Biochemistry Division

2. Biochemistry Division

　Genetic alterations, including genomic instability, and complex interactions between the genetic backgrounds of hosts and various environmental factors play important roles in multi-stage human carcinogenesis. The Biochemistry Division seeks to clarify the genetic and epigenetic alterations that are implicated in various stages of carcinogenesis by using animal models. Elucidation of the biological roles of poly(ADP-ribosyl)ation of nuclear proteins with respect to DNA damage responses and the molecular mechanisms involved in the stable maintenance of genomic integrity are also currently being undertaken.
1. Linkage Mapping of the Genetic Susceptibility to ACF Formation by PhIP on Rat Chromosome 16

　2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is one of the most abundant mutagenic heterocyclic amines (HCAs) contained in cooked meat and fish. PhIP induces colon tumors in rats when administered orally. Using aberrant crypt foci (ACF), a precancerous legion in the colon, as a surrogate marker for colon carcinogenesis, genetic linkage analysis has been carried out to identify various genetic traits regulating the susceptibility to colon carcinogenesis. F344 rats possess the dominant trait over ACI rats for the induction of ACF by PhIP.⁽⁴⁴⁾By linkage analysis using 170 progeny of (F344 x ACI)F1 x ACI backcross rats,⁽⁴⁵⁾ a susceptibility gene in F344 rats was mapped between D16Rat40 and D16Rat60 on Chromosome 16, giving a lod score of more than 4.0. No other locus gave a significant lod score of more than 2.0. Congenic rat strains which carry the susceptibility genes in F344 rats are now being generated to facilitate the identification of the responsible genes.
2. Carcinogenicity of PhIP in Various Mice Strains

　To investigate the carcinogenic actions of PhIP on various organs in mice, PhIP was administered to two different strains of mice. PhIP induced intestinal tumors in C57BL/6N mice in a dose-dependent manner, mainly at the small intestine. However, no tumor induction was observed in BALB/c mice by PhIP-administration, although spontaneous tumors developed in the small intestine. In addition, PhIP also induced lymphomas in male C57BL/6N mice, but not in females, at an incidence that was significantly higher than in the untreated control group. The susceptibility of mice to PhIP-induced carcinogenesis was therefore demonstrated to be affected by their genetic background. Reports related to this work can be found in the attached list of publications.⁽⁴⁶⁾
3. Molecular Mechanisms for the Induction of Minisatellite Mutations

　Minisatellite DNA sequences (MN), which are comprised of 5-100bp tandem repeats, are frequently mutated in various tumors of both humans and experimental animals. However, the underlying mechanisms for the induction of MN mutations are largely unknown. A mouse MN, Pc-1, contains (5ﾕGGGCA3ﾕ)n repeats, and this GC-rich repeat was revealed to form an intrastrand anti-parallel quartet (G4ﾕ-structure) by NMR and CD spectrum analyses. This specific, more complex structure of the repeats could be partially implicated in the hypermutable characters of MNs in cells. We have also identified and purified six MN-binding proteins from NIH3T3 cells. The biological function of these proteins, specifically in the maintenance of MN stability, is currently being evaluated.
4. Gene-disruption Study of Poly (ADP-ribose) Polymerase in Mice

　Poly(ADP-ribose) polymerase (Parp) plays an important role in the repair process of DNA strand breaks and maintenance of genomic integrity.⁽⁴⁷⁾Parp^-/- mice were generated by disrupting both of the alleles of exon 1 using the homologous recombination method, and the sensitivity of Parp^-/- mice to streptozotocin (STZ), which induces NAD depletion in isolated islet b-cells in vitro, was then analyzed. Progressive atrophy and loss of functional b-cells in islets were observed, however, the extent of b-cell death in Parp^-/- mice was markedly less than in wild type Parp^+/+ mice. These findings indicate an essential role for Parp in islet b-cell damage and the induction of diabetes as a consequence of treatment by STZ.
5. STGC Induction in Parp-Knockout ES Cell Tumors

　The effects of Parp deficiency on the tumorigenic activity of mouse embryonic stem (ES) cells were analyzed using Parp^+/+, Parp^+/- and Parp^-/- ES cells.⁽⁴⁸⁾ These ES cells showed similar tumorigenicity when injected subcutaneously into nude mice, however, microscopic examination of tumors derived from Parp^-/- clones showed the presence of syncytiotrophoblastic giant cells (STGC) containing single or multiple megalo-nuclei. These cells were present within large areas of intratumoral hemorrhage. No STGCs were observed in tumors of either Parp^+/- clones or the wild type Parp^+/+ cells. Therefore, the lack of Parp expression in ES cells contributes to the induction of STGCs during the development of teratocarcinoma.




