Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric ...

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Oncogene (2000) 19, 1868 ± 1874 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric polyposis and cancer in mice Xiaoling Xu1,3, Steven G Brodie1,3, Xiao Yang1,3, Young-Hyuck Im2, W Tony Parks2, Lin Chen1, Yong-Xing Zhou1, Michael Weinstein1, Seong-Jin Kim2 and Chu-Xia Deng*,1 1

Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, MD 20892, USA; 2Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, MD 20892, USA

The tumor suppressor SMAD4, also known as DPC4, deleted in pancreatic cancer, is a central mediator of TGF-b signaling. It was previously shown that mice homozygous for a null mutation of Smad4 (Smad47/7) died prior to gastrulation displaying impaired extraembryonic membrane formation and endoderm dierentiation. Here we show that Smad4+/7 mice began to develop polyposis in the fundus and antrum when they were over 6 ± 12 months old, and in the duodenum and cecum in older animals at a lower frequency. With increasing age, polyps in the antrum show sequential changes from hyperplasia, to dysplasia, in-situ carcinoma, and ®nally invasion. These alterations are initiated by a dramatic expansion of the gastric epithelium where Smad4 is expressed. However, loss of the remaining Smad4 wild-type allele was detected only in later stages of tumor progression, suggesting that haploinsuciency of Smad4 is sucient for tumor initiation. Our data also showed that overexpression of TGF-b1 and Cyclin D1 was associated with increased proliferation of gastric polyps and tumors. These studies demonstrate that Smad4 functions as a tumor suppressor in the gastrointestinal tract and also provide a valuable model for screening factors that promote or prevent gastric tumorigenesis. Oncogene (2000) 19, 1868 ± 1874. Keywords: Smad4; Dpc4; Juvenile polyposis; gastric cancer; TGFb1; Cyclin D1 Introduction Gastric carcinomas are a leading cause of cancer incidence and mortality worldwide (Kinzler and Vogelstein, 1996; Pisani et al., 1993). As in many tumor types, gastric carcinogenesis re¯ects a multistep process of morphologic abnormalities associated with the accumulation of genetic alterations, including activation of oncogenes and/or inactivation of tumor suppressor genes (reviewed in Correa, 1992; Kinzler and Vogelstein, 1996; Moss, 1998). It was recently demonstrated that one mechanism of gastric cancer progression is the inactivation of the TGF-b signaling pathway (Markowitz and Roberts, 1996; Yang et al., 1999a), where SMAD4/DPC4 is the central mediator (Heldin et al., 1997; Massague, 1998).

*Correspondence: C-X Deng, 3 The ®rst three authors contributed equally to this study Received 9 November 1999; revised 21 January 2000; accepted 31 January 2000

Human SMAD4/DPC4 is located in chromosome 18q21.1, in which loss of heterozygosity (LOH) is frequently demonstrated in a wide variety of tumors, including pancreatic, colon and gastric adenocarcinomas (Hahn et al., 1996; Howe et al., 1998a; Tamura et al., 1996). SMAD4/DPC4 was ®rst cloned as a tumor suppressor that is deleted or mutated in about 40 ± 50% of pancreatic cancers (Hahn et al., 1996). Mutations in Smad4 have also been identi®ed in about 30% of colon carcinomas and less than 10% of other cancers (Nagatake et al., 1996; Schutte et al., 1996). Recently, it was reported that germline mutation of human SMAD4 contributes to familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer (Friedl et al., 1999; Howe et al., 1998b). In addition to SMAD4, eight other SMAD proteins have been described in the TGF-b signaling pathways (reviewed in Heldin et al., 1997; Massague, 1998). The functions of several SMAD family members have been studied using gene targeting. By introducing mutations into dierent functional domains in these genes, we and others have demonstrated that Smad2 plays multiple roles in mesoderm induction, left-right symmetry, and anterior-posterior axis formation (Nomura and Li, 1998; Waldrip et al., 1998; Weinstein et al., 1998). Smad3 mediates TGF-b signals for mucosal immunity and tumor suppressor activity in colon cancer (Yang et al., 1999c; Zhu et al., 1998). Smad5 is indispensable for normal angiogenesis during early embryogenesis (Chang et al., 1999; Yang et al., 1999b). However, the most severe phenotype was observed in Smad4-null embryos which died at embryonic day 6.5 without signs of gastrulation (Sirard et al., 1998; Yang et al., 1998). They also exhibited impaired extraembryonic membrane formation and decreased epiblast proliferation (Yang et al., 1998). Interestingly, when Smad4 heterozygous mice were bred with Apc heterozygous mice, the cis-compound Smad4+/7; Apc+/7 mice developed intestinal carcinomas with all the tumors showing LOH for both wild-type alleles (Takaku et al., 1998). This observation indicates that reduced TGF-b/ Smad4 signals in combination with other factors predispose mice to cancer. To study whether loss of one Smad4 allele can initiate tumorigenesis, we closely monitored our Smad4 heterozygous mice (referred to as Smad48+/7 because of the targeted disruption in exon 8 of Smad4) (Yang et al., 1998) for up to 20 months. We found that Smad48+/7 mice began to develop polyps in the fundus, antrum, duodenum and cecum of the gastrointestinal tract at dierent ages. The antral polyps in older mice

