Biochemistry Division

2.BIOCHEMISTRY DIVISION

　Multistep genetic alterations, genomic instability and complex interactions between the genetic backgrounds and various environmental factors play important roles in human carcinogenesis.⁽³⁷⁾ The Biochemistry Division has been conducting investigations to clarify the genetic alterations involved in various stages of carcinogenesis using animal models. Elucidation of the biological roles of poly(ADP- ribose)polymerase and glycohydrolase with respect to carcinogenesis and tumor phenotypes, and the molecular mechanisms underlying the stable maintenance of genomic integrity are also currently under investigation.
1. Genetic Traits for the Susceptibility to ACF Induction by PhIP

　2- Amino- 1- methyl- 6- phenylimidazo [ 4,5- b] pyridine (PhIP)is one of the most abundant carcinogenic heterocyclic amines contained in cooked meat and fish. In rats, PhIP induces colon cancers and aberrant crypt foci (ACF), a precancerous lesion of the colon.^(38,³⁹⁾ Using ACF as a marker, QTL analysis has been employed to identify genetic traits that regulate the susceptibility to colon carcinogenesis. The susceptibility trait in F344 rats was mapped to chromosome 16 between D16Rat40 and D16Rat60.⁽⁴⁰⁾ Congenic rat strains with this genetic region were more sensitive to ACF induction by PhIP than ACI. Potential resistant traits in ACI were also mapped to chromosomes 6 and 9 by preliminary genome- wide linkage analysis, using a subset of (BUF x ACI)F1 x BUF backcross progeny with extreme phenotypes. Investigation into the genetic interactions between these traits is currently ongoing.
2. Molecular Mechanisms for the Induction of Minisatellite Mutations

　Minisatellite DNA sequences (MN), comprising 5- 100- bp tandem repeats, are frequently mutated in various tumors, however, the molecular mechanisms underlying the induction of MN mutations are largely unknown. The minisatellite repeat (5 'GGGCA3 ')n in the mouse MN Pc- 1 was found to form an intrastrand anti- parallel quadruplex (G4 '- quartet)by NMR and CD spectrum analyses.⁽⁴¹⁾ Since DNA polymerase progression in vitro is blocked at d(GGG) sites, the higher structure of these repeats could be partially implicated in the hypermutable characteristic of MNs in vivo. One of the MN- binding proteins, MNBP- B, was demonstrated to bind to a (5 'GGGCA3 ')n sequence with a high affinity, and partly unfolds the G4'- structure.
3. Functional Analysis of Serine/ Threonine Phosphatase

　The biological functions of serine/ threonine phosphatases are regulated by forming molecular complexes with other cellular components. Genetic alterations in these regulatory subunits in colon cancers are being investigated. PP5 has recently been demonstrated to bind CDC16 and CDC27, which are essential components in the APC complex. In a recent study, PP5 was shown to interact with the HMG BOX Protein- 1 in a yeast two- hybrid system. Since this HMG BOX protein was known to interact with the Rb protein, PP5 could also play an important role in cell- cycle machinery. Reports related to this work can be found in the attached list of publications.⁽⁴²⁾
4. Involvement of Parp in Carcinogenesis and Tumor Phenotype

　Poly(ADP- ribose)polymerase (Parp) is involved in the recovery from various types of DNA damage.^(43-⁴⁵⁾ The role of Parp in N- nitrosobis(2- hydroxypropyl) amine (BHP)- induced carcinogenesis was studied.Parp^-/- and Parp^+/+ male mice were administered BHP through drinking water for 20 weeks. The incidence and numbers of liver hemangiomas induced by BHP were significantly higher in Parp^-/- mice than in Parp^+/+ mice.Parp^-/- mice also developed hemangiosarcomas in the liver. Lung adenomas were also induced in Parp^-/- mice, but not in Parp^+/+ mice. These findings indicate that Parp- deficiency could be a risk factor for carcinogenesis. Parp^-/- embryonic stem (ES)cells developed teratocarcinoma- like tumors with syncyticotrophoblastic giant cells (STGCs), when injected subcutaneously into nude mice.⁽⁴⁶⁾ These STGCs possessed micro- villi on their surfaces and were positively stained with an anti- prolactin antibody, which implies that loss of Parp induces trophoblast differentiation of ES cells during tumor formation.⁽⁴⁷⁾
5. Function of Poly(ADP-ribose)Glycohydrolase (Parg)

　Parg is the main enzyme engaged in poly(ADP- ribose)degradation. Parg was purified as a 60- kDa protein from rat testes. A rat Parg cDNA encoding a 109- kDa protein was isolated and it was found that the C- terminal half functions as a catalytic domain. A potential sequence for a leucine- rich type nuclear export signal was found at the N- terminal half.⁽⁴⁸⁾ The rat Parg gene was mapped to the distal region of chr 16p at the same position as D16Mit2 by linkage analysis using a (F344xACI)F1xACI backcross panel.⁽⁴⁹⁾ The human Parg gene was also mapped by fluorescence in situ hybridization to Chr 10q11.23- 21.1,⁽⁵⁰⁾ where loss of heterozygosity and chromosomal rearrangements were reported in glioblastomas and thyroid papillary carcinomas, respectively.




