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Molecular Biology and Genetics |
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan [T. U., M. S., T. K., Y. Y., T. N., T. S., M. N.], and Department of Pathology, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan [M. Y., M. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rat stomach carcinoma induced by administration of
MNNG3
in drinking water provides a good model for human stomach cancer of a
differentiated type (5)
. The histology of stomach
carcinomas induced in the model closely resembles that of human
differentiated-type stomach carcinomas, and their carcinogenic
processes are modulated by various promoting and preventive agents in a
way similar to that observed in human populations (6, 7, 8)
.
Differences in the susceptibility to MNNG-induced stomach carcinomas
have been known among rat strains (9, 10, 11, 12)
. Stomach
carcinomas are induced in the ACI/N (ACI) strain at a rate of 
80%,
and in the BUF/Nac (BUF) strain at 20% (11)
. The
incidences of stomach carcinomas in F1 and
F2 rats of ACI and BUF rats indicated involvement
of an autosomal gene whose BUF allele is dominant over the ACI allele
(11)
.
As the mechanism of the different susceptibilities, different levels of cell proliferation in response to chronic mucosal damage by MNNG, superimposed with a strong inflammatory reaction, have been implicated (13) . Whereas ACI rats show a large increase in the cell proliferation rate after MNNG treatment, BUF rats show only a small increase. It is likely that a high rate of cell proliferation leads to a high rate of mutations, and finally to a high incidence of stomach carcinomas. MNNG is a direct-acting carcinogen, excluding differences in the capacity of metabolic activation as a mechanism for the different cancer susceptibilities between ACI and BUF rats. Moreover, DNA adduct levels in the pylorus of the two strains are the same (5) , excluding differences in the local distribution of MNNG as a mechanism. These previous findings suggest that the mechanisms involved in the different susceptibilities between the two strains are not restricted to MNNG-induced stomach carcinogenesis.
To identify the gene(s) involved in the different susceptibility between ACI and BUF rats, where involvement of an autosomal dominant gene of the BUF type has been suggested, we produced 117 male ACI x (ACIxBUF)F1 backcross rats in this study, and performed a carcinogenicity test for 80 weeks. "Carcinoma development" in individual rats and, when a rat was bearing one or more tumors, the histological grade of malignancy, depth of tumor invasion, tumor size, and number of tumors per rat were determined. The results of linkage analysis are reported.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ACI, BUF, and backcross rats were caged in groups of three to four rats and fed Oriental MF diet (Oriental Yeast, Tokyo, Japan). The cages were kept in an air-conditioned room at 25°C and 55% humidity with 12-h light and dark cycles. The rats were administered 83 mg/l MNNG in drinking water ad libitum from the age of 8 weeks through the age of 40 weeks. MNNG was purchased from Sigma-Aldrich (St. Louis, MO), and a fresh 830 mg/l (deionized water) stock solution was prepared once a week. The stock solution was diluted 10-fold with deionized water twice a week. Rats that became moribund or reached 80 weeks of age were sacrificed.
The stomach was opened along the greater curvature, and sections of the glandular stomach were prepared by cutting along the sagittal axis. When a macroscopic tumor was observed, the tumor was cut into two pieces, one of which underwent histological examination and the other was kept frozen. When a macroscopic tumor was absent, slices were made at 2-mm intervals underwent histological examination. Histological examination was performed by an experienced pathologist (M. T.), and when stomach carcinoma was present, its histological grade of malignancy, depth of tumor invasion, maximal diameter, and number of tumors per rat were determined (14) . When two or more carcinomas were present in a rat, the most malignant phenotype from one of the carcinomas was adopted as the phenotype of the rat.
The liver and/or tail of each rat was kept frozen, and DNA for genotyping was extracted by serial extraction with phenol and chloroform (15) .
Genotyping.
Genotypes of 161 loci, consisting of 146 microsatellite markers, 5
B1-RDA markers (16)
, and 10 AP-RDA markers
(17)
, were determined for all of the 100 effective rats.