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progressed to adenocarcinoma through a multistep process of morphological changes, including hyperplasia, dysplasia, and in-situ and invasive carcinoma. These morphological changes were accompanied by a series of alterations including increased epithelial proliferation and upregulation of TGF-b1 and Cyclin D1. Of note, LOH of the remaining Smad4 wild-type allele occurred at later stages of tumorigenesis. This study provides compelling evidence that SMAD4 is a tumor suppressor gene in the gastrointestinal (GI) tract and also provides an animal model to study the mechanisms of gastric cancer formation.

Results Smad48+/7mice developed polyposis in the GI tract We have previously showed that the Smad487/7 mice died prior to gastrulation, while the Smad48+/7 mice were phenotypically normal up to 8 months in terms of growth rate, morphology, health and fertility (Yang et al., 1998). Because human SMAD4/DPC4 has been implicated in the formation of pancreatic and GI tract cancers or polyps at varying frequencies (Friedl et al., 1999; Hahn et al., 1996; Howe et al., 1998b; Nagatake et al., 1996; Schutte et al., 1996), we followed Smad48+/7 mice to determine the eect of gene dosage on tumor formation. After examination of the GI tracts of Smad48+/7 mice over 1 year of age, we identi®ed masses that developed in the lower region of the stomach (Figure 1a). Upon dissection, it was clear that the masses were arising above the pyloric sphincter, and that the enlargement was caused by polyps formed in the antrum (Figure 1b ± d). Next, we carefully examined Smad48+/7 mice of varying ages in an attempt to describe and follow tumor progression. Histologically, polyps were ®rst detected in the fundus region of some six month old Smad48+/7 mice. The aected areas increased in size as the mice aged. The fundal polyps were very small in size and clustered in the aected area (Figure 1f), and rarely developed into adenomas, remaining the same size in most older animals. Antral polyps with varying sizes were found in all Smad48+/7 mice that were over one year of age (n=25). Small polyps were sometimes found in younger animals (Figure 1b). Compared with fundal polyps, the antral polyps were much larger, but fewer in number. Polyps and tumors were also found in the cecum (2/25, Figure 1g), and duodenum (5/25, Figure 1h) of older animals at lower frequencies. This was an intriguing ®nding since heterozygous SMAD4 mutations were recently identi®ed in human colorectal cancers and juvenile polyposis (Friedl et al., 1999; Howe et al., 1998b; Miyaki et al., 1999). To further analyse the antral tumors, we performed histologic analysis on Smad48+/7 mice and littermate controls. We analysed antral tissues for changes in glandular architecture and cellular morphology. As Smad48+/7 mice increased in age, we found the antral glands became more severely tortuous, dilated, and elongated compared to controls (Figure 2a ± g). These tumors progressed with age from simple hyperplasia (Figure 2b) to atypical hyperplasia (Figure 2c), dysplasia (Figure 2d), carcinoma in situ (Figure 2e), and ultimately to invasive carcinoma (Figure 2f, g). Increases in stromal

