Biochemistry Division

2.BIOCHEMISTRY DIVISION

    Research in the Biochemistry Division is focused on elucidating multi-step genetic alterations in carcinogenesis and genetic factors that influence the susceptibility of individuals to the development of cancers using animal models. The biological roles of poly (ADP-ribose) metabolism in carcinogenesis and the molecular mechanisms underlying the maintenance of genomic integrity are also under investigation.
Genetic Alterations in Rat Colon Cancers Induced by PhIP

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces aberrant crypt foci (ACF) and colon cancers in rats.^(48-⁵⁰⁾ Approximately one-fourth of ACF were diagnosed as dysplastic, and they demonstrated b-catenin accumulation and one-fourth harbored mutations in the same way as colon tumors. These dysplastic ACF were strongly considered as candidates for preneoplastic lesions of PhIP-induced colon carcinogenesis. Gene expression profiles in PhIP-induced colon tumors were analyzed by DNA Chip technology. Thirty-one of 8,740 genes or ESTs were up-regulated (3-fold or more) commonly in colon tumors of both F344 and ACI rats, and 41 were down-regulated (3-fold or less). Some genes were differentially regulated between F344 and ACI, and were considered to be involved in the differential susceptibility to colon carcinogenesis among rat strains.
Exploration of Susceptibility Genes for Colon Carcinogenesis by PhIP

    The congenic rat strain, harboring the F344-derived susceptibility gene (Sct) to colon carcinogenesis on rat chromosome 16, showed higher susceptibility to ACF induction by PhIP. Differentially expressed genes in the colon between the progeny with homozygous Sct alleles of F344 (F_Sct/F_Sct) and homozygous Sct alleles of ACI (A_Sct/A_Sct) were investigated using GeneChip (Affymetrix). Thirty of 8,740 genes were differentially expressed between F_Sct/F_Sct and A_Sct/A_Sct by 2-fold or more. Four of them were mapped on chr 16, and a gene encoding N-acetyltransferase, which is required for metabolic activation of PhIP, was also included.
Molecular Mechanisms Underlying Induction of Minisatellite Mutations

    The minisatellite sequences (MNs) are frequently altered in various types of tumors. The tandem repeat of d(GGCAG)n in the mouse hypervariable MN Pc-1 was demonstrated to form an intramolecular quadruplex (G4') structure and to cause arrest of DNA synthesis at d(GGG)n sites. The G4' structure of Pc-1 and other Pc-1-like MN repeats could be responsible for the hypermutable feature of these MNs in vivo. Six MN Pc-1 binding proteins (MNBPs) were isolated from NIH3T3 cells.^(51,⁵²⁾ One of them, MNBP-B, bound to single-stranded d(GGCAG)n and other G-rich repetitive sequences, such as d(GTTAGG)n. In addition, MNBP-B unfolded the G4' structure of d(GGCAG)₅ and abrogated the arrest of DNA synthesis at d(GGG)n sites. MNBP-B also unfolded the G4' structure of the telomeric d(TTAGGG)₄ repeat. MNBP-F, which binds to the C-rich strand of Pc-1, was located in the nucleoli and on some fiber-like structures in the cytoplasm. Transient expression of the full length of MNBP-F induced nuclear shape deformities, suggesting its involvement in karyokinesis.
Involvement of Poly(ADP-ribose) Metabolism in Carcinogenesis

    Among the poly(ADP-ribose)polymerase family of proteins, Parp-1 plays a dominant role in DNA damage response. The incidence of tumors induced by alkylating agents, N-nitroso-bis(2-hydroxypropyl)amine and azoxymethane, was significantly higher in Parp-1 ^-/- mice than in Parp-1 ^+/+ mice.⁽⁵³⁾ Parp-1 ^-/- and Parp-1 ^+/+ embryonic fibroblasts (EFs) were immortalized spontaneously during in vitro culture. Parp-1 ^-/- EFs showed extensive hyperploidy compared to Parp-1 ^+/+ EFs.⁽⁵⁴⁾ Most of the Parp-1 ^-/- and Parp-1 ^+/+ EFs contained a single nucleus, suggesting that Parp-1 -deficiency could result in the perturbation of ploidy control through endoreduplication. In relation to this, the induction of trophoblast giant cells (TGCs) was observed during the development of teratocarcinoma-like tumors from Parp-1 ^-/- ES cells grafted in nude mice.⁽⁵⁵⁾ TGCs are present in the placenta and differentiate from spongiotrophoblasts through repeated endoreduplication. Disruption of Parp-1 may promote endoreduplication and result in the induction of TGCs from spongiotrophoblasts. Syncytiotrophoblastic giant cells (STGCs) observed in human germ cell tumors are very similar to TGCs. Alteration of Parp-1 gene in human tumors, including germ cell tumors is now ongoing. To understand the role of poly(ADP-ribose) degradation by poly(ADP-ribose) glycohydrolase (Parg) in carcinogenesis, specific Parg inhibitors were screened from a fermentation broth of actinomycetes. A cyclic hexadepsipeptide antibiotic, PD124, 966, and a novel cyclic peptide antibiotic, pargamicin, were found to inhibit Parg activity.




