Global expression analysis of N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide microarrays

Masanobu Abe1,3, Satoshi Yamashita1, Takashi Kuramoto1, Yoshikazu Hirayama1, Tetsuya Tsukamoto4, Tsutomu Ohta2, Masae Tatematsu4, Misao Ohki2, Tsuyoshi Takato3, Takashi Sugimura1 and Toshikazu Ushijima1,5

1 Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan
2 Center for Medical Genomics, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan
3 Department of Oral Surgery, University of Tokyo Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 135-8655, Japan
4 Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa, Nagoya 464-8681, Japan

5 To whom correspondence should be addressed Email: tushijim{at}gan2.res.ncc.go.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rat stomach carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model for differentiated-type human stomach carcinomas. Here, we analyzed expression profiles in five MNNG-induced rat stomach carcinomas by the high-density oligonucleotide microarray containing ~8000 probe sets. 244 and 208 genes were up- and down-regulated, respectively, by 3-fold and over in four or five carcinomas. Up-regulated genes included those involved in the extracellular matrix remodeling (i.e. Collagen types I, III, V, MMP3), immune response (i.e. lysozyme, complements) and in ossification (i.e. Osteoblast-specific factor). Genes down-regulated included those related to hydrocarbon metabolism (i.e. aldose A, aldehyde dehydrogenase), gastric juice (ion transporter genes) and mucous production (Mucin 5) and gastric hormones (gastrin and somatostatin). The expression profile of the MNNG-induced rat stomach carcinomas shared many features with human stomach carcinomas while cyclin D1 was down-regulated in rat stomach carcinomas but up-regulated in human stomach carcinomas. When the expression profile of the MNNG-induced rat stomach carcinomas was compared with those of two kinds of rat mammary carcinomas, only 13 genes were commonly altered. These results showed that MNNG-induced stomach carcinomas possessed infiltrating capacity and had lost differentiated phenotypes of the stomach, in the same way as human stomach carcinomas, and could be used as a good model for them from the viewpoint of molecular expression profile.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; ECM, extracellular matrix; EST, expressed sequence tag; MNNG, N-methyl-N'-nitro- N-nitrosoguanidine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rat stomach carcinomas induced by administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water are used as a good model for human stomach carcinoma of the differentiated type (1,2). They respond to tumor promoters in a manner accordant with epidemiological studies in human (3,4), and show histological structures similar to differentiated-type human stomach carcinomas (5). As for their molecular basis, infrequent occurrence of p53 mutations and absence of K-ras and ß-catenin mutations were observed (6), and these were also in accordance with the majority of differentiated-type human stomach carcinomas. Therefore, analysis of MNNG-induced rat stomach carcinomas is expected to yield information applicable to human stomach carcinomas, which is one of the most common malignancies worldwide (7).

Global expression analysis by cDNA microarray has been performed in human stomach cancers (8,9). Genes related to cell cycle, growth factor, cell motility, cell adhesion and matrix remodeling were found to be altered in human stomach carcinomas. Global expression analysis of MNNG-induced rat stomach carcinomas enables us to clarify their molecular features common with and distinct from human stomach carcinomas. The common molecular features are expected to include those essential for stomach carcinogenesis. Also, the expression profiles in the MNNG-induced stomach carcinomas can be compared with those in rat mammary carcinomas (10). Comparison of cancers of different tissues will help us to speculate expression changes related to cancer phenotypes and those related to differentiated phenotypes of each tissue.

In this study, we analyzed five primary rat stomach carcinomas with a high-density oligonucleotide microarray representing ~8000 probe sets for rat genes and expressed sequence tags (ESTs), and compared the obtained profiles with those of human stomach carcinomas and with those of rat mammary carcinomas.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals, carcinoma induction and RNA extraction
Male ACI/NJcl (ACI) rats were purchased from CLEA Japan (Tokyo, Japan) and fed a CE-2 diet (CLEA Japan). The rats were administered 83 mg/l MNNG (Sigma-Aldrich, St Louis, MO) in drinking water ad libitum from the age of 8 weeks through to 40 weeks, and were killed at the age of 65 weeks. Stomach carcinomas were found in 22 of 28 MNNG-induced rats. For nine carcinomas, which had diameters >5 mm, half of the mass was snap-frozen in liquid nitrogen for RNA extraction, and the other half was used for routine histological diagnosis. Five samples with good quality RNA were selected for the microarray analysis. The five samples showed various degrees of invasion (sm, two samples; m, two samples; se, one sample), which was similar to the distribution among the 22 carcinomas. Histological diagnoses were made by experienced pathologists (T.T. and M.T.).