Figure 1 GI polyps and tumor formation in Smad48+/7 mice. (a ± d) Representative samples of antral polyps/tumors (arrows) found in 8 month (a,b), 15 month (c), and 18 month (d) old Smad48+/7 mice. (An) Antrum, (De) duodenum, (Fu) fundus. (e, f) Hyperplastic polyps found in the fundus of a 7 month old mouse (f), but not in its littermate control (e). (g) Polyposis found in the cecum (Ce) of a 8 month old Smad48+/7 mouse (arrow). (h) A tumor (arrow) found in duodenum of a 15 month old Smad48+/7 mouse. Magni®cations: 3.26for a ± c, g and h; 26for d; 106for e and f

tissue and leukocyte in®ltration were also observed (Figure 2e,f). Most animals over 12 months exhibited polyps with severe dysplasia (Figure 2d). The in situ carcinoma had characteristic changes including prominent nucleoli, increased mitoses, decreased gland spacing, and anaplastic cells with nuclear enlargement and hyperchromasia (Figure 2e). The ®ndings were identi®ed in three animals over 18 months of age. Two of these animals also had regions suspicious for invasion, and one animal had a de®nitive invasive carcinoma (Figure 2f,g). Testing for H. felis infection, which presents an important carcinogenic risk factor in gastric cancer (Fox et al., 1996, 1997), was negative, indicating that the gastric tumors are indeed caused by genetic changes and not by environmental insult. In the fundal region we found variously increased proliferation of foveolar cells, pariental cells and neck cells (data not shown). Hyperplasias were identi®ed in both the duodenal crypts and colonic glands (Figure 2h). One duodenal lesion proved to be an invasive adenocarcinoma (Figure 2i). LOH of Smad4 wild-type allele occurs late in gastric tumorigenesis Loss of heterozygosity is a common mechanism for inactivation of tumor suppressor genes. Therefore, we were interested in determining the status of the wildtype Smad4 allele during tumor progression. We Oncogene


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Figure 3 Southern blot and mutation analyses. (a) LOH in the Smad4 locus by Southern blot using EcoRV digestion which produces a fragment of 9.5 kb and 3.6 kb from wild-type and mutant allele, respectively. Genotype as indicated. Lanes 1 and 2 are DNA's from spleen of a wild-type and a Smad48+/7 mouse, respectively. Others are either from antral tumors (T) or polyps (P). (b) SSCP mutation analysis of GI tumor hot spots. Representative SSCP samples from three advanced tumors (T) and control (N) animals. Exon 5 of p53, exon 1 of K-ras, exon 9 of Smad4, and Apc around Min mutation site were examined in this study. No mutations were detected

Figure 2 Histologic analysis of polyps and tumors found in the antrum, fundus, and cecum of Smad48+/7 mice. (a ± f) Progression of antral tumor formation demonstrating hyperplasia (b), atypical hyperplasia (c), dysplasia (d). (e ± g) Representative in-situ carcinoma (e,f) and invasive carcinoma (g) with prominent nucleoli, increased mitoses decreased cytoplasmic to nuclear ratio [arrows point to glands, which are losing their normal structure, and arrowheads point to in®ltrating lymphocytes (e), and invasive cancer cells (f,g)]. These abnormalities were not found in wildtype mice (a). (h ± i) Dysplasia (h) and invasive carcinoma of the duodenum (i). Arrow in (i) points to muscle. Magni®cations: 296for a ± d; 976for e and f; 1456for g; and 486for h and i

performed Southern blot analysis using antral tumors from Smad48+/7 animals ranging in age from 3 ± 18 months. Five of eight tumors having diameters of over 0.4 cm demonstrated loss of heterozygosity (LOH) at the Smad4 locus (Figure 3a). In contrast, only one of ®ve tumors (or polyps) of smaller sizes showed LOH. In addition, we have also performed micro-dissection followed by PCR and con®rmed that the polyps/ tumors that did not show LOH contained the wild-type allele of Smad4 (data not shown). Histologic analysis indicated that tumors showing LOH were from animals with severe antral dysplasia and/or carcinoma (see Figure 2e ± g). Consistent with the LOH data, these tumors showed no Smad4 protein as revealed by immunohistochemical staining (see Figure 4a,b). Altogether, these results indicate LOH of Smad4 occurs as Oncogene

