Profiling and selection of genes differentially expressed in the pylorus of rat strains with different proliferative responses and stomach cancer susceptibility
Satoshi Yamashita,
Kuniko Wakazono,
Takashi Sugimura and
Toshikazu Ushijima,1
Carcinogenesis Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
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Abstract
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Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/NJcl (ACI) rats show persistent and strong cell proliferation in response to gastric mucosal damage by MNNG while BUF/NacJcl (BUF) rats show transient and limited cell proliferation. This difference is considered as one of the mechanisms for the high susceptibility of ACI rats to MNNG-induced stomach carcinogenesis. To identify genes involved in the differential induction of cell proliferation, cDNA subtraction was performed using RNA isolated from the pylorus of ACI and BUF rats treated with MNNG. By the temporal patterns of their expressions, the isolated 16 genes were overviewed and clustered into groups. Expression of the genes in group 1 (such as MHC class I and class II genes and interferon-inducible genes Iigp, Mx2 and Ubd) was induced by MNNG treatment, and the genes in group 2 (such as cellular retinoic acid-binding protein II (CrabpII)) were constantly expressed regardless of MNNG treatment. Then, expression profiles among multiple rat strains were compared with the extents of induction of cell proliferation. Iigp, CrabpII and EST222005 were found to show relatively good accordance, and these three genes were considered as candidates for genes that control differential induction of cell proliferation. Presence of polymorphisms at the genomic DNA level was indicated for CrabpII and EST222005, and these two genes were considered to be better candidates than Iigp. It was shown that the temporal profiles and profiles among strains, taking advantage of animal models, are useful to select candidate genes from a collection of genes isolated by various genome-wide scanning methods.
Abbreviations: CrabpII, cellular retinoic acid-binding protein II; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; RDA, representational difference analysis; RDA-WEEC, RDA with elimination of excessive clones.
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Introduction
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N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat stomach cancers are known as a good model of differentiated-type human stomach cancers (1,2). When MNNG is administered to rats in the drinking water, erosions with severe inflammatory reaction are rapidly induced in weeks 12, and gradually subside in weeks 68 (3,4). Regenerative changes are mainly observed in weeks 612, atypical changes are then observed in weeks 1532, and adenocarcinomas of the glandular stomach are finally induced in weeks 3572 (4). MNNG-induced stomach cancers respond to tumor promoters in a manner accordant with epidemiological studies in human (5,6), and show histological structures similar to differentiated-type human stomach cancers (7). As for molecular bases for MNNG-induced rat stomach cancers, infrequent occurrence of p53 mutations and absence of K-ras and ß-catenin mutations were observed (8), and this profile is again in accordance with the majority of differentiated-type human stomach cancers.
Bralow et al. (9) found that different strains of rats showed significantly different susceptibility to MNNG-induced gastric carcinogenesis. ACI/NJcl (ACI) rats are susceptible strains and BUF/NacJcl (BUF) rats are resistant strains (9,10). By linkage analysis using male ACIx (ACI x BUF)F1 backcross rats, we mapped one definitive quantitative trait locus (QTL) and three suggestive QTLs that are involved in the stomach cancer susceptibility. A definitive QTL on rat chromosome (chr.) 15, Gastric cancer susceptibility gene 1 (Gcs1), gave BUF-dominant susceptibility to MNNG-induced stomach carcinogenesis, and three suggestive QTLs on rat chr. 4, 3 and 15 gave BUF-dominant resistance (11).
As for a mechanism of the different stomach cancer susceptibilities, differential induction of cell proliferation after mucosal damage has been implicated (12). MNNG does not require metabolic activation steps, and directly attacks DNA and cellular proteins (13,14). The degrees of erosions were almost the same in the pyloric mucosae of the two strains, or a little more severe in BUF rats (4), and DNA adduct levels were at the same levels (2). These showed that MNNG delivery to its target tissue and the overall level of DNA damage and repair are at similar levels in the two strains. However, in ACI rats, cell proliferation in reaction to mucosal damage is much stronger, and the upward shift of the proliferative zone is more prominent (12). In addition, (ACI x BUF)F1 rats show induction of cell proliferation similar to BUF rats, and show stomach cancer susceptibility again similar to BUF rats (10,12). Considering the important roles of cell proliferation in carcinogenesis (15,16), the difference between ACI and BUF rats in induction of cell proliferation has been considered as one of the mechanisms for the different stomach cancer susceptibilities between the two strains. It can be hypothesized that the genes responsible for stomach cancer susceptibility overlap the genes responsible for the differential induction of cell proliferation.
