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EGFR Induoed-Tyrosine Phosphorylation of ZO-1 May Regulate the Liver Metastasis of Coloractal Cancer Tsukasa I<aihara, Toshihiro Kusaka, Hitoshi Kawamata, Oept of Pathel, Ookkyo Univ Sch of Medicine, Tochigi Japan; Yasushi Oda, Internal Medicine, Kumamoto Regional Medical Ctr, Kumamoto Japan; Jyouji Imura, Shigehiko Fujii, Yoshihiko Ueda, Takahiro Fujimori, Oept of Pathol, Dokkyo Univ Sch of Medicine, Tochigi Japan

BEGIN Dysfunction of the adherence junction is known to be associated with the invasion and metastasis in various tumors, while it is little known whether or not dysfunction of the tight junction is associated with tumor metastasis. ZO-1 is a critical regulator of epithel~ tight junction. Several reports described that reduced expression of ZO-1 was associated with the invasion of the breast and gastrointestinal cancers. It was also reported that EGFR induced tyrosine-phesphelylabon of ZO-1 and reorganized ZO-1 at the tight junction in A431 cells. To explore the role of ZO-1 in coloractal cancer metastasis, we carried out immunohistochemlstzy and immunoprecipitation-Western blotting for ZO-1 in primary coloreotal cancers (Pr-CRC) and liver metastasized colorectal cancers. Methods A total of 42 cases (24 Pr-CRC with liver metastasis, 18 Pr-CRC without liver metastasis) were examined ZO-1 expression by an immunohistochemical staining. Further- more, lysates from the samples were immunoprecipitated with antioZO-1 or anti-EGFR antibody, and detected with anti-phesphotyrosine, anti-ZO-1 and ardi-EGFR antibndk~. Results The membrane staining of ZO-1 was observed in normal colon mucosa of all spicie- mens. The same staining pattern of ZO-1 was observed in only 18/42 Pr-CRC (43%. The ratio of the membrane staining of ZO-1 in Pr-CRC with liver metastasis (8/24:33.3 is somewhat lower than that in Pr-CRC without liver metastasis (10/18: 55.6%. However it did not reach the significant level (p = 0.14). The ratio of membrane staining of ZO-1 in the liver metastasized cancer (15/24: 62.5% is significantly higher than that in the corresponding Pr-CRC (p = 0.04). By an Immunoprecipilation-Western blotting, ZO-1 bound to EGFR irrespectively in the status of EGFR, pheshelylated or not. Interestingly, tyrosine-phespholylation of EGFR and ZO-1 in Pr-CRC with fiver metastasis was apparently higher than those in normal mucosa, Pr-CRC without liver metastasis, and the liver-metastasized cancer. Conclusions Our observations suggest that the expression of ZO-1 at the tight junction (lumioer side of the membrane) may maintain the tubular structure of colon cancer, and dysfunction of ZO-1 by EGFR-induced tyrosine-phosphelylation may enhance the migration of the cells at the primary site. Then, ZO-1 may gain the function again at the liver, and the tumor cells form the tubular structure again at the liver.

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Anti-VEGFR-3 Antibody Inhibits bFGF-dependent N e o v a e a o f a r ~ Kenji Shimizu, Kazuhiko Kawashima, Yoshihide Ueda, Hiroki Hashida, Meaeald Awane, Kyoto Univ graduate Sch of medicine, Kyoto Japan; Michiyuki Kanal, Admichi Takahayashi, Kitano Hosp, Osaka Japan; Yoshio Yamaoka, Kyoto Univ graduate Sch of medicine, Kyoto Japan