a late event in antral tumor progression, but is not an obligate step in tumor initiation. Many oncogenes and tumor suppressor genes have been implicated in the pathogenesis of gastric and colon cancer, and are also altered in other forms of cancer. These genetic changes include the inactivation of p53, mutations in K-ras, decreased expression of p21, ampli®cation of c-met and c-ErbB-2, and microsatellite instability (reviewed in Kinzler and Vogelstein, 1996; Moss, 1998; Tahara, 1995). In an attempt to further characterize tumor progression, we performed PCR and SSCP analysis on genes commonly mutated in gastrointestinal cancers. We analysed Smad4 (exon 9), p53 (exons 5, 6, 7), K-ras (exon 1 and 2), H-ras (exon 1) and Apc, and found no detectable changes (Figure 3c). In addition, we performed immunohistochemistry using antibodies speci®c for cmyc and ErbB-2 but found no signi®cant dierences in staining of antral tumors compared to control tissue. These data suggest that many of the tumorigenic mechanisms involving these oncogenes and tumor suppressor genes are not contributing factors for antral tumor progression in Smad48+/7 mice. Gastric polyps and tumors exhibited increased proliferation Tumor progression involves an increase in cellular proliferation and/or decrease in apoptosis. Because


TGF-bs are known to inhibit cellular proliferation and induce apoptosis in many cell types through Smad signaling cascade (Ko et al., 1998; Yamamoto et al., 1996), we were interested in determining whether one of

these two mechanisms or a combination was responsible for antral tumor progression. To assess proliferation, we performed 3H-thymidine labeling in control and Smad48+/7 mice. In control antrum and non-hyperplastic regions of tumors, we identi®ed [3H]thymidine positive cells (S-phase cells) primarily at the base of the gastric glands (Figure 4c,e). In contrast, [3H]thymidine positive cells were detected throughout the expanded hyperplastic or dysplastic regions of antral tumors (Figure 4c,d, and not shown). To determine whether decreased apoptosis also occurred, we performed TUNEL assays and found no signi®cant change in apoptotic ®gures between tumors and controls (not shown). These data suggest that increased cellular proliferation is a major factor contributing to antral polyps and tumor progression in Smad48+/7 mice.

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Gastric polyps and tumors exhibited increased expression of TGF-b1 and cyclin D1

Figure 4 Proliferation and immunohistochemical analyses of polyps/tumors in Smad48+/7 mice. (a,b) Immunohistochemistry using an antibody to Smad4 to show its expression in control antrum epithelium (a, arrow) and a tumor (b), which is negative for the staining. (c) 3H-thymidine labeling of a highly proliferative hyperplasic antral polyp (arrow) and adjacent non-hyperplastic region (arrowhead) from a 12 month old Smad48+/7 mouse. (d) An enlarged image of hyperplastic antrum shown in (c). (e) 3Hthymidine labeling of the antrum from an age matched control mouse, showing proliferative cells near the base of gastric glands. (f,g) TGF-b1 expression near the apical region in control antrum epithelium (f), and in a tumor from Smad48+/7 mouse at an increased level (g, arrow). (h,i) Cyclin D1 expression in isthmus of control antrum (h, arrow), and in tumor showing higher intensity (i). Magni®cations: 506for a, b, d, e, f and g; 156for c; and 37.56for h and i

TGF-b signals are mediated through the interactions of Smad2 and Smad3 with the common mediator Smad4. This signaling cascade ultimately leads to activation of genes responsible for regulation of growth, dierentiation, embryonic development, and tissue morphogenesis (Massague, 1990). In order to understand tumorigenesis associated with Smad4 loss, we used immunohistochemistry to screen for proteins expressed in gastric tissues to monitor changes associated with tumorigenesis. TGF-b1 is normally expressed in the gastric epithelial cells (Figure 4f). Its expression was increased in antral tumors in a non-uniform fashion (Figure 4g). Similar non-uniform increased expression of TGF-b RII in tumors was also observed (not shown). These are interesting ®ndings, since TGF-b1 overexpression is found in some human gastric cancers (Naef et al., 1997; Yoshida et al., 1989). TGF-b1 inhibits proliferation of most epithelial cell types by blocking progression of the G1 phase of the cell cycle and inhibiting Cyclin D1 expression (Ko et al., 1998). Overexpression of Cyclin D1 was shown to attenuate the responsiveness of cells to TGF-b through down-regulation of TGF-b RII expression (Okamoto et al., 1994). Our data showed that Cyclin D1 is normally expressed at a low level in the gastric epithelial cells (Figure 5h). In Smad48+/7 mice, slightly increased expression of Cyclin D1 was found in areas of hyperplasia (not shown). However, stronger staining was found in advanced tumors (Figure 4i). We showed earlier that the advanced tumors exhibited LOH of Smad4 (Figure 3a), and failed to express Smad4