In this study, we approached the genes that control differential induction of cell proliferation from expression profiles among rat strains. The genes differentially expressed in the pylorus of ACI and BUF rats after MNNG exposure were isolated by cDNA subtraction. The isolated genes were characterized by the temporal profiles of their expression. The candidate genes that control cell proliferation were selected by analyzing rat strains both for induction of cell proliferation and for expression levels of the isolated genes.
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Materials and methods
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Rat strains, MNNG treatment and RNA extraction
ACI, BUF, and their F1 rats were purchased from CLEA Japan (Tokyo, Japan). LEW/Crj (LEW), WKY/NCrj (WKY), SHR/NCrj (SHR) and F344/DuCrj (F344) rats were purchased from Charles River Japan (Yokohama, Japan). WKAH, BN and WTC rats were donated by Dr Serikawa of Kyoto University. After acclimatization for 1 week, rats at 8 weeks of age were given MNNG (Aldrich Chemical, Milwaukee, WI) in the drinking water at a concentration of 83 mg/l for a specified period, and were given distilled water for 1 day before being killed. Pyloric mucosa was scraped off with blades, and total RNA was extracted using ISOGEN (NIPPON GENE, Tokyo, Japan). mRNA was isolated from the total RNAs after treatment with DNase I (Promega, Madison, WI) using Oligotex-dT30 Super (Takara Shuzo, Kyoto, Japan).
cDNA subtraction
cDNA subtraction was performed by cDNA-representational difference analysis (cDNA-RDA) (17,18) and cDNA-RDA with elimination of excessive clones (cDNA-RDA-WEEC) (19). First strand cDNA was synthesized from 4 µg of mRNA by SuperScript II reverse transcriptase (Life Technologies, Rockville, MD) and 5'-TTTGGATCC(T)30VN-3' oligo dT primer. Second strand cDNA was synthesized by DNA polymerase I and RNase H. The double-strand cDNA was then digested with MboI (Takara Shuzo). To 1 µg of digest, the RBam adaptor (17) was ligated, and the ligation product was amplified by PCR for 20 cycles with the RBam24 oligonucleotide to prepare an amplicon. The RBam adaptor was removed by digestion with MboI, and the JBam adaptor was ligated only to the tester amplicon. One µg of the tester amplicon was mixed with 40 µg of the driver amplicon, and the mixture underwent denaturation and reannealing. One tenth of the reannealed product was amplified by PCR with the JBam24 oligonucleotide. The second cycle was performed using the product of the first cycle, after replacing the JBam adaptor with the NBam adaptor, as the tester, and the driver amplicon as the driver. The PCR product after the second cycle was cloned into a plasmid. Further, cDNA-RDA-WEEC was performed to isolate genes that were expressed at low levels but still differentially. In cDNA-RDA-WEEC, 6 µl of PCR solutions of the differential clones isolated in the first cDNA-RDA were added to 20 µg of driver amplicon, and RDA was performed similarly. For each subtraction experiment, 96 clones were analyzed for their independence by cross-hybridization. Independent clones were screened by dot-blot hybridization with the amplicons of ACI and BUF rats to examine their differential expression. Differential expression was finally confirmed by Northern blot analysis.
Sequencing and chromosomal mapping
Cycle sequencing was performed using BigDye Terminator kit and ABI310 DNA sequencer (Applied Biosystems, Foster City, CA). Homology searches were performed with the BLAST program at a GenBank Web site. When a clone had a homology with a mouse or human gene and the rat orthologue had not been cloned, the clone was considered to be the rat orthologue. The genes, whose chromosomal positions had not been known, were mapped using the Rat/Hamster Radiation Hybrid Panel (Research Genetics, Huntsville, AL) and the RH Mapping Service in OLETF Project (http://ratmap.ims.u-tokyo.ac.jp/menu/RH.html).
