Cycleoxygeases-2 (COX-2) accelerates gastric epithelial restitution via increasing cell preliferation and enhancement of cell migration

2556

proteins (hypophosphorylatedforms) becameapparentand significant by day 1 post-confluence,correlating with G1 arrest. 2- The expressionof E2F1,which was elevatedin subconfluent HIEC, was significantly down-regulated in confluent HIEC while there was no significant modulation of E2F4expression.3- However,analysisof Rh/E2Fcomplexesformation demonstrated that E2F4, which appears to be mostly "free" in subcontluent growing HIEC, was sequesteredby pRb proteins in confluent quiescentcells. 4- Transienttranstection experiments demonstratedthat the forced expressionof E2F1and E2F4stimulated dihydrotolate-reductase gene expression (S-phase marker) by 2- and 7-fold, respectively. 5- Immonofluorescence studies on intestinal sections revealedthat while E2F4was weakly detectablein the nuclei of most crypt celts, strong staining was observed in a few ceils; double staining experiments revealed that cells which expressed high levels of E2F4 were all positive for Ki67 staining. Expressionof E2F1 was detected in the nuclei of all crypt cells along with a decreasein stain intensity as the cells migrated towards the villus. Conclusion. Our data demonstratea much more important role for E2F4 in cell cycle progression of intestinal crypt cells and that sequestration of E2Fs by Rb proteins is a critical step in initiating cell cycle exit. Supported by CIHR of Canada.

Cyste(i)ne Stimulates Intestinal Ceil Proliferation Independent Of Glutatbione Synthesis Takahiro Noda, LSU Health Science Ctr, Shreveport, LA; Ryuichi Iwakiri, KazumaFujimoto, Saga Medical Sch Internal Medicine, Saga Japan; Tak Yee Aw, LSU Health Science Ctr, Shreveport, LA BACKGROUND:Cysteine, an important amino acid thiol, and its disulfide, cystine, have cell growth potential. Glufathione (GSH), an ubiquitous intraCelluiarthiol, is also related to cell proliferation. Since cysteine is rate limiting for GSH synthesis, if is unclear whether the proliferative effects of cyste(i)ne are mediated by a change in the intracellular GSH status. The objective of this study was to delineate the relationship of cyste(i)ne and intraceilular GSH and cell proliferation in the human colonic CaCo-2 cell line. METHODS:CaCo-2 cells were cultured in cyste(i)ne free DMEM 21013 medium (minus cyste(i)ne and serum). Cells were treated with several concentrations (200/~M-400#.M) of cysteine and/or cystine with or without 5mM DL-bnthionine-[S, R]-suffoximine (BSO), an inhibitor of GSH synthesis. 3Hthymidino incorporation, protein content, and intracellular GSH were measured. Cell cycle analysis and western blot analysis were also performed. RESULTS: Baseline GSH levels in these cells were significantly lower than the levels in cells cultured in standard medium (DMEM 12800 contains 200pM cystine and 10% serum). Addition of cyste(i)ne increased GSH levels that wore attenuated by BSO. In the absence of BSO, exposure to cyste(i)ne exhibited no proliterat~ effects in spite of GSH increases. In the presenceof BSO, (i.e, GSH depletion), cysto(i)ne stimulated cell proliferation in dose independentmanner, but did not change the GSH status. Taken together, these data show that cysteine and cystine stimulate CaCo-2 proliferative activity independentof GSH synthesis. Cell cycle analysis showed that the proliferative effects of cyste(i)ne were due to stimulation of progression of G1 cells into the S phase. This cell cycle progression was correlated with increased expression of cyclin D1 and the phoepho~iation of the retinoblastomaprotein (pRb). CONCLUSION:Exogenously supplied cyste(i)ne to GSH depleted intestinal cells exerted GSH independent proliferative effect by inducing cell cycle progression that was mediated by cyclin D1 expressionand pRb phoephoryla~on.Supported by NIH DK44510.