Distances between markers summed up to 1637 cM, covering almost the
entire genome of the rat. The average of the distances between two
markers was 11.6 cM.
Microsatellite primers were synthesized by Sawady Technology (Tokyo, Japan) based on previous reports (18) , or purchased from Research Genetics (Huntsville, AZ; Ref. 19 ). Annealing temperatures of 60°C and 55°C and Mg2+ concentrations of 1.5 and 2.5 mM were first tested for each pair of primers, and PCR for genotyping was performed in the optimized condition using 25 ng of template DNA. PCR products were run in 3% NuSieve gels or in 6% polyacrylamide gels.
Genotyping with B1-RDA and AP-RDA markers was performed as reported previously (16 , 17) . "B1-amplicons" and "AP-amplicons" were prepared by amplifying 100 ng of genomic DNA with an appropriate B1- or AP-primer and then dot-blotted onto a nylon membrane after denaturation. The filters were hybridized with B1-RDA or AP-RDA markers, which had been labeled with random hexamers and the Klenow fragment (Multiprime; Amersham-Pharmacia), and purified by gel filtration chromatography (Microspin; Amersham-Pharmacia). Prehybridization, hybridization, wash, and signal detection were performed as reported (16 , 17) . All of the markers used were selected on the basis that they give signals with BUF but not with ACI.
Linkage Analysis.
A genome map for the backcross rats was calculated with Mapmaker/EXP
software (20)
. A genome-wide survey for QTLs was performed
with the Mapmaker/QTL software (20)
. For the carcinoma
development phenotype, a rat was given a digit of 0 or 2, depending on
the diagnosis. For the "histological grade" and "depth of tumor
invasion" phenotypes, a rat was given scores ranging from 1 to 5. For
the "tumor size" phenotype, the maximal diameter of the carcinoma
(in mm) in histological sections was used. To assess the effects of the
genotypes, 
2 tests and t tests for
unequal distribution were performed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The first stomach carcinoma in ACI rats was observed at the age of 52
weeks. Therefore, backcross rats that survived 52 weeks or more and
whose stomach histology was available were counted as effective
animals. Deaths of the ACI and backcross rats before 52 weeks were
caused mainly by hydronephrosis and resultant renal failure, which is
known to take place in ACI rats at an incidence of 5–20% (21
, 22) . Deaths of the ACI, BUF, and backcross rats after 52 weeks
were due to stomach carcinomas, duodenal sarcomas, pneumonia, and other
causes. Incidences of duodenal sarcomas are known to be in the same
range in ACI and BUF rats (11)
. Similar to previous
studies (10
, 11)
, stomach carcinomas were observed in 16
of 21 effective ACI rats (76%) and 3 of 22 effective BUF rats (14%).
In the backcross rats, 48 of 100 effective rats (48%) developed
stomach carcinomas (Table 1)
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. The
diagnosis on their susceptibility was made by mimicking the sizes and
ages observed in inbred strains. Because it was impossible to make a
definitive classification before performing linkage analyses, we tested
12 combinations of three criteria for the susceptible rats (S1, S2, and
S3) and four criteria for the resistant rats (R1, R2, R3, and R4), as
shown in Table 1
. For the rats that survived the full-term of the carcinogenicity test (79–80 weeks of age), rats with stomach carcinomas of a large size could be diagnosed as "susceptible" and those without a carcinoma could be diagnosed as "resistant." The diameter of the smallest carcinoma in the ACI rats at this period was 5 mm in this study. Therefore, a backcross rat with a carcinoma 3 mm in diameter was not classified as susceptible, and those with carcinomas 4 mm in diameter were classified as susceptible in one criterion (S3) and not in the others (S1, S2). For the rats that were sacrificed at early periods of the carcinogenicity test, only those with a stomach carcinoma could be diagnosed as susceptible, but those without a carcinoma could not be diagnosed as susceptible or resistant because they might have developed a stomach carcinoma if they had survived the full-term. To explore what age was appropriate to mark "early periods," we tested different criteria for resistant rats (R1–R4).
