat Genome U34A array, 5 were differentially expressed between F/F and A/A without PhIP-treatment and 25 after 2-week administration of PhIP. These genes are possible candidates for the gene governing susceptibility to colon carcinogenesis by PhIP. Molecular Mechanisms of the Induction of Minisatellite Mutations Minisatellite DNA sequences (MNs) are frequently mutated in various types of tumors. The minisatellite repeat (5'-GGGCA-3')n in the mouse MN Pc-1 was found, by NMR and CD spectrum analyses, to form an intrastrand anti-parallel quadruplex (G4') structure. Since DNA polymerase progression in vitro is blocked at d(GGG)sites, the higher structure of these repeats could be partially responsible for the hypermutability of MNs in vivo. Several MN Pc-1 binding proteins have been isolated from a cultured mouse cell line. MNBP-B was demonstrated to bind to a (5'-GGCAG- 3')n sequence and to partly unfold the G4'-structure. MNBP-F was localized in the cytoplasm and transient expression of MNBP-F in HeLa cells induced nuclear shape deformities, indicating its possible involvement in the mitotic phase. Reports related to this work can be found in the attached list of publications.⁽⁴⁵⁾ Role of Poly(ADP-ribose) Polymerase-1 in Carcinogenesis Poly(ADP-ribose) polymerase-1 (Parp-1) is activated in nuclei and catalyzes poly(ADP-ribosyl)ation of proteins using NAD as a substrate, ⁽⁴⁶⁾ and is involved in recovery from various types of DNA damage.⁽⁴⁷⁾ Synthesized poly(ADP-ribose) is mainly degraded by poly(ADP-ribose) glycohydrolase (Parg).⁽⁴⁸⁾ To investigate the role of Parp-1 in carcinogenesis, Parp-1 knockout mice were subjected to carcinogenesis experiments, using N-nitrosobis(2-hydroxypropyl) amine (BHP) and azoxymethane (AOM). The incidence and numbers of liver hemangiosarcomas and lung adenomas induced by BHP and of colon cancers by AOM were significantly higher in Parp-1^-/- mice than in Parp-1^+/+ mice. These findings indicate Parp-deficiency to possibly be a risk factor for carcinogenesis. The relationship of Parg to carcinogenesis is now under investigation. Lack of Parp-1 Confers Tumor Phenotype and Trophoblast Differentiation Parp-1^-/- ES cells developed teratocarcinoma-like tumors with trophoblastic giant cells (TGCs) which possess single or multiple megalo-nuclei, when injected subcutaneously into nude mice. The TGCs were present within large intratumoral hemorrhages. In situ hybridization analysis revealed that the tumors from Parp-1^-/- ES cell clones expressed placental lactogen I and 4311, indicating that they consisted of heterogeneous trophoblast cell subtypes. TGCs also expressed an angiogenic factor, proliferin, as well as the vasodilatory factors, adrenomedullin and eNOS. These findings indicated that a full spectrum of trophoblast differentiation may occur during tumor development specifically in the Parp-1 deficient state and that lack of Parp might promote differentiation of ES cells into spongiotrophoblast.