2.BIOCHEMISTRY DIVISION



	Multistep genetic alterations, genomic instability and complex interactions between the genetic backgrounds and various environmental factors play important roles in human carcinogenesis.⁽³⁷⁾ The Biochemistry Division has been conducting investigations to clarify the genetic alterations involved in various stages of carcinogenesis using animal models. Elucidation of the biological roles of poly(ADP- ribose)polymerase and glycohydrolase with respect to carcinogenesis and tumor phenotypes, and the molecular mechanisms underlying the stable maintenance of genomic integrity are also currently under investigation. 1. Genetic Traits for the Susceptibility to ACF Induction by PhIP 　2- Amino- 1- methyl- 6- phenylimidazo [ 4,5- b] pyridine (PhIP)is one of the most abundant carcinogenic heterocyclic amines contained in cooked meat and fish. In rats, PhIP induces colon cancers and aberrant crypt foci (ACF), a precancerous lesion of the colon.^(38,³⁹⁾ Using ACF as a marker, QTL analysis has been employed to identify genetic traits that regulate the susceptibility to colon carcinogenesis. The susceptibility trait in F344 rats was mapped to chromosome 16 between D16Rat40 and D16Rat60.⁽⁴⁰⁾ Congenic rat strains with this genetic region were more sensitive to ACF induction by PhIP than ACI. Potential resistant traits in ACI were also mapped to chromosomes 6 and 9 by preliminary genome- wide linkage analysis, using a subset of (BUF x ACI)F1 x BUF backcross progeny with extreme phenotypes. Investigation into the genetic interactions between these traits is currently ongoing. 2. Molecular Mechanisms for the Induction of Minisatellite Mutations 　Minisatellite DNA sequences (MN), comprising 5- 100- bp tandem repeats, are frequently mutated in various tumors, however, the molecular mechanisms underlying the induction of MN mutations are largely unknown. The minisatellite repeat (5 'GGGCA3 ')n in the mouse MN Pc- 1 was found to form an intrastrand anti- parallel quadruplex (G4 '- quartet)by NMR and CD spectrum analyses.⁽⁴¹⁾ Since DNA polymerase progression in vitro is blocked at d(GGG) sites, the higher structure of these repeats could be partially implicated in the hypermutable characteristic of MNs in vivo. One of the MN- binding proteins, MNBP- B, was demonstrated to bind to a (5 'GGGCA3 ')n sequence with a high affinity, and partly unfolds the G4'- structure. 3. Functional Analysis of Serine/ Threonine Phosphatase 　The biological functions of serine/ threonine phosphatases are regulated by forming molecular complexes with other cellular components. Genetic alterations in these regulatory subunits in colon cancers are being investigated. PP5 has recently been demonstrated to bind CDC16 and CDC27, which are essential components in the APC complex. In a recent study, PP5 was shown to interact with the HMG BOX Protein- 1 in a yeast two- hybrid system. Since this HMG BOX protein was known to interact with the Rb protein, PP5 could also play an important role in cell- cycle machinery. Reports related to this work can be found in the attached list of publications.⁽⁴²⁾ 4. Involvement of Parp in Carcinogenesis and Tumor Phenotype 　Poly(ADP- ribose)polymerase (Parp) is involved in the recovery from various types of DNA damage.^(43-⁴⁵⁾ The role of Parp in N- nitrosobis(2- hydroxypropyl) amine (BHP)- induced carcinogenesis was studied.Parp^-/- and Parp^+/+ male mice were administered BHP through drinking water for 20 weeks. The incidence and numbers of liver hemangiomas induced by BHP were significantly higher in Parp^-/- mice than in Parp^+/+ mice.Parp^-/- mice also developed hemangiosarcomas in the liver. Lung adenomas were also induced in Parp^-/- mice, but not in Parp^+/+ mice. These findings indicate that Parp- deficiency could be a risk factor for carcinogenesis. Parp^-/- embryonic stem (ES)cells developed teratocarcinoma- like tumors with syncyticotrophoblastic giant cells (STGCs), when injected subcutaneously into nude mice.⁽⁴⁶⁾ These STGCs possessed micro- villi on their surfaces and were positively stained with an anti- prolactin antibody, which implies that loss of Parp induces trophoblast differentiation of ES cells during tumor formation.⁽⁴⁷⁾ 5. Function of Poly(ADP-ribose)Glycohydrolase (Parg) 　Parg is the main enzyme engaged in poly(ADP- ribose)degradation. Parg was purified as a 60- kDa protein from rat testes. A rat Parg cDNA encoding a 109- kDa protein was isolated and it was found that the C- terminal half functions as a catalytic domain. A potential sequence for a leucine- rich type nuclear export signal was found at the N- terminal half.⁽⁴⁸⁾ The rat Parg gene was mapped to the distal region of chr 16p at the same position as D16Mit2 by linkage analysis using a (F344xACI)F1xACI backcross panel.⁽⁴⁹⁾ The human Parg gene was also mapped by fluorescence in situ hybridization to Chr 10q11.23- 21.1,⁽⁵⁰⁾ where loss of heterozygosity and chromosomal rearrangements were reported in glioblastomas and thyroid papillary carcinomas, respectively.