gi67 positive b,mphoid cells increased in the medulla during 24 to 48 h. The number of cax~cer cells in the marginal sinus reached the maximmu at 6 h, then declined until 48 h, but significant proliferation was observed front 4 days atier inoculation. The marginal sinus was then filled with cancer cells and expanded. At this period, the ED3 macrophages accumulated and intermingled with cancer ceils, Some cancer ceils resulted in apoptosis, being incorporated by the ED3 macrophages. In LN depleted of macrophages with liposomes containing DMDP, cancer cells massivdy invade the LN parenchyma TNF-e~ and IL-I[3 in the LN significanfly increased at ] and 3 h, respectively [1-2 increased significantly during 6 to 24 h. These cytokines returned to the basal level thereaher. 1FN-y or IL-12 did not increase at earlier periods, but significantly decreased from 24 to 96 h Localization of IL- 113 positive cells was consistent with that of ED2 macrophage in the medulla, but not of ED3 macrophage. TNF-cx positive cells were found in the germinal centen Conclusions: Immune responses in thks study include germinal center activation, followed by lymphoid cell pmlileration in the medulla and sinus histiocytosis, which mimic histological maniIesta- tions of regional LN in cancer patients with improved survival Thus, LN did have capability to eliminate carmer cells temporarily. Depiction of immunocompetent cells due to deteriora- non of cytokine induction may be responsible for later loss of the capabihty to prevent metastasis Macmphages contribute as a first line defense against cancer invasion. There is a different role in preventing metastasis between marginal atrd medullary sinus macrophages. W969 Up-regulation of Muc4 and Its Complex Formation With ErbB2 in Murine Gallbladder Carcinoma - a Potential Mechanism fur Gallbladder Carcinogenesis and/or Tumor Growth Naoki Miyahara, J~michi Shoda, Tetsuya Ueda, Tom Kawamoto. Naomi Tanaka, Tatsuro Irnimra Yoshihiro Akimoto, Hayato Kawakami, John Digiovanni Kaom Kiguchi Background: BKS.ErbB*2 transgenic (Tg) mice overexpressing ErbB-2 (Cancer Res 2001), coupled with the induction of COX-2 overexpression, develop GB carcinoma (GBCa) at a high incidence. However, infurmation on ligands for ErbB-2 activation and their ErbB-2 signaling pathways important for the development of GBCa has not been fully obtained. Recently, Mac4 (siafunmcin complex) is known to influence ErbB-2 (tyrosine kinase) activity through direct interaction with the receptor in mammarian carcinoma cells. Aims: "17o study the expression level of Muc4 and its interaction with ErbB-2 in the Tg mouse GBCa for a better understandirtg o[ the mechanism involved in the development of GBCa Methods: The expression level of Muc4 in Tg mouse GBCa was demrmined by competitive PCR The tissue distribution of Muc4 protein and mR.",;A was evaluated by immunofluorescence staining using CLSM and in situ hybridization, respectively. The complex tbrmation of Muc4 with ErbB-2 was a~ssed by immunoprecipitation. Elfects of a selective COX-2 inhibitor, cele- coxtb, on the Muc4 expression leveI and the complex furmation in Fg mouse GBCa were studied Tissue PGE2 concentration was determined by EL& Results: In Tg mmlse GBCa, the Muc4 mRNA level was up-regnlated (799-1732% of non-Tg mouse GB) Both Muc4 mRNA and protein were strongly positive in the cancerous epithelia of the GBCa, whereas they were detected in only trace amounts in the non-Tg mouse GB In heterozygous Tg mice, only a modest upregnlation of Muc4 expression (175%) was observed in the GK The magnitude of }efuc4 up-regulation in the GB appeared to be dependent on the ErbB-2 expressmn levels there in the immunofluorescence images, Muc4 was observed to co-localize with the ErbB-2 overexpressed in the GBCa, The data on immunoprecipitation confirmed the direct interaction between Muc4 and ErbB-2. Treatment with celecoxib did not cause any significant changes in the Muc4 protein and mRNA levels (2041-2755%). A selective irfhibition of the COX-2 activity, which was confirmed by the decreased PGE2 level, may not solely be able to attemtate the Muc4 up-regnlatirat. Conclusions: In BKS.ErbB-2 mice, up-regulation of Muc4 and its complex formation with the ErbB-2 may play an important role in the