Northern blot analysis and quantitative RT-PCR
A pool of total RNA (25 µg) was prepared from three rats that underwent the same treatment. Two pools from ACI and two from BUF were run in a 1% agarose gel containing 2.2 M formaldehyde. The RNA was transferred to Hybond-XL membrane (Amersham Pharmacia Biotech, Uppsala, Sweden), and the filter was hybridized with a 32P-labelled probe. Signals were quantified by BAS-2000II scanner (Fuji Photo Film, Tokyo, Japan).
For quantitative RT-PCR, cDNA was synthesized from total RNA with oligo (dT)1218 primer and Superscript II reverse transcriptase (Life Technologies). Real-time PCR analysis was performed using a iCycler iQ detection system (Bio-Rad Laboratories, Hercules, CA) with SYBR Green PCR Core Reagents (Applied Biosystems) and 200 nM of primers. The PCR conditions were 3 min at 50°C, 10 min at 95°C, followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 30 s at specified temperature, and extension for 30 s at 72°C. The sequences of the primers and annealing temperature are listed in Table I
. The absence of non-specific amplification was confirmed by analyzing the PCR products with agarose gel electrophoresis and melting curve. To quantify the number of molecules of a specific gene in the sample, a standard curve was generated using templates that contained 101 to 106 copies of the gene. The amount of ß-actin of each cDNA solution was also quantified, and the amount of the gene of interest was normalized to the amount of ß-actin.
Analysis of induction of cell proliferation
Male rats of each strain at 8 weeks of age were given 63 mg/l MNNG for 2 weeks. They were intraperitoneally injected with 200 mg/kg of BrdU (Sigma Chemical, St. Louis, MO) 1 h before being killed. The stomach was fixed with formalin, and cells with BrdU were stained with anti-BrdU antibody (DAKO A/S, Glostrup, Denmark) and ABC vector staining kit. Numbers of labelled cells were counted in 30 pits in each rat. Four ACI, eight WKY, 11 LEW, nine F344 and six BUF rats were analyzed, and the number for a strain was calculated as the average ± standard deviation.
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Results
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Isolation of differentially expressed genes by cDNA subtraction
Three male ACI rats and three male BUF rats were treated with MNNG for 2 weeks, and RNA was extracted from their pyloric epithelium. cDNA subtraction was performed using a pool of RNA from the three male ACI rats as the tester and a pool of RNA from the three male BUF rats as the driver, and nine clones overexpressed three-fold or more in ACI rats were isolated (representative results in Figure 1
). In cDNA subtraction using BUF as the tester and ACI as the driver, seven clones overexpressed three-fold or more in BUF rats were isolated (Figure 1
). The nine clones overexpressed in ACI rats included mouse Iigp gene, rat endogenous retroviral sequence, rat aldehyde dehydrogenase gene, rat MHC class II RT1.B-1 ß chain (RT1.B-1ß), rat Mx2 gene (also called MxB), rat mucin-like protein gene, rat diubiquitin gene (Ubd, also called Fat10), rat MHC class II RT1.B-1
chain (RT1.B-1
), and rat EST448076 (Table II
). The seven clones overexpressed in BUF rats included the rat pancreatic lipase gene, rat cellular retinoic acid-binding protein II gene (CrabpII), mouse ma40a113, clone bA7, rat schlafen-4 gene (Slfn-4), rat EST222005, and rat MHC class I RT1.A1b (RT1.A1b) (Table II
). Clone bA7 did not have any homology in GenBank, but still had an open reading frame longer than 200 bp.

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Fig. 1. Representative results in northern blot analysis using clones obtained by cDNA-RDA. RNAs from three ACI or BUF rats were pooled for one lane, and two pools for each strain were run.
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Temporal profiles of the differentially expressed genes
RNA was collected from the pyloric glands of ACI and BUF rats at seven time points (before MNNG treatment and 1, 2, 3, 5, 7 and 10 weeks after initiation of MNNG treatment). Expression levels of the 16 isolated genes and three other related genes, MHC class II invariant chain (Ii), MHC class II transactivator (CIITA) and PCNA, were quantified by quantitative RT-PCR (Figure 2
). According to the induction levels by MNNG, the 16 genes could be clustered into two groups. Expressions of the genes in group 1 (Figure 2A and 2B
) were maximally induced at 1 and 2 weeks after initiation of MNNG treatment. They could be further clustered by the presence of differences between ACI and BUF rats before MNNG treatment. Genes in group 1a (Figure 2A
) were not differentially expressed before MNNG treatment and the strain differences were induced by MNNG treatment. The differences (fold change) were maintained till 10 weeks after initiation of MNNG treatment. On the other hand, genes in group 1b (Figure 2B
) were differentially expressed even before MNNG treatment, and, even when their expression was induced by MNNG, the strain differences were maintained. The genes in group 2 (Figure 2C
) were expressed at relatively constant levels, and strain differences were accordingly constant.