Figure 5 Summary of morphological and molecular changes associated with antral tumor progression in Smad48+/7 mice. Smad4 mediated signals are required to maintain normal growth of gastric epithelium. However, haploinsuciency caused by the loss of one Smad4 allele results in increased proliferation of the antral epithelium, which initiates polyp formation. Other events, including increases in Cyclin D1 lead to further increased rates of proliferation and the loss of TGF-b1 cell cycle control, leading to genetic alterations such as LOH for Smad4 and subsequent dysplasia. Additional genetic changes would eventually result in carcinoma formation Oncogene


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protein (Figure 4b), thus, the higher expression of Cyclin D1 in these tumors is consistent with a view that TGF-b/Smad4 signals inhibit cell proliferation through repressing the activity of this gene. Discussion The human SMAD4/DPC4 gene was initially cloned as a tumor suppressor in patients with cancers of the pancreas and other tissues (Hahn et al., 1996). However, homozygous disruption of Smad4 in mice caused early embryonic lethality, preventing studies of Smad4's functions during later stages of development and tumorigenesis. In this study, we assessed the tumor suppressor function of Smad4 in the GI tract using Smad48+/7 mice. We showed that all Smad48+/7 mice developed hyperplasia of the fundus and antrum. The fundal hyperplasia rarely developed into tumors while the polyps found in the antrum increased in size and number, eventually developing into adenocarcinoma as mice aged. Polyps were also found in the duodenum and cecum of Smad48+/7 mice but they occurred at lower frequencies. These observations indicated that Smad4 is a major tumor suppressor gene in the GI tract, especially in the stomach. The development of gastric cancer in Smad48+/7 mice showed a multistep process of morphological changes ranging from hyperplasia to dysplasia, in-situ and likely invasive carcinoma. This prolonged complex process shares striking similarity with the adenocarcinoma sequence (Kinzler and Vogelstein, 1996; Vogelstein, 1990). However, SSCP analysis and immunohistochemical studies for genes involved in colorectal tumorigenesis showed no changes in structure (p53, K-ras, H-ras, Apc and Smad4) and/or expression (c-Myc, ErbB-2, E-cad and b-catenin), suggesting dierences in molecular mechanisms between gastric and colorectal tumor progression. In addition, Smad4 LOH was not detected until the later stages of antral carcinogenesis suggesting that the haploid loss of Smad4 initiated polyps and tumors. However, further tumor progression required the loss of Smad4, which serves as a rate-limiting step for advanced tumor formation. Germline mutations in SMAD4 have been found in a subset of families with familial juvenile polyposis (FJP) (Howe et al., 1998b). FJP is an autosomal dominant disease, characterized by predisposition to hamartomatous polyps and gastrointestinal cancer (Jarvinen and Franssila, 1984). In a large Iowa FJP kindred, all 13 aected individuals carried a 4 bp deletion in SMAD4 (exon 9), which created a premature stop codon leading to protein truncation. In addition, SMAD4 mutation analysis of nine unrelated FJP patients demonstrated that three patients carried the same 4 bp deletion, one carried a 2 bp deletion in exon 8 and one carried a 1 bp insertion in exon 5. The most interesting ®nding was that only one of 11 tumors examined demonstrated LOH of the wild-type SMAD4 allele, suggesting that FJP is caused by reduced SMAD4 signals. Therefore, both the murine and human forms of GI polyposis share similar mechanisms in that they are both initiated by SMAD4 haploinsuciency. It is important to gain an understanding of how haploinsuciency of SMAD4 results in tumor initia-