Normal pyloric epithelial cells were collected from two untreated 66-week-old male rats using the gland isolation technique (11). A whole layer of stomach tissue was washed in PBS, and incubated at 37°C in calcium-free Hank's balanced salt solution (Invitrogen, San Diego, CA) with 30 mM EDTA for 15 min. Stomach epithelium was peeled off, and collected by centrifugation. Two RNA samples were pooled for microarray analysis.

Total RNA was extracted using ISOGEN (NIPPON GENE, Tokyo, Japan) and purified using an RNeasy Mini kit (Qiagen, Valencia, CA).

High-density oligonucleotide microarray analysis
GeneChip Rat Genome U34A arrays, which contained 6000 probe sets of known rat genes and 2000 probe sets of rat ESTs, were purchased from Affymetrix (Santa Clara, CA). From 8 µg of total RNA, the first-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen, Groningen, the Netherlands) and a T7-(dT)24 primer (Amersham Bioscience, Buckinghamshire, UK), and then the double-strand cDNA was synthesized with Escherichia coli RNase H, E.coli DNA polymerase I and E.coli DNA ligase (Toyobo, Tokyo, Japan). From the double-strand cDNA, biotin-labeled cRNA was prepared using the HighYield RNA transcript labeling kit (Affymetrix). Twenty micrograms of labeled cRNA was fragmented, and the U34A arrays were hybridized. The arrays were stained with streptavidin-phycoerythrin conjugate (Molecular Probes, Eugene, OR) and then scanned with a GeneArray scanner (Hewlett-Packard, Palo Alto, CA). The scanned images were processed using an Affymetrix GeneChip Analysis Suite (version 4.0.1).

Microarray data analysis
The expression of each gene was normalized so that the average intensity of all the genes would be 100. The ‘average fold change’ of a gene was calculated from the ratio of the average intensity in one group and the average in the other group. Genes differentially expressed in the MNNG-induced rat stomach carcinomas were selected as those differentially expressed in four or five of the five carcinomas with a cut-off value of three. Homology searches were performed with the BLAST program at a GenBank Web site. When a clone had a significant homology with a mouse gene (e value < e-100), the clone was considered to be its rat ortholog (shown by ‘Mm’ in tables). Chromosomal location and functional annotation of genes were searched for using web databases (LocusLink and RATMAP).

The expression profiles reported previously for mammary carcinomas (10) were obtained using GeneChip Rat Genome U34A arrays, as in this study. The mammary carcinomas were induced in female (F344 x SD)F1 rats.

Quantitative RT–PCR
cDNA was synthesized from 2.5 µg of total RNA with oligo (dT)12-18 primer and SuperScript II reverse transcriptase (Invitrogen). Quantitative RT–PCR analysis was performed using an iCycler iQ detection system (Bio-Rad Laboratories, Hercules, CA) with SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA). The sequences of the primers are listed in Table I. The number of molecules of a specific gene in a sample was measured by comparing its amplification with the amplifications of standard samples that contained 101 to 106 copies of the gene, and was normalized to that of cyclophilin (10,12). A fold change of the gene in a carcinoma was calculated by dividing the normalized value of the carcinoma by that of the normal control.


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Table I. List of primers used for quantitative RT–PCR

 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gene expression profiles of rat stomach carcinomas
Genes differentially expressed in the stomach carcinomas, compared with the normal pyloric epithelial cells, were first selected with a criterion that the average fold change of a gene in the five stomach carcinoma samples was larger than three and that the change was commonly observed in four or five of them. With this criterion, 244 and 208 genes were found to be up- and down-regulated, respectively, in the carcinomas, compared with the normal samples, and the complete data set is available at our web site (http://www.ncc.go.jp/research/rat-genome/).

Then, a stricter criterion of the average fold change being larger than 20 was adopted, and 50 and 25 genes were found to be up- and down-regulated, respectively (Table II). Among the up-regulated genes with the stricter criterion, genes involved in cell adhesion, those involved in extracellular matrix (ECM) remodeling, immune response and ossification were present. Among the down-regulated genes, those related to hydrocarbon metabolism, gastric mucous production and gastric hormones were present.