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Fig. 2. Expression levels of the genes in group 1a (A); those in group 1b (B); those in group 2 (C); and other related genes (D). For each gene, the expression profiles among rat strains in the pylorus were analyzed after MNNG treatment for 2 weeks (left panels). Temporal profiles (right panels) were measured just before initiation of MNNG treatment (week 0) for 2 weeks, and at weeks 1, 2, 3, 5, 7 and 10. Expression level of a gene was quantified by normalizing the copy number of the gene, measured by real-time RT-PCR, to that of ß-actin. fACI, female ACI rats; F1, F1 rats of ACI and BUF.
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Throughout the analytical periods, RT1.B-1ß and EST448076 were expressed only in ACI rats, while rat pancreatic lipase, CrabpII and EST222005 were expressed only in BUF rats. The PCNA gene, which is expressed in late G1/S-phase and reflects the number of cells in cell cycle (20), were expressed at relatively constant levels, and difference between ACI and BUF rats was slight (Figure 2D
). Therefore, ß-actin was used for normalization of mRNA amount in this study.
Expression profiles among rat strains
To select genes that control induction of cell proliferation, the number of BrdU-labelled cells in a pit was measured in additional rat strains after treatment with MNNG for 2 weeks. The numbers obtained for male ACI, WKY, LEW, F344 and BUF rats were 6.9 ± 0.5, 7.5 ± 0.3, 8.5 ± 1.0, 4.6 ± 1.1 and 3.7 ± 0.7 cells/pit, respectively. The former three strains showed relatively large numbers of labelled cells while the latter two strains showed smaller numbers (P < 0.001 by Student's t-test). The number of labelled cells in (ACI x BUF)F1 (F1) rats was reported to be similar to that of BUF rats (12).
The expression levels of the 16 genes isolated and the three other related genes were measured in the pylorus of male rats of six strains, ACI, WKY, LEW, F344, BUF and F1, after MNNG treatment for 2 weeks (Figure 2
, left panels). Additional data for SHR, WKAH, BN and WTC strains can be found at our Web (www.ncc.go.jp/research/rat-genome). Sex differences between male ACI (ACI) and female ACI (fACI) rats were slight in all genes. Iigp, CrabpII and EST222005 showed profiles among the strains almost concordant with the induction of cell proliferation. Since most of MHC genes are highly polymorphic, direct comparison of their expression levels is not possible between the rat strains with different haplotypes, such as ACI and BUF rats. Therefore, we analyzed expression level of CIITA to estimate the intensity of the signal to transcribe MHC genes (21), and that of Ii, nonpolymorphic and co-expressed with class II genes (22), to estimate the response of class II genes (Figure 2D
). The expression profiles of Ii and CIITA among rat strains were not in accordance with the extents of induction of cell proliferation.
Screening for polymorphisms at the genomic DNA level
As a simple procedure for screening of polymorphisms at the genomic DNA level, genomic DNA of ACI and BUF rats was amplified by PCR with the primers used for RT-PCR analysis. PCR product was obtained for 15 of the 16 genes analyzed (Table II
), and three of them showed polymorphisms. For RT1.B-1ß and EST448076, PCR product was obtained only from ACI rats. For RT1-A1b, PCR products of different sizes were obtained for ACI and BUF rats.