<Objective> Neovasculanzation is an essential eventr for the tumor growth.Vascular endothe- lial growth factor (VEGF) and basic fibrobiact growthtactor (bFGF) are major factor which facilitate tumor vascuiarization. VEGFreceptor-3 (VEGFR-3), a receptor for VEGF-C and -D, has been shown to befunctionally expressed in tumor blood vessels and in vivo experiments havedemonstrated that anti-VEGFR-3 antagonistic antibody inhibited tumor growth. In the present study, mechanism of inhibition of tumor growth by anti-VEGFR-3 At) was analyzed in vivo and in vitro. <Methods> (in vivo) 0.5 ml of matrigel supplemented with 500 no of bFGF was injected subcutaneously to the abdominal midline of nude mice. Intra-peritoneal administration of anti-VEGFR-3 Ab at a concentration of 600 microgram/ml or the came concentration of control Ab was done every other day after matrigel injection. Matrigel plugs were excised at 14 days alter injection and the expression of VEGF-C or VEGFR-3 was assessed by imunohistocbemistry using respective antibody. The number of blood vessels neovascuiarized in matrigel was defined as CD31-positive cells. (in vitro) F-2 cell, tumorigenic mouse endothelial cell line, was seed onto polymerized matrigel and anti-VEGFR-3 At) or control IgG was added to the culture, then cells were culterud for 6-12 h and photographed to assess the endothelial tube formation. <Result>The number of CD31-positive cells in mat~'igel plugs supplemented with bFGF remarkably increased at 14 days after implantation. The increased expression of VEGF-C and VEGFR-3 were observed in matrigel plugs during the period of neovascularization induced by bFGF. This increased neovascularization in the matrigel plugs were reduced by peritoneal injection of anti-VEGFR-3 Ab. F-2 cells culturing on matrigel rapidly aligned and formed a tube-like structure and this endothelial tube formation was markedly inhibited by the addition of anti-VEGFR-3 Ab to the culture medium. <Conclu- sion>These results suggested that VEGFR-3 signals mediated by binding of its cognate preferred ligand VEGF-C might have a critical role in bFGF-dependent neovascuiarization. Blockade of VEGFR-3 mediated signals by anti-VEGFR-3 antagonistic Ab might serve as a new approach for anti-angiogenic cancer therapy.

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VEGF Expression and Neovascularination in Barralt's Cerslnoma and its Pramalignant Lesions Christian Moebius, Dept of Surg, Muencben Germany; Ingrid Backer, Oept of Pathology, Muenchen Germany; Marcus Feith, Joerg-Ruediger Siewert, Oept of Surg, Muenchen Germany; Hubert J. Stein

Introduction: Angiogenesis is essential for tumor growth and metastasis and of prognostic value in many different tumor entities. In Barrett s carcinoma (Be), the results are controversial. However, immature vessels in tumors are structurally and functionally different from mature vessels. The relation between mature and immature vessels as prognostic factor in Ba and in the metaplasia-dysplasia-sarcinoma (m-d-a)sequence has not been assessed. Method: 45 Ba (23 with associated metapiasia, 27 with associated high grade dysplasia) were immuno- stained for VEGF, CO 31 and ~-sm actin to discriminate between mature and immature vessels. VEGF stained slights were quantitatively evaluated measuring optical density with a

computer based program and expressed as percentage of control slides staining ( placental tissue). The neovasculadeation coefficient (nc)was estimated with a new, interactive analytic computer program. Results: The median survival of the study group was 45,73 months ( 0 to 111,34). VEGF was 23,52% (_ 1t,58) in metapiasia, 63,57 % (+_34,31) in high grade dyspiasia, 57,9% (+_ 25,02) in T1 carcinoma and 83,21% (+_ 51,14) in advanced carcinoma. The nc was 3, 42 (+- 0,9) in metapiasia, 11, 49 (+-1,79) in high grade dysplasia, 12, 41 (+3,13) in T1 carcinoma and 29, 39 (± 8,5) in advanced carcinoma (p< 0,001). By univariant analyses VEGF did not correlate with clinicopethological data, but nc did correlate with histopathological staging (p<O,O01). Survival time in patients with low nc was significantly higher than in patients with high nc (p<0,021). Conclusions: Based on a new, strong quantita- tive evaluation program, the present study indicates, that the "angingenetic switch" is an early event in the m-d-c sequence of Be. In add/hon, the nc correlates with conventional prognostic factors. High aogiogonesis score is associated with short survival. These findings might offer new, anti-angiogenetic therapeutic strategies in treatment of Ba and Barrett s metaplaala to prevent malignant progression,

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Rqofofion of Vasmilar Endothelial Growth Factor (VEGF) Production by Both Coil Smlface Membrane and Nuclear Pfos(aolandin (PG) Receptors

Hirorni Bamha, Saltama Medical Ctr, Saitama Medical Sch, Kawagoe Japan; Shinichi Ota, Saltama Medical Sch, Iruma-oun Japan; Akira Kato, Chiaki Kawamoto, Saitama Medical Ctr, Saltama Medical Sch, Kawagce Japan; Kenji Fujiwara, Saitama Medical $ch, iruma-gun Japan

