Biochemistry Division

2. Biochemistry Division

Genetic background, genomic stability and environmental factors play important roles during human carcinogenesis. The Biochemistry Division seeks to clarify the interactions between genetic background and environmental factors in multi- stage carcinogenesis using animal models, and to elucidate the biological roles of cellular protein modification by ADP-ribosylation with respect to DNA-damage responses. Investigation of the cellular machinery involved in maintaining genomic stability is also undertaken.
1. Carcinogenicity of PhIP in Mice

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is one of the most abundant mutagenic heterocyclic amines (HCAs) present in cooked food. Two different strains of mice, C57BL/6N (B6) and BALB/cA (Balb), were used in a carcinogenesis experiment, based on the fact that B6 animals are sensitive to colon carcinogenesis and Balb mice to mammary carcinogenesis by synthetic carcinogens, DMH and DMBA, respectively. PhIP induced lymphoma, small intestinal and cecal tumors in B6 mice. Tumor incidence in the small intestine were 22% and 50%, when mice were fed 300 ppm of PhIP for 12 and 40 weeks, respectively. In Balb, lymphomas, small intestinal and mammary tumors were frequently observed, however, tumor incidence was not significantly different from that in the untreated group. Differences in genetic background between B6 and Balb may be implicated in the different susceptibility to carcinogenesis by PhIP. Reports related to this work can be found in the attached list of publications.^{(33, 34)}
2. Genetic Susceptibility of Rats to Colon Carcinogenesis

PhIP induces colon tumors in F344 male rats when administered orally.⁽²⁵³⁾PhIP was administered to various rat strains, and susceptibility to the induction of aberrant crypt foci (ACFs) in the colon was analyzed as a marker for colon carcinogenesis. F344 rats were susceptible, giving rise to 3.5亇1.8 ACFs/rat, and ACI was resistant (0.9亇0.7 ACFs/rat). The susceptibility to ACF induction of F344 was demonstrated to be a dominant trait over that of ACI using (F344 x ACI)F1 rats, and genetic linkage analysis was performed using 170 (F344 x ACI)F1 x ACI backcross rats. A locus with a high LOD score of 3.5 was identified as a susceptible locus on chromosome 16, and any other locus did not give a significant lod score of more than 2.0. The major susceptible gene is considered to reside on this locus.
3. Genetic Alteration at Minisatellite DNA Sequences

In an attempt to clarify the molecular mechanisms underlying the tumor- promoting activity of okadaic acid (OA), a genetic alteration at minisatellite DNA sequences (MSs) was identified. MS is an array of 5-100 bp tandem repeats and is considered to be a hot spot for meiotic recombination. When NIH3T3 cells were treated with 7.5 nM of OA for six days and subsequently subcloned, 29% (9 of 31) of the analyzed subclones harbored minisatellite mutation (MSM) as shown by DNA fingerprint analysis using the Pc-1 minisatellite sequence as a probe. In contrast, only 3% (1 of 30) of untreated cells exhibited MSM. Moreover, tumors obtained by inoculating OA-treated NIH3T3 cells into nude mice also harbored MSM. The induction of MSM could be implicated in the tumor-promoting activity of OA.⁽³⁵⁾
4. The role of Poly(ADP-ribose) polymerase in Response to DNA Damage and Carcinogenesis

BALB/c3T3 A31 fibroblasts and a variant A31-1-1 were used to study cellular response to DNA damage. G1 arrest was abrogated in A31-1-1 cells after g-irradiation, although G2 arrest was observed. As p53 stabilization and p21 mRNA induction were observed after g-irradiation, a p53-independent pathway may be involved in the abrogation of G1 arrest.⁽³⁶⁾ Parp is one of the key molecules implicated in DNA-damage responses.⁽³⁷⁾ By disrupting both alleles of the mouse Parp gene by homologous recombination, Parp-deficient ES cell lines were produced. Parp^-/-ES cells showed increased sensitivity to methyl methanesulfonate and g-irradiation, suggesting that Parp is required for recovery from DNA damage. Parp^+/- or^-/-ES cells gave rise to teratocarcinoma as well as Parp^+/+ ES cells when implanted into nude mice, and tumors obtained from Parp^-/- cells had the characteristic features of intra-tumoral hemorrhages along with multinucleated syncytiotrophoblastic giant cells. Parp may be involved in determining genomic stability, and further investigation of Parp is now ongoing by generation of Parp knockout mice.
5. The Possibility of a Mono(ADP-ribosyl)ation Reaction by Helicobacter pylori

The issue of toxins produced by Helicobacter pylori (H. pylori) needs to be urgently clarified, given that H. pylori causes gastric epithelial cell damage which may lead to cancerous changes. During an investigation of the possibility of mono(ADP-ribosyl)ation by H. pylori as observed for other bacterial toxins, incorporation of radioactivity derived from [adenylate-³²P]NAD into the 80 kDa protein was observed. However, this modification reaction was proved to be adenylation of the DNA ligase of H. pylori.⁽³⁸⁾