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Discussion
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The 16 genes, which had been isolated by cDNA-RDA using RNA isolated from the pylorus of ACI and BUF rats after MNNG exposure, were clustered into three groups, 1a, 1b and 2, by their temporal profiles of expression. Genes in groups 1a and 1b were maximally induced at weeks 1 and 2, which was in accordance with the time course of reactive inflammation (4). Genes with known functions in groups 1a and 1b included one MHC class I gene, two MHC class II genes and three interferon-inducible genes, Iigp (23), Mx2 (MxB) (24) and Ubd (Fat10) (25). All these genes were known to be involved in inflammatory response, and genes with unknown functions in groups 1a and 1b, EST448076 and EST222005, were speculated to be also involved in inflammatory response. Before MNNG treatment, genes in group 1a were not differentially expressed while those in group 1b were differentially expressed. It was expected that differential expression of genes in group 1a after MNNG treatment were due to differential induction of their upstream signals.
The extents of induction of cell proliferation were measured in 10 rat strains, including (ACI x BUF)F1 rats. As for the six strains whose stomach cancer susceptibilities had been known (10,26), the extents of induction of cell proliferation were in accordance with the susceptibility. ACI, WKY and LEW rats showed strong induction in cell proliferation and were susceptible to stomach cancer, while F344, BUF and F1 showed weak induction and were resistant. These profiles among strains were used to select genes that control differential induction of cell proliferation. Among the multiple genes related to the regulation of cell proliferation induction, the same set of genes were expected to be involved in the difference between ACI and BUF rats and in the difference between ACI and F1 rats. Therefore, expression level in F1 rats comparable with BUF rats was used as a requisite criterion. The accordance in strains other than ACI, BUF and F1 rats were additionally used. Based on these criteria, Iigp, CrabpII and EST222005 were selected as candidate genes that control differential induction of cell proliferation after MNNG treatment. Although Ubd (Fat10) was known to modulate cell growth during B cell or dendritic cell development and activation (27), its expression profile among strains was against its being a candidate gene.
CrabpII encodes a transcriptional regulator that is involved in retinoic acid (RA) signalling (28), and plays a key role in the synergistic growth suppressive effect by RA and
-interferon in breast cancer cells (29). This key role of CrabpII in cell proliferation suggested that its polymorphism could cause differential response to mucosal damage caused by MNNG. As for EST222005, no function has been reported. Iigp was cloned as an interferon-inducible GTPase (23), but its function has not been characterized yet. By use of temporal profiles and profiles among rat strains, we were able to select not only a well characterized gene, CrabpII, but also poorly characterized genes, EST222005 and Iigp, as candidate genes. Analysis of cell types where these candidate genes are expressed and linkage mapping using the induction of cell proliferation as a phenotypic marker will further contribute to find out critical genes for induction of cell proliferation. None of the genes selected were in the chromosomal positions where QTLs for stomach cancer susceptibility had been mapped using ACI x (ACI x BUF)F1 backcross rats (11). However, there is still a possibility that new QTLs for stomach cancer susceptibility will be mapped using intercross rats and co-localized with some of the genes selected in this study.
From the MHC region on rat chromosome 20, three MHC genes and two genes, Ubd (Fat10) and EST222005 were isolated. A previous report that Ii, the MHC class II gene and the MHC class I gene are induced after MNNG exposure (30,31) was confirmed. However, the expression profiles among rat strains of Ii and CIITA suggested that the total expression levels of the MHC class II genes were unlikely to control differential induction of cell proliferation. There remains a possibility that the haplotypes of MHC genes are involved in differential induction of cell proliferation.
RT1.B-1ß, EST448076, CrabpII and EST222005 were expressed only in one strain at any time point, and were not expressed at all in some strains. They were analyzed by PCR using primers for RT-PCR and genomic DNA, and presence of polymorphisms in genomic DNA was confirmed for RT1.B-1ß and EST448076. These suggested that CrabpII and EST222005 could also have polymorphisms at the genomic DNA level. Theoretically, differential expression of a gene between two strains can be induced by two mechanisms. One is by different intensities of upstream signal, and the other is by differences intrinsic to the gene, such as a polymorphism in its promoter or deletion of its coding sequence. Genes that are differentially induced by the latter mechanism should have much better chances of working as primary causes. Therefore, among the three genes that showed accordance among the strains, CrabpII and EST222005 were selected as those with priority.
In this study, temporal profiles and profiles among rat strains offered a non-biased method to evaluate the isolated genes. The selection strategy contributed to the selection of CrabpII and EST222005 as candidates for genes that control differential induction of cell proliferation. The selection strategy taken here can be applied to search for genes responsible for various interesting phenotypes in animal models.