Oncogene

tion. SMAD4 is a common mediator for all TGF-b family members, which constitute a large family with over 40 members. Therefore, loss of one SMAD4 allele could signi®cantly reduce signals from all family members involved in gastric epithelial proliferation. In addition, the TGF-b signaling pathway represents a direct pathway, through which membrane bound TGFb RI phosphorylates the SMADs, which translocate to the nucleus and trigger transcription of target genes. Because there are no ampli®cation steps between membrane signals and downstream transcription targets, the relative quantity of the intracellular mediator (SMAD4) becomes critical. Indeed, developmental haploinsuciency is also observed in other Smads. Nomura and Li (1998) recently reported that approximately 10% of Smad2+/7 mice displayed abnormalities in mesoderm induction. In another study, mice doubly heterozygous for Smad2 and Smad3 mutations died during gestation caused by reduced signaling levels of these two genes (Weinstein and Deng, unpublished observation). Indeed, tumor initiation without the loss of the wild-type allele has been reported for a number of tumor suppressor genes, including p53 (Venkatachalam et al., 1998), p27 (Fero et al., 1998), and TGF-b1 (Tang et al., 1998), suggesting that haploinsuciency of tumor suppressor genes will become a common phenomenon associated with various tumor types as a consequence of improvement of the detection approaches. Of all TGF-b family members, TGF-b1 and its receptors have been most strongly implicated in gastric cancer formation. TGF-b1 is produced by gastric epithelial cells which, in turn, acts through an autocrine loop to inhibit cellular proliferation by decreasing Cyclin D1 expression and cyclin-cdk4 association (Ko et al., 1998; Naef et al., 1997; Sandhu et al., 1997; Yoshida et al., 1989). The TGF-b1 signals were also found to induce apoptosis in cultured gastric cancer cells after they were arrested in the G1/S phase of the cell cycle (Yamamoto et al., 1996). However, many cultured gastric cells lines lose their ability to respond to TGF-b1 inhibition by reducing expression of TGF-b RI (Ito et al., 1992) or generating mutations in TGF-b RII (Park et al., 1994; Yang et al., 1999a). Microsatellite instability in TGF-b RII, resulting in the functional loss of the receptor, is also found in many gastric tumor cells (Grady et al., 1999; Myero et al., 1995). The Smad4 heterozygous and homozygous tumor cells (LOH of Smad4) from our study are similar to human gastric cancer cells carrying reduced or ineective TGF-b receptors, in that they both suer downstream disruptions in the TGF-b signaling pathway. We have detected increased TGF-b1 and Cyclin D1 expression in both antral polyps and tumors. TGFb RII staining was generally enhanced in hyperplastic and dysplastic regions of pyloric tumors. The increased expression of TGF-b1 and its receptor may be a consequence of a feedback response caused by blockage in downstream pathways. This overexpression is also observed in human gastric cancers (Naef et al., 1997; Yoshida et al., 1989). On the other hand, the increases in Cyclin D1 signaling may be important for further tumor progression. Cyclin D1 is an important component of G1/S cell cycle check point. Its overexpression is thought to inactivate Rb and trigger E2F release resulting in increased cellular proliferation (Oswald et al., 1994; Schulze et al., 1994; Sherr,


1996). Thus, it is conceivable that upregulation of Cyclin D1, whose expression and activity are normally inhibited by TGF-b1 (Ko et al., 1998; Sandhu et al., 1997), is responsible for the over proliferation of epithelial cells in antral hyperplasia and tumors. In summary, we have shown that Smad4 is expressed in gastric epithelium and serves as a negative regulator of epithelial cell proliferation. The haploinsuciency due to the loss of one wild-type allele in Smad48+/7 mice, over time, causes an upregulation in epithelial proliferation, which is in part mediated by increased levels of Cyclin D1 (Figure 5). This lack of cell cycle control leads to hyperplasia and dysplasia. The increases in TGF-b1 and its receptors may be triggered by epithelial overgrowth leading to compensation or increased expression as a means to prevent cellular proliferation. However, this overexpression may not be eective due to the reduced quantity of the down stream mediator (Smad4). The LOH of Smad4 ultimately leads to further genetic changes and invasive tumor formation. Because metastasis was not observed in Smad48+/7 mice, we suggest that further genetic changes, in addition to loss of Smad4, would be required. The demonstration that Smad4 is a tumor suppressor in gastric antral cancer does not exclude the possibility that Smad4 also plays a role in repressing tumor formation along the entire GI tract. However, the requirement of Smad4 functions in other segments of the GI tract appears not as critical as that of gastric tissues. For examples, colon cancer does not initiate in Smad4+/7 mice until an Apc heterozygous mutation is introduced (Takaku et al., 1998). Similarly, other unknown factors must exist to account for the lower frequencies of duodenal and cecal polyps and tumors in Smad48+/7 mice. Even in stomach, polyps did not develop until several months after birth, suggesting additional hits, besides the reduced Smad4 signals, must occur to initiate this multistep gastric cancer series. The identi®cation of these factors deserves a full investigation in the near future. Materials and methods Genotype and LOH analysis Genotyping of Smad48+/7 and control mice, which were in 129/Black Swiss or 129/C57 B6 background, was performed using PCR, and Smad4 LOH studies were performed using Southern blot analysis as described (Yang et al., 1998). Preparation for histologic evaluation and antibody staining Control and transgenic mice were dissected through midline incision of the abdomen. The stomach was isolated in whole by cutting both ends of the stomach after ligation of the lower esophageal end and proximal part of duodenum. One ml of 10% formalin was injected into gastric cavity using a 25 gauge needle, in¯ating the stomach. The in¯ated stomach was then submerged in ®xatives for 10 min. Next, stomach tissue was cut alongside the greater curvature, washed with