	2. Biochemistry Division



		Genetic background, genomic stability and environmental factors play important roles during human carcinogenesis. The Biochemistry Division seeks to clarify the interactions between genetic background and environmental factors in multi- stage carcinogenesis using animal models, and to elucidate the biological roles of cellular protein modification by ADP-ribosylation with respect to DNA-damage responses. Investigation of the cellular machinery involved in maintaining genomic stability is also undertaken. 1. Carcinogenicity of PhIP in Mice 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is one of the most abundant mutagenic heterocyclic amines (HCAs) present in cooked food. Two different strains of mice, C57BL/6N (B6) and BALB/cA (Balb), were used in a carcinogenesis experiment, based on the fact that B6 animals are sensitive to colon carcinogenesis and Balb mice to mammary carcinogenesis by synthetic carcinogens, DMH and DMBA, respectively. PhIP induced lymphoma, small intestinal and cecal tumors in B6 mice. Tumor incidence in the small intestine were 22% and 50%, when mice were fed 300 ppm of PhIP for 12 and 40 weeks, respectively. In Balb, lymphomas, small intestinal and mammary tumors were frequently observed, however, tumor incidence was not significantly different from that in the untreated group. Differences in genetic background between B6 and Balb may be implicated in the different susceptibility to carcinogenesis by PhIP. Reports related to this work can be found in the attached list of publications.^{(33, 34)} 2. Genetic Susceptibility of Rats to Colon Carcinogenesis PhIP induces colon tumors in F344 male rats when administered orally.⁽²⁵³⁾PhIP was administered to various rat strains, and susceptibility to the induction of aberrant crypt foci (ACFs) in the colon was analyzed as a marker for colon carcinogenesis. F344 rats were susceptible, giving rise to 3.5亇1.8 ACFs/rat, and ACI was resistant (0.9亇0.7 ACFs/rat). The susceptibility to ACF induction of F344 was demonstrated to be a dominant trait over that of ACI using (F344 x ACI)F1 rats, and genetic linkage analysis was performed using 170 (F344 x ACI)F1 x ACI backcross rats. A locus with a high LOD score of 3.5 was identified as a susceptible locus on chromosome 16, and any other locus did not give a significant lod score of more than 2.0. The major susceptible gene is considered to reside on this locus. 3. Genetic Alteration at Minisatellite DNA Sequences In an attempt to clarify the molecular mechanisms underlying the tumor- promoting activity of okadaic acid (OA), a genetic alteration at minisatellite DNA sequences (MSs) was identified. MS is an array of 5-100 bp tandem repeats and is considered to be a hot spot for meiotic recombination. When NIH3T3 cells were treated with 7.5 nM of OA for six days and subsequently subcloned, 29% (9 of 31) of the analyzed subclones harbored minisatellite mutation (MSM) as shown by DNA fingerprint analysis using the Pc-1 minisatellite sequence as a probe. In contrast, only 3% (1 of 30) of untreated cells exhibited MSM. Moreover, tumors obtained by inoculating OA-treated NIH3T3 cells into nude mice also harbored MSM. The induction of MSM could be implicated in the tumor-promoting activity of OA.⁽³⁵⁾ 4. The role of Poly(ADP-ribose) polymerase in Response to DNA Damage and Carcinogenesis BALB/c3T3 A31 fibroblasts and a variant A31-1-1 were used to study cellular response to DNA damage. G1 arrest was abrogated in A31-1-1 cells after g-irradiation, although G2 arrest was observed. As p53 stabilization and p21 mRNA induction were observed after g-irradiation, a p53-independent pathway may be involved in the abrogation of G1 arrest.⁽³⁶⁾ Parp is one of the key molecules implicated in DNA-damage responses.⁽³⁷⁾ By disrupting both alleles of the mouse Parp gene by homologous recombination, Parp-deficient ES cell lines were produced. Parp^-/-ES cells showed increased sensitivity to methyl methanesulfonate and g-irradiation, suggesting that Parp is required for recovery from DNA damage. Parp^+/- or^-/-ES cells gave rise to teratocarcinoma as well as Parp^+/+ ES cells when implanted into nude mice, and tumors obtained from Parp^-/- cells had the characteristic features of intra-tumoral hemorrhages along with multinucleated syncytiotrophoblastic giant cells. Parp may be involved in determining genomic stability, and further investigation of Parp is now ongoing by generation of Parp knockout mice. 5. The Possibility of a Mono(ADP-ribosyl)ation Reaction by Helicobacter pylori The issue of toxins produced by Helicobacter pylori (H. pylori) needs to be urgently clarified, given that H. pylori causes gastric epithelial cell damage which may lead to cancerous changes. During an investigation of the possibility of mono(ADP-ribosyl)ation by H. pylori as observed for other bacterial toxins, incorporation of radioactivity derived from [adenylate-³²P]NAD into the 80 kDa protein was observed. However, this modification reaction was proved to be adenylation of the DNA ligase of H. pylori.⁽³⁸⁾

2. Biochemistry Division

1. Carcinogenicity of PhIP in Mice

2. Genetic Susceptibility of Rats to Colon Carcinogenesis

3. Genetic Alteration at Minisatellite DNA Sequences

4. The role of Poly(ADP-ribose) polymerase in Response to DNA Damage and Carcinogenesis

5. The Possibility of a Mono(ADP-ribosyl)ation Reaction by Helicobacter pylori