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Notes
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1 To whom correspondence should be addressed Email: tushijim{at}ncc.go.jp 
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Acknowledgments
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This work was supported by Grants-in-Aid for the 2nd-term Cancer Control Strategy and for Cancer Research from the Ministry of Health, Labor and Welfare; by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research (OPSR); and by a grant from the Princess Takamatsu Cancer Research Fund.
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References
|
---|
-
Sugimura,T. and Fujimura,S. (1967) Tumor production in glandular stomach of rats by N-methyl-N'-nitro-N-nitrosoguanidine. Nature, 216, 943944.[ISI][Medline]
-
Ohgaki,H. and Sugimura,T. (1997) Experimental gastric cancer. In Sugimura,T. and Sasako,M. (eds) Gastric Cancer. Oxford University Press, New York, pp. 7386.
-
Matsukura,N., Kawachi,T., Sasajima,K., Sano,T., Sugimura,T. and Hirota,T. (1978) Induction of intestinal metaplasia in the stomachs of rats by N-methyl-N'-nitro-N-nitrosoguanidine. J. Natl Cancer Inst., 61, 141144.[ISI][Medline]
-
Ohgaki,H., Kusama,K., Hasegawa,H., Sato,S., Takayama,S. and Sugimura,T. (1986) Sequential histologic changes during gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI and resistant BUF rats. J. Natl Cancer Inst., 77, 747755.[ISI][Medline]
-
Tatematsu,M., Takahashi,M., Fukushima,S., Hananouchi,M. and Shirai,T. (1975) Effects in rats of sodium chloride on experimental gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. J. Natl Cancer Inst., 55, 101106.[ISI][Medline]
-
Kobori,O., Shimizu,T., Maeda,M., Atomi,Y., Watanabe,J., Shoji,M. and Morioka Y. (1984) Enhancing effect of bile and bile acid on stomach tumorigenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats. J. Natl Cancer Inst., 73, 853861.[ISI][Medline]
-
Saito,T., Inokuchi,K., Takayama,S. and Sugimura,T. (1970) Sequential morphological changes in N-methyl-N'-nitro-N-nitrosoguanidine carcinogenesis in the glandular stomach of rats. J. Natl Cancer Inst., 44, 769783.[ISI][Medline]
-
Hirayama,Y., Wakazono,K., Yamamoto,M., Kitano,M., Tatematsu,M., Nagao,M., Sugimura,T. and Ushijima,T. (1999) Rare mutations of p53, Ki-ras and beta-catenin genes and absence of K-sam and c-erbB-2 amplification in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach cancers. Mol. Carcinogen., 25, 4247.[ISI][Medline]
-
Bralow,S.P., Gruenstein,M. and Meranze,D.R. (1973) Host resistance to gastric adenocarcinomatosis in three strains of rats injesting N-methyl-N'-nitro-N-nitrosoguanidine. Oncology, 27, 168180.[ISI][Medline]
-
Ohgaki,H., Kawachi,T., Matsukura,N., Morino,K., Miyamoto,M. and Sugimura,T. (1983) Genetic control of susceptibility of rats to gastric carcinoma. Cancer Res., 43, 36633667.[Abstract]
-
Ushijima,T., Yamamoto,M., Suzui,M., Kuramoto,T., Yoshida,Y., Nomoto,T., Tatematsu,M., Sugimura,T. and Nagao,M. (2000) Chromosomal mapping of genes controlling development, histological grade, depth of invasion and size of rat stomach carcinomas. Cancer Res., 60, 10921096.[Abstract/Free Full Text]
-
Ohgaki,H., Tomihari,M., Sato,S., Kleihues,P. and Sugimura,T. (1988) Differential proliferative response of gastric mucosa during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI rats, resistant Buffalo rats and their hybrid F1 cross. Cancer Res., 48, 52755279.[Abstract]
-
Sugimura,T., Fujimura,S., Nagao,M., Yokoshima,T. and Hasegawa,S. (1968) Reaction of N-methyl-N'-nitro-N-nitrosoguanidine with protein. Biochem. Biophys. Acta, 170, 427429.[ISI][Medline]
-
Lawley,P.D. and Shah,S.A. (1972) Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels. Biochem. J., 128, 117132.[ISI][Medline]
-
Ames,B.N. and Gold,L.S. (1990) Too many rodent carcinogens: mitogenesis increases mutagenesis [published erratum appears in Science 1990, Sep 28; 249 (4976):1487]. Science, 249, 970971.[ISI][Medline]
-
Cohen,S.M. and Ellwein,L.B. (1990) Cell proliferation in carcinogenesis. Science, 249, 10071011.[ISI][Medline]
-
Lisitsyn,N., Lisitsyn,N. and Wigler,M. (1993) Cloning the differences between two complex genomes. Science, 259, 946951.[ISI][Medline]
-
Hubank,M. and Schatz,D.G. (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res., 22, 56405648.[Abstract]
-
Ushijima,T., Morimura,K., Hosoya,Y., Okonogi,H., Tatematsu,M., Sugimura,T. and Nagao,M. (1997) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc. Natl Acad. Sci. USA, 94, 22842289.