	2. Biochemistry Division



		Genetic alterations, including genomic instability, and complex interactions between the genetic backgrounds of hosts and various environmental factors play important roles in multi-stage human carcinogenesis. The Biochemistry Division seeks to clarify the genetic and epigenetic alterations that are implicated in various stages of carcinogenesis by using animal models. Elucidation of the biological roles of poly(ADP-ribosyl)ation of nuclear proteins with respect to DNA damage responses and the molecular mechanisms involved in the stable maintenance of genomic integrity are also currently being undertaken. 1. Linkage Mapping of the Genetic Susceptibility to ACF Formation by PhIP on Rat Chromosome 16 　2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is one of the most abundant mutagenic heterocyclic amines (HCAs) contained in cooked meat and fish. PhIP induces colon tumors in rats when administered orally. Using aberrant crypt foci (ACF), a precancerous legion in the colon, as a surrogate marker for colon carcinogenesis, genetic linkage analysis has been carried out to identify various genetic traits regulating the susceptibility to colon carcinogenesis. F344 rats possess the dominant trait over ACI rats for the induction of ACF by PhIP.⁽⁴⁴⁾By linkage analysis using 170 progeny of (F344 x ACI)F1 x ACI backcross rats,⁽⁴⁵⁾ a susceptibility gene in F344 rats was mapped between D16Rat40 and D16Rat60 on Chromosome 16, giving a lod score of more than 4.0. No other locus gave a significant lod score of more than 2.0. Congenic rat strains which carry the susceptibility genes in F344 rats are now being generated to facilitate the identification of the responsible genes. 2. Carcinogenicity of PhIP in Various Mice Strains 　To investigate the carcinogenic actions of PhIP on various organs in mice, PhIP was administered to two different strains of mice. PhIP induced intestinal tumors in C57BL/6N mice in a dose-dependent manner, mainly at the small intestine. However, no tumor induction was observed in BALB/c mice by PhIP-administration, although spontaneous tumors developed in the small intestine. In addition, PhIP also induced lymphomas in male C57BL/6N mice, but not in females, at an incidence that was significantly higher than in the untreated control group. The susceptibility of mice to PhIP-induced carcinogenesis was therefore demonstrated to be affected by their genetic background. Reports related to this work can be found in the attached list of publications.⁽⁴⁶⁾ 3. Molecular Mechanisms for the Induction of Minisatellite Mutations 　Minisatellite DNA sequences (MN), which are comprised of 5-100bp tandem repeats, are frequently mutated in various tumors of both humans and experimental animals. However, the underlying mechanisms for the induction of MN mutations are largely unknown. A mouse MN, Pc-1, contains (5ﾕGGGCA3ﾕ)n repeats, and this GC-rich repeat was revealed to form an intrastrand anti-parallel quartet (G4ﾕ-structure) by NMR and CD spectrum analyses. This specific, more complex structure of the repeats could be partially implicated in the hypermutable characters of MNs in cells. We have also identified and purified six MN-binding proteins from NIH3T3 cells. The biological function of these proteins, specifically in the maintenance of MN stability, is currently being evaluated. 4. Gene-disruption Study of Poly (ADP-ribose) Polymerase in Mice 　Poly(ADP-ribose) polymerase (Parp) plays an important role in the repair process of DNA strand breaks and maintenance of genomic integrity.⁽⁴⁷⁾Parp^-/- mice were generated by disrupting both of the alleles of exon 1 using the homologous recombination method, and the sensitivity of Parp^-/- mice to streptozotocin (STZ), which induces NAD depletion in isolated islet b-cells in vitro, was then analyzed. Progressive atrophy and loss of functional b-cells in islets were observed, however, the extent of b-cell death in Parp^-/- mice was markedly less than in wild type Parp^+/+ mice. These findings indicate an essential role for Parp in islet b-cell damage and the induction of diabetes as a consequence of treatment by STZ. 5. STGC Induction in Parp-Knockout ES Cell Tumors 　The effects of Parp deficiency on the tumorigenic activity of mouse embryonic stem (ES) cells were analyzed using Parp^+/+, Parp^+/- and Parp^-/- ES cells.⁽⁴⁸⁾ These ES cells showed similar tumorigenicity when injected subcutaneously into nude mice, however, microscopic examination of tumors derived from Parp^-/- clones showed the presence of syncytiotrophoblastic giant cells (STGC) containing single or multiple megalo-nuclei. These cells were present within large areas of intratumoral hemorrhage. No STGCs were observed in tumors of either Parp^+/- clones or the wild type Parp^+/+ cells. Therefore, the lack of Parp expression in ES cells contributes to the induction of STGCs during the development of teratocarcinoma.

2. Biochemistry Division

1. Linkage Mapping of the Genetic Susceptibility to ACF Formation by PhIP on Rat Chromosome 16

2. Carcinogenicity of PhIP in Various Mice Strains

3. Molecular Mechanisms for the Induction of Minisatellite Mutations

4. Gene-disruption Study of Poly (ADP-ribose) Polymerase in Mice

5. STGC Induction in Parp-Knockout ES Cell Tumors