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Table II. Genes whose expression especially changed in MNNG-induced stomach carcinoma

 
As for known oncogenes, PDGF receptor {alpha} was highly up-regulated, and Gro oncogene (Gro1) was found to be up-regulated at 14.6-fold. As for known tumor-suppressor genes, few were present among the down-regulated genes, but Metastasis-suppressor gene Kangai 1 (Kai1, EST227901 in U34A) was found to be down-regulated at 7.2-fold.

Confirmation of microarray data by quantitative RT–PCR
To confirm the data obtained by the microarray analysis, the expression levels of 18 representative genes were analyzed by quantitative RT–PCR. The 18 genes were selected for their wide variety of fold changes between the carcinoma and control samples, and included five Cathepsin genes.

As a whole, the expression changes identified by the microarray analysis were well reproduced by the quantitative RT–PCR analysis (Figure 1). The only exception was the expression level of the p41-Arc gene, whose expression change was detected in the opposite manner by the two methods. This indicated that expression changes in the microarray analysis of <2-fold need confirmation by quantitative RT–PCR analysis. Fold changes in the microarray analysis >10 were found to be underestimated compared to their changes detected by quantitative RT–PCR analysis.



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Fig. 1. Comparison of the expression levels of 18 genes analyzed by the GeneChip and by the quantitative RT–PCR.

 
Comparison with human stomach carcinomas
Genes differentially expressed in the MNNG-induced rat stomach carcinoma were compared with those reported to be differentially expressed in human stomach carcinomas (8,9) (Table III). Commonly observed up-regulated genes were those related to ECM remodeling and cellular trafficking. Commonly observed down-regulated genes were those related to differentiated phenotypes of the stomach, such as enzymes involved in HCl production and proteases for food digestion.


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Table III. Genes altered in both rat and human stomach carcinomas

 
Comparison with rat mammary carcinomas
The expression profiles in the MNNG-induced stomach carcinomas were compared with those in rat mammary adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and those induced by 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine (PhIP) (10). Genes differentially expressed in the mammary carcinomas were selected as those differentially expressed in three or four of four DMBA-induced mammary carcinoma and in two or three of three PhIP-induced mammary carcinoma with a cut-off value of 3-fold.

DMBA-induced mammary carcinomas and PhIP-induced mammary carcinomas shared 126 and 149 genes that were commonly up- and down-regulated, respectively. However, only eight and five genes were found to be commonly up- and down-regulated, respectively, in MNNG-induced stomach carcinomas and the two types of mammary carcinomas (Table IV).


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Table IV. Genes altered in both rat stomach and rat mammary carcinomas

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Global expression analysis of ~8000 probe sets in five MNNG-induced rat stomach carcinomas was performed. Among the genes up-regulated in the MNNG-induced rat stomach carcinomas, genes involved in remodeling of ECM constituted a major fraction. Matrix metalloproteinase 10 (Mmp10), Mmp13, Mmp2, Mmp3 and Cathepsin S (CatS), which are known to be involved in degradation of the ECM (1315), were highly up-regulated (Table II). Sparc, Decorin (Dcn) and Lumican, glycoproteins present in the ECM and involved in cancer invasion (1618), were also highly up-regulated. Tissue plasminogen activator (tPa), which plays an important part in cancer spread and metastasis (19), was also up-regulated (9.5-fold). These results indicated that active remodeling of the ECM was present in MNNG-induced rat stomach carcinomas. Many genes involved in the immune response, such as Cd74, Interferon-induced mRNA, Igkc, RT-1D, C1qb, Cxcl14, Thy1, CatS, C1 s, Gpx3 and C1r, were also highly up-regulated. Most of these genes were considered to be expressed by immune cells infiltrating into the stomach carcinomas. Interestingly, among the up-regulated genes, those related to ossification and bone remodeling, such as Matrix G1a protein, Sparc, Col1a1, Dcn and Osteoblast-specific factor 2 and CatK, were found. Ossification was reported previously to be occasionally observed in MNNG-induced rat stomach carcinomas (20) although ossification was absent in the carcinomas analyzed here.

Among the genes down-regulated, those involved in detoxification were observed. UDP-glucuronosyltransferases (UGTs) are known to be involved in the conjugation of small lipophilic agents (21). NAD(P)H quinone oxidoreductase 1 (DT-diaphorase, Nqo1) catalyzes the reduction of various quinone substrates that exert their toxicity through DNA damage. Genes related to differentiated phenotypes of the stomach, such as Mucin 5 subtypes A and C (Muc5ac), Gastrin and Somatostatin, were also highly down-regulated. A decrease of Muc5ac was considered to reflect a difference between normal gastric epithelial cells and cancer cells while a decrease of Gastrin and Somatostatin was considered to be due to lack of neuroendocrine cells in stomach carcinoma tissues. Genes involved in the metabolism of sugars, alcohols, fatty acids and cholesterols, such as Aldolase A, Aldehyde dehydrogenase, Intestinal fatty acid binding protein and 3-hydroxy-3- methylglutaryl-Coenzyme A synthase 2, were found to be highly down-regulated.