Linkage analysis was performed using these 12 combinations of the
carcinoma development phenotype, and three loci were found to give LOD
scores >2.0 with at least one of the 12 combinations (Table 2
and Fig. 2
). Because criteria S1, S2, and S3 gave similar LOD score curves,
results with S3 and R1-R4 are shown in Fig. 2
. The most
influential gene, Gcs1, was mapped on chr. 15 with
the highest LOD score of 3.8 (criterion R1S3; n = 69), and was found to have a paradoxical effect on the
development of stomach carcinomas. Despite the fact that the BUF rat is
resistant as an inbred strain, the BUF allele at Gcs1
conferred dominant susceptibility to MNNG-induced stomach
carcinogenesis. Whereas 13 of 30 rats with the AA genotype
(43%) at Gcs1 (represented by D15Rat102)
developed stomach carcinomas, 34 of 39 rats with the AB
genotype (87%) developed carcinomas (P = 0.0001; Table 3
).
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), 33 of 40 rats with the
AA genotype (83%) developed stomach carcinomas, whereas 14
of 33 rats with the AB genotype (42%) developed carcinomas
(P = 0.0004). For Gcr2
(represented by D3Rat55), 28 of 34 rats with the
AA genotype (82%) developed stomach carcinomas, whereas 19
of 39 rats with the AB genotype (49%) developed carcinomas
(P = 0.003). When Gcr1 and
Gcr2 were combined, 21 of 25 rats (84%) with the
AA genotypes at both loci developed stomach carcinomas,
whereas 7 of 24 rats (29%) with the AB genotypes at both
loci developed carcinomas (P = 0.0002). The
effects of the two genes on the development of stomach carcinomas were
considered as additive. Gcs1 also showed an additive effect
with Gcr1 and Gcr2, but in an opposite direction.
All of the 30 rats (100%) with the susceptible genotype at
Gcs1 (AB) and at either Gcr1 or at
Gcr2 (AA) developed stomach carcinomas.
Linkage with the Histological Grade.
All of the 80 rats with stomach tumors were given a score based on the
histology of the tumors they bore (Table 4)
. Linkage analysis with the score showed a strong linkage with chr. 15,
which was considered to be the same with Gcs1 for the
rats with tumors (LOD, 3.6; n = 80) but not
for those with carcinomas (n = 48; Table 2
).
This indicated that Gcs1 exerts its effect mainly at the
stage of conversion from an adenoma to a carcinoma but that it does not
have an effect on the progression of the histological grade of a
carcinoma (see "Discussion"). When the backcross rats were
classified by the genotypes of Gcs1 (represented by
D15Rat102), a major difference was found in the
number of rats with an adenoma (Table 4)
. The LOD score obtained for
the rats with tumors (ET group) was 3.6, whereas that obtained for the
full-term rats with tumors (FT group) was 3.0.
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. Using the full-term rats, we found that the two phenotypes were
linked to the same locus on chr. 16, designated as Gcr3,
although the LOD scores were not definitive (Table 2)
.
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). For the FT group,
the average score of depth of tumor was 3.3 in rats with the
AA genotype in Gcr3 (represented by
D16Rat17), and 2.2 in those with the AB
genotype (Table 5)
. The BUF allele at Gcr3 was considered to reduce the depth
of tumor invasion by a score of 1.1 in Table 5
.
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). For the FC group, the average diameter was 21
mm in rats with the AA genotype in Gcr3, and 8 mm
in those with the AB genotype (Fig. 3B)
. The BUF
allele at this locus was considered to reduce the diameter of
carcinomas by an average of 13 mm. The average tumor diameters were
significantly different between the rats with the AA
genotype and those with the AB genotype, for rats in the FC
group (P = 0.009) or those in the FT group
(P = 0.007).
Linkage with the Number of Tumors.