PBS, and opened to lie ¯at on the Whatman paper. The stomach was wrapped with Whatman paper securely enclosing the sample, then submerged in ®xatives overnight for histologic preparation. Sections (5 mm) were stained with hematoxylin and eosin (H&E), or subject to immunohistochemical analysis with antibodies speci®c for TGF-b1, TGFb RII, Smad4, E-cadherin, ErbB-2, b-catenin, Cyclin D1 (Santa Cruz, Santa Cruz, CA, USA). Antibody detection was performed using histomouse SP kit (Zymed Laboratories, South San Francisco, CA, USA).

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In situ hybridization, thymidine labeling and TUNEL assay In situ hybridization using Smad4 probes was performed as described (Yang et al., 1998). [3H]Thymidine (Amersham) was IP injected at a dosage at 20 mCi/gram. Animals were sacri®ced 2 h post injection. Slides were dipped in emulsion (Kodak NTB-2) and exposed for 4 ± 15 days before developing. TUNEL staining was performed using ApopTag in situ apoptosis detection kit (Oncor). PCR ± SSCP DNA was extracted from the gastric tumor tissue and agematched control tissue using standard proteinase K digestion and phenol/chloroform extraction for examining mutations in the p53, Ras, Apc, and Smad4 genes. The PCR primer pairs for the ampli®cation of these genes are showing below. p53 exon 5: 5'-TCTCTTCCAGTACTCTCCTC-3'/5'-AGGCGGTGTTGAGGGCTTAC-3'. Exon 6: 5'-GGCTTCTGACTTATTCTTGC-3'/5'-CAACTGTCTCTAAGACGCAC-3'. Exon 7: 5'-TCACCTGGATCCTGTGTCTT-3'/5'-CAGGCTAACCTAACCTACCA-3'. K-ras exon 1: 5'-ATGACTGAGTATA AA CT TGT-3'/5'-GC AG CG TT AC CT CT ATCGTA-5'. Exon 2: 5'-TTCTCAGGACTCCTACAGGA-3'/5'-GATTTAGTATTATTTATGGC-3'. H-ras exon 1: 5'-GATTGGCAG CCGCTG TAGAA-3'/5'-GGCA GAGCTCACCTCTATAG-3'. Apc (150 pb fragment containing the Min mutation): 5'-GTTCTCGTTCTGAGAAAGAC-3'/5'-CTTCCATAACTTTGGCTATC-3'. Smad4 exon 9: 5'-GAGGTTGCACATAGGCAAAG-3'/5'-ACCTTTATATACGCGCTTGG-3'. One hundred ng of genomic DNA was suspended in a 20 ml total volume reaction mixture containing 10 mM Tris HCl (pH 8.3), 50 mM KCl, 200 ng of each primer, 200 mM of each dNTP, 1.5 mM MgCl2, 0.25 ml of [a-32P] dCTP (3000 Ci/ mmol, 10 Ci/ml), and 0.1 unit of Taq DNA polymerase. PCR was performed using the following conditions: 35 cycles of denaturation at 958C for 1 min, annealing at 54 ± 588C for 1 min and extension at 728C for 1 min. Following the PCR reaction, 40 ml of SSCP loading buer (95% formamide, 10 mM NaOH, 0.025% bromophenol blue, and 0.025% xylene cyanol) were added to the 4 ml aliquots of the radioactive PCR products. The reaction mixture was heat denatured for 5 min at 958C, and then chilled on ice. Next, electrophoretic analysis was performed using 2 ml; of the reaction mixture on a 6% nondenaturing gel containing 10% glycerol for 12 ± 16 h at 10 W in a cold room. The gel was dried and exposed to X-ray ®lm overnight at 7808C.

Acknowledgments We thank W Qiao for technical assistance and A Roberts and L Ferrin for critically reading the manuscript and helpful discussion.

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