CARCINOGENESIS DIVISION

13.CARCINOGENESIS DIVISION

    Carcinogenesis is a complex process, with complex interactions between host and environmental factors. This division focuses on (1) DNA methylation as a mechanism of carcinogenesis, (2) genes defining stomach cancer susceptibility as a host factor, and (3) single nucleotide instability (SNI) as a mechanism that promotes carcinogenesis.
DNA Methylation and Cancer

    Methylation of CpG islands in promoter regions is known to play important roles in switching on and off various genes. This division developed the methylation-sensitive-representational difference analysis (MS-RDA) method, which enables us to isolate DNA fragments differentially methylated in two genomes. Taking advantage of the human genome sequence, genes located near the DNA fragments can now be easily identified. Silencing of the HTR1B gene and the reduced expression of the Endothelin-1 gene were previously identified in human lung cancers by MS-RDA.
    MS-RDA was applied to human stomach cancers ^(205,²⁰⁶⁾. First, to avoid isolation of artifacts during establishment of cell lines, a primary stomach cancer was used as a source of the DNA for MS-RDA. Limited numbers of DNA fragments were isolated as differentially methylated, and few were from promoter regions. However, differential methylation was associated with reduced expressions of the insulin-induced protein 1 and p41Arc genes ⁽²⁰⁵⁾.
    Considering the heterogeneity of DNA methylation profiles among different cell types, stomach cancer cell lines were then used as a source of DNA. Ample numbers of DNA fragments were isolated from the 5' regions of genes, and the silencing of nine genes was newly discovered. The nine genes included those with reported tumor-suppressor activities ⁽²⁰⁶⁾.
    MS-RDA is also being performed using human sporadic breast cancers. In addition, methylation of an established tumor-suppressor gene, BRCA1, was examined. BRCA1 protein expression was found to be decreased due to its promoter methylation and also due to a post-transcriptional mechanism(s) ⁽²⁰⁷⁾. Interestingly, in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat breast cancers, Brca1 protein was also decreased due to a post-transcriptional mechanism(s) ⁽²⁰⁸⁾. p16 methylation was absent in the PhIP-induced breast cancers ⁽²⁰⁹⁾.
    Altered fidelity of maintenance methylation in cancers is being investigated. An assay system for environmental factors that alter DNA methylation is being developed. Involvement of DNA methylation in aberrant tissue differentiation is also being analyzed.
Cancer Susceptibility and the Rat Genome

    Differences in cancer susceptibility observed in animal strains provide models for those observed in the human population. The ACI rat shows a high susceptibility to N-methyl-N’-nitro- N-nitrosoguanidine (MNNG)-induced stomach carcinogenesis, while the BUF rat is resistant. To identify the susceptibility genes, we previously mapped them to rat chromosomes 15, 3, 4 and 16. Congenic rat strains for each locus were constructed, and their susceptibilities are being tested.
    As a mechanism for the varying susceptibility to stomach cancer, differential induction of cell proliferation after mucosal damage in the two strains has been implicated. To identify the responsible gene(s) for the differential reaction, cDNA subtraction was performed using RNA extracted from the pylorus of the two strains. Sixteen genes were shown to be differentially expressed between the two strains. Expression levels of three genes showed a good accordance with the extents of cell proliferation in six rat strains ⁽²¹⁰⁾.
    To employ other unique disease models in the rat, we have devoted ourselves to developing new genetic markers for the rat. The AP-RDA markers, which were developed for high-throughput genotyping, were used to map a responsible gene and a modifier gene in the UPL cataract rats ⁽²¹¹⁾. Involvement of the Connexin genes was strongly suggested. The mv mutant rat with myelin vacuolation was shown to be due to a mutation in the Attractin gene ⁽²¹²⁾. Its mutation was also demonstrated in a mutant strain of hamster with black hair and tremor ⁽²¹³⁾.
    Animal cancer models offer a simple yet sophisticated system for analysis of what cannot be analyzed in human ones. Expression profiles of two groups of breast cancers, those induced by PhIP and those induced by 7,12-dimethylbenz [a]anthracene (DMBA), were compared. Although the two groups of cancers were histologically indistinguishable, they were shown to have distinct expression profiles. The expression profiles were considered to have the potential to reveal carcinogens involved in specific cancers ⁽²¹⁴⁾.
Single Nucleotide Instability

    Genetic instability plays important roles in carcinogenesis. Two cell lines were established from PhIP-induced rat mammary carcinomas, and spontaneous point mutation rates (MRs) were found to be increased in these cell lines. Since the two cell lines lacked microsatellite instability, the increase of MRs was considered due to a mechanism(s) different from mismatch repair insufficiency, and it was termed single nucleotide instability (SNI). The presence of SNI was further demonstrated in five of six human breast cancer cell lines ⁽²¹⁵⁾.
    Since SNI in the rat breast cancers was characterized by an increase of A:T to C:G transversions, two known mechanisms that increase this type of mutations were examined in the PhIP-induced breast cancers. However, no mutation or decreased expression of the MutT gene was observed, and the expression of the DNA polymerase kappa gene was not increased ⁽²¹⁶⁾.
Other Research Projects

    In collaboration with Kyoto University, mutations of the RUNX3 gene were searched for in human stomach cancers. One mutation identified played a key role in demonstrating the causal role of the RUNX3 in stomach cancers ⁽²¹⁷⁾.
    cDNA-RDA was performed in collaboration with Nagoya City University. Down regulation of hXBP-1 and Lsm1 was shown to be important for the progression of human prostate cancers ^(218,²¹⁹⁾.