Among the 100 effective rats, 72 rats developed one tumor, and 8 rats
developed two tumors. Although linkage analysis was performed using the
number of stomach carcinomas per rat, no linkage was observed.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rat chr. 15 around Gcs1 is known to have synteny with human chr. 13q (24 , 25) , where the endothelin receptor type B (Ednrb) is located. Rat chr. 4 around Gcr1 has synteny with human 2p13, and it is speculated that the Msh6 gene, encoding the G/T mismatch-binding protein, is located in the region. Although MNNG-induced stomach cancers do not display microsatellite instability (26) , the Msh6 gene is one of the candidate genes for Gcr1. The MSH2 gene is also located on 2p in humans, but rat Msh2 was mapped to chr. 6 (27) , ruling out Msh2 as a candidate for Gcr1. Rat chr. 3 around Gcr2 has synteny with human 9q34 and harbors the prostaglandin G/H synthase gene (Cox1) and prostaglandin D2 synthase gene. Cox1 is known to be involved in the cytoprotection of gastric mucosa and to be expressed in the stem cells of the pylorus (28) . Considering that the suggested mechanism for the different susceptibilities between the two rat strains is the difference in the rates of cell proliferation in response to mucosal damage, Cox1 is a candidate for Gcr2. Rat chr. 16 around Gcr3 has synteny with human chr. 4q or 8p, where loss of heterozygosity is reported in human stomach cancers (29) .
Gcs1 is the major gene that controls the development of carcinomas. The histological grade of tumors was also linked to Gcs1 in rats with tumors, but the linkage was much weaker in rats with only carcinomas. This suggested that Gcs1 is mainly involved in the conversion from adenomas to carcinomas. The distribution of the histological grades of the tumors in the rats classified by the genotype of Gcs1 also supports this idea. Linkage of the histological grade with Gcs1 was observed both in the rats of all experimental periods and in the full-term rats. This indicated that the effect of Gcs1 is expressed from early periods to the end of the carcinogenicity test. On the other hand, linkage of the depth of tumor invasion and tumor size with Gcr3 was observed only in the full-term rats, not in the rats of all experimental periods. This was considered because these two parameters change as a tumor grows.
Although LOD scores obtained for Gcr1, Gcr2, and Gcr3 exceeded 1.9, the criterion for "suggested linkage" in backcross rats, they did not exceed 3.3, the criterion for "definitive linkage" (30) . After we found that as many as four genes were involved, we considered the number of effective rats, 100, relatively small. However, classification of the backcross rats by the genotypes of one of the four genes clearly showed significant differences in phenotypes. The facts that both the development of carcinoma and histological grade were linked to Gcs1 and that both depth of tumor invasion and sizes of tumors were linked to Gcr3 further indicate that the possibility of false positives is very low.
In summary, we mapped three genes involved in the completion of malignant transformation of a stomach epithelium cell and one gene involved in the growth of the completed cancer cell. The suggested mechanism for the different susceptibilities between ACI and BUF leads us to expect that some of the four genes are also involved in human cancer susceptibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Program for Promotion of
Fundamental Studies in Health Sciences of the Organization for
Pharmaceutical Safety and Research (OPSR); by a Grant-in-Aid from the
Ministry of Health and Welfare for the 2nd Term Comprehensive 10-Year
Strategy for Cancer Control; and a grant from Princess Takamatsu Cancer
Research Fund. M. S. is a recipient of Research Resident
Fellowship from the Foundation for Promotion of Cancer Research,
Japan. 
2 To whom requests for reprints should be
addressed, at Carcinogenesis Division, National Cancer Center Research
Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone:
81-3-3542-2511, ext. 4520; Fax: 81-3-5565-1753; E-mail: tushijim{at}ncc.go.jp
3 The abbreviations used are: MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
ACI, ACI/N strain; BUF, BUF/Nac strain; QTL, quantitative trait locus;
Gcs, gastric cancer susceptibility gene; chr.,
chromosome; Gcr, gastric cancer resistance gene.
Received 9/13/99. Accepted 12/16/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, no tumors;
, adenomas; 
, carcinomas; B, effect of Gcr3, represented by D16rat17, on the size of carcinomas (n = 27).