development of the GBCa. In tire ErbB-2 signaling pathways, the Muc4 up- regulation may not be govented by the COX-2 activity. This stud), was supported by G~.75520, CA16672, ES07784, and LS02~6809MM (Phannacia) W970 Pressure activates colon cancer cell adhesion by inside-out FAK, src and cytoskeletal signaling Vijayalakshmi Thamilselvan. Richard J. Gilbert, Marc D. Bass0n Although viable colon cancer cells are frequently recovered tronr the venous circulation relatively few implant and cause clinical metastasis. Since intravascular pressure initiates signals in endothdial cells, we hypothesized that venous or lynrphatic pressure might activate intracellular signals governing the adhesion of circulating colon cancer cells. Colon cancer ceils isdated from 30 patients exhiNted an ~2 fold increase in adhesion m matrix proteins when subjected to i5 mm Hg pressure fur 30 minutes. In additiou, adhesion of puma13, colon cancer cells was inhibited by 10 mM Ca 2 ~ and stimulated by 10 mM Mg2+ and Mn> , consistent with the p~esence of a divalent cation binding site on the extracelhilar domain of cdlM integrin. These observations were replicated in human SW620 colon cancer cells that then served as a modal for further study. As the effect was matrbxdndependent, subse- quent studies were peribrmed using a collagen I substrate. Pressure did not alter integrin subunit surface expression, based on flow cytometric analyses Detachment assays demon- strated that pressure altered integrin binding affiint~/for matrix more substantially than did cations Pressure activated SW520 FAK and Src ~l.5-{old, based on Western blots and in vitro kmase assays; Mn e~ and Mg~' did not. In contrast, Ca z+ actually stimulated FAK (but not arc) although Ca + inhibited adhesion. Src inhibition by pp2 prevented pressure- stimulated adhesion but did not bhick modulation of adhesion by extracellular divalent cations FAK inhibition, either by transli:ction veith a dominant negative construct (FAK- 397) to inhibit FAK signaling or by reducing FAK protein lords ~60% using an antisense interierence strategy,', also prevented pressure-stimulated adhesion but not the effects of the dwalent cations. Pretreatment wifh phalloidin to block actm rearrangement ago prevented pressure~stimulated adhesion, FAK autophosphoryIation and FAR phosphorylation by Src hut not Src activation itself. These results suggest that extraceltular pressure activates colon cancer cell adhesion by increasing integnn affinity. In contrast to the direct efli:cts of divalent cations binding to integrin extracelfular domains, the pressure eftect seems mediated by inside-out signals at the focal adhesion complex, specifically cytoskeletally mediated mechani- cal activation of FAK potentiated by activated Sre. We suggest that this mechano-transduced signal pathway may be a key regulator of the adhesion of metastasizing cancer cells. W971 Prevention of Colorectal Carcinoma in Azoxymethane Treated Rats by Non- Steroidal AntMnflammatory Drugs (NSAIDs) is Dependent on Histologic Type Jonathan Martin, Richard Le Leu, Ying Hu, Graeme Young Introduction/Aims: Carcinomas, resulting fi'om treatment with the carcinogen azoxymethane (AOM) m rats, fall into two main histologic types. Non-mucinous carcinomas are mostly distal, corrdare with numbers of aberrant crypt loci (ACF) and seem to follow an adenoma- carcinoma sequence, similar to most human colorectal carcinomas. Mucmous carcinomas are mostly proximal, associated with lymphoid aggregates, and appear to arise de-novo. We aimed to determine if the preventive effect of NSAIDs on AOM derived tumours was influenced by histologic type. Methods: Groups of 50 Sprague-Dawley rats were given drag by daily garage After I and 2 weeks, AOM was given at a dose of 15mg/kg. Groups were controls, sulindac (cycloox3,'genase (COX) inhibiting) at 5mg/kg and 20mg/kg per day and R-tlurbiprofen (non-COX inhibinng) at 30mg/kg per day. Rats were killed after 30 weeks, the colon examined for mmours using a dissecting microscope and tumours classified histologically. The proportion of rats developing carcinomas was compared between groups. Results: The overall proportion of rats developing tumours was reduced significantly by aulindac 20mg/kg and R-flurbiproti:n 