-
Iwatani,Y., Kamigaki,T., Suzuki,S., Ohno,M., Nakamura,T. and Kuroda,Y. (1999) Proliferating cell nuclear antigen as a predictor of therapeutic effect of continuous 5-fluorouracil administration in gastric cancer. Int. J. Oncol., 14, 965970.[ISI][Medline]
-
Mach,B. (1999) Perspectives: immunology. Regulating the regulator. Science, 285, 1367.[Free Full Text]
-
Moore,B.B., Cao,Z.A., McRae,T.L., Woo,C.H., Conley,S. and Jones,P.P. (1998) The invariant chain gene intronic enhancer shows homology to class II promoter elements. J. Immunol., 161, 18441852.[Abstract/Free Full Text]
-
Boehm,U., Guethlein,L., Klamp,T., Ozbek,K., Schaub,A., Futterer,A., Pfeffer,K. and Howard,J.C. (1998) Two families of GTPases dominate the complex cellular response to IFN-gamma. J. Immunol., 161, 67156723.[Abstract/Free Full Text]
-
Melen,K., Keskinen,P., Ronni,T., Sareneva,T., Lounatmaa,K. and Julkunen,I. (1996) Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope. J. Biol. Chem., 271, 2347823486.[Abstract/Free Full Text]
-
Raasi,S., Schmidtke,G. and Groettrup,M. (2001) The ubiquitin-like protein fat10 forms covalent conjugates and induces apoptosis. J. Biol. Chem., 276, 3533435343.[Abstract/Free Full Text]
-
Tatematsu,M., Aoki,T., Inoue,T., Mutai,M., Furihata,C. and Ito,N. (1988) Coefficient induction of pepsinogen 1-decreased pyloric glands and gastric cancers in five different strains of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis, 9, 495498.[Abstract]
-
Liu,Y.C., Pan,J., Zhang,C., Fan,W., Collinge,M., Bender,J.R. and Weissman,S.M. (1999) A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2. Proc. Natl Acad. Sci. USA, 96, 43134318.[Abstract/Free Full Text]
-
Delva,L., Bastie,J.N., Rochette-Egly,C., Kraiba,R., Balitrand,N., Despouy,G., Chambon,P. and Chomienne,C. (1999) Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex. Mol. Cell Biol., 19, 71587167.[Abstract/Free Full Text]
-
Widschwendter,M., Daxenbichler,G., Dapunt,O. and Marth,C. (1995) Effects of retinoic acid and gamma-interferon on expression of retinoic acid receptor and cellular retinoic acid-binding protein in breast cancer cells. Cancer Res., 55, 21352139.[Abstract]
-
Furihata,C., Oka,M., Yamamoto,M., Ito,T., Ichinose,M., Miki,K., Tatematsu,M., Sakaki,Y. and Reske,K. (1997) Differentially expressed MHC class II-associated invariant chain in rat stomach pyloric mucosa with N-methyl-N'-nitro-N-nitrosoguanidine exposure. Cancer Res., 57, 14161418.[Abstract]
-
Oka,M., Furihata,C., Kitoh,K., et al. (1998) Involvement of dendritic cell response to resistance of stomach carcinogenesis caused by N-methyl-N'-nitro-N-nitrosoguanidine in rats. Cancer Res., 58, 41074112.[Abstract]
Received December 28, 2001;
revised March 18, 2002;
accepted March 18, 2002.