We previously reported that cyclooxygenase-2 was predominantly expressed in macrophages of sporadic human colonic adenomas (int. J. Cancer 83: 470,1999). Furthermore, we showed that PGs dramatically increased VEGF, a known tumor angiogenic factor, production by activated macrophages through specific PGE receptor and PPAR-t (a nuclear receptor of PG) mediated process (6BRC 273: 485, 2000). On the other hand, PPAR,~, a subtype of PPAR, was induced by APCJ p-catenin/Tcf-4 pathway and thereby may associate with colon carcino- genesis (Cell 99: 335,1999). To characterize the targets and roles of PGs in colon carcinogene- sis, we examined the effect of each agonist of PG receptors on VEGF production by macro- phages and epithelial tumor cells. METHODS: PMA-differantiated U937 cells and HT-29 (a human colon cancer cell line) were used as a human macrophage model (H-Mac) and an epithelial tumor cell model, respectively. PGE1 for PGE receptors, and cicaprost and iloprost for PGI receptor ware used as potent agonists of membrane receptors. For nuclear receptors, 15-deoxy-/\m4-PGJ2 (a potent agonist of PPAR'y, 15d-PGJz) and L-165041 (a potent agonist of PPARo~ ware used. As an agonist of both PGI receptor and PPAR~, carbaprostacyclin (cPGI) was used. VEGF was measured by EIA. RESULTS: PGE1, cicaprost, and iloprost significonUy induced VEGF production in a dose-dependent manner by H-Mac, but not that by HT-29. The order potency at 10 ~ M was PGE~ > cicaprost > iloprost. 15d-PGJ2 (10 -5 M) signiflcanby increased VEGF production by each model, whereas L-165041 (10 -s M) modestly but significantly increased that by HT-29 and did not modulate that by H-Mac. Induction of VEGF production by 15d-PGJz was stronger than that by L-165041. cPGI increased VEGF production by H-Mac, while this agent increased VEGF production by HT-29 only at 10 .5 M. Combinations of each specific membrane receptor agonist and 15d-PGJ2 were additively increased VEGF prnduction by H-Mac. Taken together, PGEI may increase VEGF production by macrophages through specific PGI receptor rather than PGE receptor, since PGE1 also bind to PGI receptor. In epithelial tumor cells, VEGF production was mainly induced by nuclear receptors, especially PPAR~, but not by membrane receptors. CONCLUSION: These results suggest that different membrane and nuclear PG receptors are involved in the induction of VEGF by macrophnges or epithelial tumor cells, which may play important roles in colon carci- nogenesis.

E x l ~ o n Of PlatolM-Oerived Endothelial Cell Growth Factor In Calorectul Adenomas And Carcinomas Francesco Franceschi, Baylor Coil of Medicine, Houston, TX; Antonia R. Sepulveda, Univ of Pittsburgh, Pittsburgh, PA; Lei Gong, Hirokazu Fukui, Baylor Coil of Medicine, Houston, TX; Antonio Gasbarrini, Giovanni Gasberrini, Catholic Univ of Rome, Rome Italy; Robert M. Genre, Baylor Coil of Medicine, Houston, TX

BACKGROUND:Tubular adenomas represent dysplastic lesions of the coloractum with malig- nant potential. Tumor survival depends on adequate angiogenesis, which is induced by several biological factors, including platelet-derived endothelial cell growth factor (PD-ECGF). The aim of this study was to determine the patterns of PO-ECGF expression in dysplastic and neoplastic coloreotal mucoeal lesions. METHODS:Formalin-fixed paraffin-embedded tissue from 14 patients with coloreotal adenonarsinoma and 18 subjects with colorectai adenomas were studied. All adenomas were tubular or tabulo-vitlous adenomas characterized by low- grade dysplasia Immunohistochemical stains were performed using monoclonal antibodies against PD-ECGF (NeoMarkers, Union City, CA; dilution 1:600).Sections of normal colorectal mucosa ware used as control. The extension of immunoreactivity was evaluated using a scoring system ranging from 0 (absence of expression or presence in <10% of cells), 1 (expression in 10-30% cells), 2 (expression in >30-50% cells), 3 (expression in >50% cells), while the intensity was assessed by a semi-quantitative scoring system (0 = absent; 1 = mild; 2 = moderate; 3 = maximum). The results were reported as a mean -+SE.RESULTS:Ten of 14 adenocarcinomas of the colon (71%) showed expression of PD-ECGF, with a mean intensity score of 129+-.29 and mean extension score of 1.36+-.30. Six carcinomas (43%) shewed extensive PO-ECGF expression present in >30% of tumour cells. PD-ECGF was only focally expressed in four of 18 adenomas (11%), and involved < 10% of cells in the lesion. CONCLUSIONS:PD-ECGF is expressed by the majority of colorectal cancers, but to a low extent in tubular adenomas. These findings suggest that PO-ECGF is associated with advanced neoplastic disease, while significantly increased expression does not appear to be required during earlier steps of coloractal carcinogenesis.

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