2.BIOCHEMISTRY DIVISION



	Research in the Biochemistry Division is focused on elucidating multi-step genetic alterations in carcinogenesis and genetic factors that influence the susceptibility of individuals to the development of cancers using animal models. The biological roles of poly (ADP-ribose) metabolism in carcinogenesis and the molecular mechanisms underlying the maintenance of genomic integrity are also under investigation. Genetic Alterations in Rat Colon Cancers Induced by PhIP 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces aberrant crypt foci (ACF) and colon cancers in rats.^(48-⁵⁰⁾ Approximately one-fourth of ACF were diagnosed as dysplastic, and they demonstrated b-catenin accumulation and one-fourth harbored mutations in the same way as colon tumors. These dysplastic ACF were strongly considered as candidates for preneoplastic lesions of PhIP-induced colon carcinogenesis. Gene expression profiles in PhIP-induced colon tumors were analyzed by DNA Chip technology. Thirty-one of 8,740 genes or ESTs were up-regulated (3-fold or more) commonly in colon tumors of both F344 and ACI rats, and 41 were down-regulated (3-fold or less). Some genes were differentially regulated between F344 and ACI, and were considered to be involved in the differential susceptibility to colon carcinogenesis among rat strains. Exploration of Susceptibility Genes for Colon Carcinogenesis by PhIP The congenic rat strain, harboring the F344-derived susceptibility gene (Sct) to colon carcinogenesis on rat chromosome 16, showed higher susceptibility to ACF induction by PhIP. Differentially expressed genes in the colon between the progeny with homozygous Sct alleles of F344 (F_Sct/F_Sct) and homozygous Sct alleles of ACI (A_Sct/A_Sct) were investigated using GeneChip (Affymetrix). Thirty of 8,740 genes were differentially expressed between F_Sct/F_Sct and A_Sct/A_Sct by 2-fold or more. Four of them were mapped on chr 16, and a gene encoding N-acetyltransferase, which is required for metabolic activation of PhIP, was also included. Molecular Mechanisms Underlying Induction of Minisatellite Mutations The minisatellite sequences (MNs) are frequently altered in various types of tumors. The tandem repeat of d(GGCAG)n in the mouse hypervariable MN Pc-1 was demonstrated to form an intramolecular quadruplex (G4') structure and to cause arrest of DNA synthesis at d(GGG)n sites. The G4' structure of Pc-1 and other Pc-1-like MN repeats could be responsible for the hypermutable feature of these MNs in vivo. Six MN Pc-1 binding proteins (MNBPs) were isolated from NIH3T3 cells.^(51,⁵²⁾ One of them, MNBP-B, bound to single-stranded d(GGCAG)n and other G-rich repetitive sequences, such as d(GTTAGG)n. In addition, MNBP-B unfolded the G4' structure of d(GGCAG)₅ and abrogated the arrest of DNA synthesis at d(GGG)n sites. MNBP-B also unfolded the G4' structure of the telomeric d(TTAGGG)₄ repeat. MNBP-F, which binds to the C-rich strand of Pc-1, was located in the nucleoli and on some fiber-like structures in the cytoplasm. Transient expression of the full length of MNBP-F induced nuclear shape deformities, suggesting its involvement in karyokinesis. Involvement of Poly(ADP-ribose) Metabolism in Carcinogenesis Among the poly(ADP-ribose)polymerase family of proteins, Parp-1 plays a dominant role in DNA damage response. The incidence of tumors induced by alkylating agents, N-nitroso-bis(2-hydroxypropyl)amine and azoxymethane, was significantly higher in Parp-1 ^-/- mice than in Parp-1 ^+/+ mice.⁽⁵³⁾ Parp-1 ^-/- and Parp-1 ^+/+ embryonic fibroblasts (EFs) were immortalized spontaneously during in vitro culture. Parp-1 ^-/- EFs showed extensive hyperploidy compared to Parp-1 ^+/+ EFs.⁽⁵⁴⁾ Most of the Parp-1 ^-/- and Parp-1 ^+/+ EFs contained a single nucleus, suggesting that Parp-1 -deficiency could result in the perturbation of ploidy control through endoreduplication. In relation to this, the induction of trophoblast giant cells (TGCs) was observed during the development of teratocarcinoma-like tumors from Parp-1 ^-/- ES cells grafted in nude mice.⁽⁵⁵⁾ TGCs are present in the placenta and differentiate from spongiotrophoblasts through repeated endoreduplication. Disruption of Parp-1 may promote endoreduplication and result in the induction of TGCs from spongiotrophoblasts. Syncytiotrophoblastic giant cells (STGCs) observed in human germ cell tumors are very similar to TGCs. Alteration of Parp-1 gene in human tumors, including germ cell tumors is now ongoing. To understand the role of poly(ADP-ribose) degradation by poly(ADP-ribose) glycohydrolase (Parg) in carcinogenesis, specific Parg inhibitors were screened from a fermentation broth of actinomycetes. A cyclic hexadepsipeptide antibiotic, PD124, 966, and a novel cyclic peptide antibiotic, pargamicin, were found to inhibit Parg activity.