30mg/kg. However, as shown in the table, when histologic type was considered this eftect was confined to non-mucinous carcinomas. There was a trend toward an increase in the pmportinn of rats developing mucinous tumours in the R-[lurbiprofen group. Conclusions: NSAIDs prevent non-mucinous colorectal carcinomas and not mucinous carcinomas in AOM treated rats. Suppression of non-mucinons carcinomas is independent of COX inhibition. Since non-mucinous tumours appear more relevant to most ht~.man colorectal carcinoma, and the eftect of preventive treatment differs with histo- logic type, mmour histology must be taken into account in future studies using AOM. Study Group PmporUon wRh Muclnous Ade- Proportion with Non.Mucinous nocarr Adenocarcinomas No drug 016 0,50 Sul~t/ac 5mg/kg 0.18 (RR 1,13, CI 0,47~2,6B) 0.34 {RR 0.68, Cf 0.42-1.09) Sulindac 20mg/kg 0.16 (RR 1.0, CI 0A1-2.46} 0,08 (RR 0.16, CI 0.06-0.43) b R-flurblpro~ 0.32 (RR 2.0 CI 0.94- 4.25) 0.20 (RR 0.40, CI 0.22-0.74), 3om~lql 1<0.01 vs no drug grcu.ip,b 1<0,001 vs no drug group (Pearson Chi-square test} RR is ~etive risk and CI is 95% confidence interval. W972 Undegraded Lambda Carrageenan Promotes Formation of MNU (N-methyl-N- nitrosourea) Induced Stem Cell Mutations in Balh/c Mouse Colon Eihsh T. Donnelly, Helen Bardwefl, Geraldine A. Thomas, Dfllwyn Williams, Margaret Hoper, Paul Crow< William G. McCluggage, Michaei Stevenson, David H. Phillips, Alan Hewer, Martin Osborne, Frederick C CampbeIl Interactions of dietary components may" promote tumorigenesis but are poorly understood Red meat increases colonic N-nitmsation. Lambda carrageenan (CgN) is a non genotoxic food additive with pro-inflammatory potential Vee hypothesize that undegraded CgN and a N-nitroso compound (N-methyl-N-nitrosourea; MNU) have additive or interactive effects upon mutational endpoints in mouse colon. CgN (1% or 4% in drinking water) and MNU were administered alone or in combination to 3 groups of female Balb/c mice. CgN treatment alone was assessed against histological scores of colonic mucosal inflammation or injury. Individual or combined CgN/MNU efti:ets were then assessed upon DNA adduct formation and crypt stem cell mutations in mouse coion. Stem cell mutations were assayed by mutant endogenous metallothionein reporter gone assay. MNU was administered as a single ip dose of 62 5mg/kg. In combined studies, single or recurrem short term or coninmons CgN intake regimens were administered with MNU. Continuous treatment by CgN alone for 10 weeks induced significantly greater colonic inflammation and ir~iury scores than drinking water control (p<0.01) Short-term CgN treatment did not enhance MNU-induced DNA adduct fbrmation. However, all patterns of CgN intake significantly increased MNU induced stem cell mutations over those of MNU alone (p<0 01). Recurrent short term or continuous 1% CgN intake had significantly greater promotional effects on MNU induced mutations than a single short term 1% CgN treatment (p<0 01) This study shows that undegraded CgN is pro-inflammatory in mouse colon and modulates MNU dosimetry ibr colonic crypt stem cell mutations W973 Polyethylene Glycol (PEG) Suppresses Transcription Factor SNAIL in Azoxymethane (AOM)-induced Colonic Aberrant Crypt Focci (ACF) and Human Colon Cancer Cell Line HCT-116 Ramesh K, Wali, Jennefer L, Koetsier, Marc B. Bissonnette, Hemant K. Roy Background: Loss of E-cadherin function has been shown to promote both initiation and progresaion phases of eolmr cancer, however, little is known about its transcripttonaI regula- tion. In breast, hepatocellular and gastric cancers, overexpression of SNAIL family of tran- scriptional repressors have been implicated in the E-cadherin downregnlation. Our prevllous data indicates that E-cadherin may be a target for chemopreventive action of variety of agents We and others have in the past demonstrated that PEG is a potent chemopreventive agent and inhibits down-regnlation of E-cadherin in AOM-induced colon cancer. We, there- fore, hypothesized that PEG treatment will dowm-egulate SNAIL expressi(m leading to increased E-cadherin production. To test this hypothesis we cmrducted both animal and cell cuhm'e studies Methods: Seventy five male Fisher 344 rats were fed AIN 76A diet for AGA Abstracts A-604