13.CARCINOGENESIS DIVISION



	Carcinogenesis is a complex process, with complex interactions between host and environmental factors. This division focuses on (1) DNA methylation as a mechanism of carcinogenesis, (2) genes defining stomach cancer susceptibility as a host factor, and (3) single nucleotide instability (SNI) as a mechanism that promotes carcinogenesis. DNA Methylation and Cancer Methylation of CpG islands in promoter regions is known to play important roles in switching on and off various genes. This division developed the methylation-sensitive-representational difference analysis (MS-RDA) method, which enables us to isolate DNA fragments differentially methylated in two genomes. Taking advantage of the human genome sequence, genes located near the DNA fragments can now be easily identified. Silencing of the HTR1B gene and the reduced expression of the Endothelin-1 gene were previously identified in human lung cancers by MS-RDA. MS-RDA was applied to human stomach cancers ^(205,²⁰⁶⁾. First, to avoid isolation of artifacts during establishment of cell lines, a primary stomach cancer was used as a source of the DNA for MS-RDA. Limited numbers of DNA fragments were isolated as differentially methylated, and few were from promoter regions. However, differential methylation was associated with reduced expressions of the insulin-induced protein 1 and p41Arc genes ⁽²⁰⁵⁾. Considering the heterogeneity of DNA methylation profiles among different cell types, stomach cancer cell lines were then used as a source of DNA. Ample numbers of DNA fragments were isolated from the 5' regions of genes, and the silencing of nine genes was newly discovered. The nine genes included those with reported tumor-suppressor activities ⁽²⁰⁶⁾. MS-RDA is also being performed using human sporadic breast cancers. In addition, methylation of an established tumor-suppressor gene, BRCA1, was examined. BRCA1 protein expression was found to be decreased due to its promoter methylation and also due to a post-transcriptional mechanism(s) ⁽²⁰⁷⁾. Interestingly, in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat breast cancers, Brca1 protein was also decreased due to a post-transcriptional mechanism(s) ⁽²⁰⁸⁾. p16 methylation was absent in the PhIP-induced breast cancers ⁽²⁰⁹⁾. Altered fidelity of maintenance methylation in cancers is being investigated. An assay system for environmental factors that alter DNA methylation is being developed. Involvement of DNA methylation in aberrant tissue differentiation is also being analyzed. Cancer Susceptibility and the Rat Genome Differences in cancer susceptibility observed in animal strains provide models for those observed in the human population. The ACI rat shows a high susceptibility to N-methyl-N’-nitro- N-nitrosoguanidine (MNNG)-induced stomach carcinogenesis, while the BUF rat is resistant. To identify the susceptibility genes, we previously mapped them to rat chromosomes 15, 3, 4 and 16. Congenic rat strains for each locus were constructed, and their susceptibilities are being tested. As a mechanism for the varying susceptibility to stomach cancer, differential induction of cell proliferation after mucosal damage in the two strains has been implicated. To identify the responsible gene(s) for the differential reaction, cDNA subtraction was performed using RNA extracted from the pylorus of the two strains. Sixteen genes were shown to be differentially expressed between the two strains. Expression levels of three genes showed a good accordance with the extents of cell proliferation in six rat strains ⁽²¹⁰⁾. To employ other unique disease models in the rat, we have devoted ourselves to developing new genetic markers for the rat. The AP-RDA markers, which were developed for high-throughput genotyping, were used to map a responsible gene and a modifier gene in the UPL cataract rats ⁽²¹¹⁾. Involvement of the Connexin genes was strongly suggested. The mv mutant rat with myelin vacuolation was shown to be due to a mutation in the Attractin gene ⁽²¹²⁾. Its mutation was also demonstrated in a mutant strain of hamster with black hair and tremor ⁽²¹³⁾. Animal cancer models offer a simple yet sophisticated system for analysis of what cannot be analyzed in human ones. Expression profiles of two groups of breast cancers, those induced by PhIP and those induced by 7,12-dimethylbenz [a]anthracene (DMBA), were compared. Although the two groups of cancers were histologically indistinguishable, they were shown to have distinct expression profiles. The expression profiles were considered to have the potential to reveal carcinogens involved in specific cancers ⁽²¹⁴⁾. Single Nucleotide Instability Genetic instability plays important roles in carcinogenesis. Two cell lines were established from PhIP-induced rat mammary carcinomas, and spontaneous point mutation rates (MRs) were found to be increased in these cell lines. Since the two cell lines lacked microsatellite instability, the increase of MRs was considered due to a mechanism(s) different from mismatch repair insufficiency, and it was termed single nucleotide instability (SNI). The presence of SNI was further demonstrated in five of six human breast cancer cell lines ⁽²¹⁵⁾. Since SNI in the rat breast cancers was characterized by an increase of A:T to C:G transversions, two known mechanisms that increase this type of mutations were examined in the PhIP-induced breast cancers. However, no mutation or decreased expression of the MutT gene was observed, and the expression of the DNA polymerase kappa gene was not increased ⁽²¹⁶⁾. Other Research Projects In collaboration with Kyoto University, mutations of the RUNX3 gene were searched for in human stomach cancers. One mutation identified played a key role in demonstrating the causal role of the RUNX3 in stomach cancers ⁽²¹⁷⁾. cDNA-RDA was performed in collaboration with Nagoya City University. Down regulation of hXBP-1 and Lsm1 was shown to be important for the progression of human prostate cancers ^(218,²¹⁹⁾.