Limited numbers of oncogenes were up-regulated in the MNNG-induced rat stomach carcinomas. PDGF receptor {alpha} was highly up-regulated, and the Gro oncogene (Gro1) was up-regulated (14.6-fold). However, expressions of the Ki-ras and ß-catenin oncogenes, whose mutations are reported in human stomach cancers (22), were not increased. Similarly, very limited numbers of tumor-suppressor genes were down-regulated. Kai1, whose down-regulation is reported to be associated with progression of human colon carcinomas (23) but not that of human stomach carcinomas (24), was down-regulated (7.2-fold). Expression of the APC gene was down-regulated at 3.0-fold on the average by the microarray analysis and at 4.7-fold by quantitative RT–PCR. The expression of the p53 gene was not down-regulated. These suggested that genes with small expression changes, although they cannot be identified by microarray approach, could be biologically meaningful.

The MNNG-induced rat stomach carcinomas had many common alterations with human stomach carcinomas, suggesting that MNNG-induced rat stomach carcinomas are a good model of human stomach carcinomas. Commonly up-regulated genes included the Gro1 oncogene and those involved in the remodeling of the ECM and in the immune response. Commonly down-regulated genes included the Kai1 tumor-suppressor gene and those related to the differentiated phenotypes of the stomach, such as Cathepsin E (Cat E), Progastricsin (Pgc). In contrast, cyclin D1 was down-regulated in the rat stomach carcinomas while up-regulated in human stomach carcinomas. This was in accordance with a previous report describing that cell proliferation in rat stomach carcinomas was slower than that of the normal glandular stomach, which is very rapid among various tissues (25). Mucin 3, which is expressed in the normal intestine and intestinal metaplasia of the stomach (26), was up-regulated in the rat stomach carcinomas and appeared down-regulated in human stomach carcinomas. This was considered to be due to the fact that non-cancerous mucosae surrounding human stomach carcinomas usually have intestinal metaplasia with high Muc3 expression while rat control samples do not. Genes associated with lymph node metastasis, such as Rbp4, Igf2 and Fn1, were specifically up-regulated in human stomach carcinoma (8). This was in accordance with the low metastatic potential of MNNG-induced rat stomach carcinoma (2). These findings indicated that, although MNNG-induced stomach carcinomas grow slowly and have low metastatic potential, they shared the capacity for invasive growth and loss of differentiated phenotypes with human stomach carcinomas.

The expression profiles in the MNNG-induced stomach carcinomas can be compared with those in rat mammary carcinomas (10). Three types of chemically induced rat carcinomas shared only a limited number of gene expression changes. This indicates that the common expression changes related to cancer phenotypes in different tissues were very limited. Up-regulation of C1qb, RT1.Mb and Mif indicated that immune cells were infiltrating into both the stomach and breast carcinomas. Scd2, which plays a key role in the synthesis of unsaturated fatty acids and maintenance of membrane fluidity (27), was up-regulated in all of the three types of carcinomas. Its human homolog, the Scd gene, is reported to be overexpressed in human colonic and esophageal carcinomas (28), and its overexpression was speculated to be related to cancer phenotype in various tissues.

The invasive phenotype and loss of differentiated phenotypes in MNNG-induced stomach carcinomas were substantiated by oligonucleotide microarray analysis. Genes whose expression changes have not been reported were picked up, and functions of unknown genes/ESTs can now be speculated from the data obtained for the known genes that showed similar expression patterns. The wealth of information obtained here will facilitate research involving these genes.


    Acknowledgments
 
The authors are grateful to Drs E.Okochi and A.Kaneda for their critical reading of the manuscript. This work was supported by a Grant-in-Aid for the Cancer Research; and a Grant-in-Aid for the Millennium Genome Project from the Ministry of Health, Labour and Welfare. M.A. and Y.H. are recipients of Research Resident Fellowships from the Foundation for Promotion of Cancer Research.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 16, 2002; revised February 20, 2003; accepted February 21, 2003.