2554 Human Enterocytic Apoptosis And Survival: Differentiation State-Specific Control Mechanisms. Remy Gauthier, Jean-Francois Drolet, Fac de Medicine, U de Sherbrooke, Sherbmoke, PQ Canada; John C. Reed, The Burnham Institute, La Julia, CA; Anne Vezina, Pierre H. Vachon, Fac de Medicine, U de Sherbrooke, Sherbrooke, PQ Canada BACKGROUND/AIMS:In this study, we investigatedthe question whether the regulation of human enterocyticcell survival along the crypt-villus axis involvesdistinct control mechanisms dependingon the state of differentiation, using the well establishedCaco-2/15 human enterocyte-like in vitro model. METHODS: We analyzed the effects of insulin, pharmacological inhibitors of Fak, MEK/Erkand PI3-K/AId, and integrin (~,/34)-blocking antibodies, on the survival of undifferentiated (-2 days postconfluence) and differentiated (30 days postconfluence) Caco-2/15 cells. Cells were exposedfor 48 h to serum free medium containing either insulin (10 p.g/ml) or one of the following inhibitors: genistein (tyrosine kinases; 150 pM), PD98059 (MEK/Erk pathway; 20/AVI), Ly294002 (PI-3-K; 5/~M), or cytochalasin D (Fak; t p.M). Integrin subunit/31- and/~4-blocking antibodies ware the mouse monoclonals P4C10 and 3E1, respectively, with non-immune mouse IgG's as control. Apoptosis was evaluated by ISEL and/or by ONA laddefing assays. Relative expression levels of six Bcl-2 homologs (Bcl-2, BcI-XL, Mcl-1, Bax, Bak, and Bad) and activated levels of Fak, Erk-2 and Akt were also analyzed by Western blot and/or immunoprecipitution. RESULTS: 1- the enterocyt¢ differentiation processof Caco-2/15 cells results in the establishmentof differentiation-distinct profiles of Bcl-2 homolog expression levels, as well as p125F=, p42~2 and p57= acthrated levels; 2- the inhibition of Fak, MEK, PI3-K, ~ integnns or/34 integrins have distinct impacts on cell survival cepending on the state of differentiation (for instance, Fak inhibition has a significantly greater impact on the survival of differentiated cells); 3- exposureto insulin and the inhibition of Fak, MEK and PI3-K resulted in differentiation state-disctinct modulations in the expression of each Bcl-2 homolog analyzed;and 4- Fak, ~ and p4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of differentiation (for example,/34 integrins and MEK/Erk are not invoked in the survival of undifferentiated cells). CONCLUSIONS:Taken together, these data indicate that regulation of human intestinal epithelial cell survival is subject to differentiation state-specific control mechanisms, strongly supporting the concept that enterocytic apoptosls and survival are distinctively regulated along the crypt-villus axis in vivo. (supported by the CIHR and the FRSQ)

2551 CVelo~2 (COX-2) Accelerates Gantdc Epitkelial Restitution Via Increasing Cell P~Hfferlml Md Enhancement Of Cell Migration Mlyoko Hirose, Osanu Kohayashi,Hanako Misawa, Hiroto Miwa, Yoshiyuki Takei, Nobuhiro Sato, Dept of Gastroenterology,Juntendo Univ, Tokyo Japan Background & Aim The expression of COX-2 increases during ulcer healing in vivo. COX-2 induced by various growth factors acceleratesgastric epithelialrestoration.Sinceprostaglandin (1:~) E~andEt up-regulats vascular endothelialgrowth factor (VEGF) production, COX-2 may also act on angiooenesls.This study was aimed to clarify the mechanism(s) by which COX2 accelerates gastric epithelial restitution. Methods To assess epithelial restitution, artificial wounds (3mn~) were created on complete monolayer of RGM-1 cells after starvation of FCS for 24h. The expression of COX-2 induced by addition of FCS was detected by immunocytochemlst~. Wound repair was evaluated in the presence or absence of a selective COX-2 inhibitor, JTE-522 (Japan Tobacco), arachidonic acid (AA) or PGE2.Production of PGE2and VEGF by RGM1 was determined by ELISA. Lamellipodia was visualized by staining actin filament with rhodamine-phalloidinmethod. Activation of MAPKand JNK/SAPKwere detected by Western blotting. Results COX-2 protein becamedetectablein RGM-f cells located at the edge of wound from 6 h after FCS addition. Concomitantly, lamellipodia formation and polymemed a~n were observed. COX-2 was sublocalized around the surface of nuclear membrane of the ceils. COX-2 staining reached a maximal intensity at 9-12 h after FCS and remained detectable until wound healing was complete. Addition of JTE-522 retarded signifieantty the wound healing in a dose dependentmanner (10-5, 3 x 10s, 104 M), resuting in almost complete inhibition of RGM1 cell proliferation at 104 M (94% inhibition, from 41.6 + 4.1 to 2.58 + 1.24 % BrdU positive cells). Surprisingly, AA or PGE2had no direct effects on wound healing in this model. Activation of MAPK, JNK/SAPKand Recl by FCS was not inhibited, but the formation of lameilipodiaand accumulation of actin were preventedby JTE522. Conclusion These results suggest that COXo2accelerateswound healing through two mechanisms: one is the increase in cell proliferation and the other enhancement of cell migration. The former appears to involve intracellular signaling pathway distinct from or downstream of the MAPKand JNK/SAPKpathway. Further, metabolitesof M are least likely involved in the cell proliferating effect of COX-2, which might propose a new role for COX2 in reguiatuion of cell proliferation.

2555 Colonic Tumor Cell Lines Undergo Growth Arrest Upon Hypotonic Stress Despite Cell Une-Variable Modulation Of Keratin Phosphorylntion Guo Zhong Tan, Li Fen(], M Bishr Omary, Palo Alto VA Medical Ctr, Palo Alto, CA Background: Intermediatefilaments (IF), microtubules and microfilaments are the three major components of the cytoskeleton. In digestive epithelia, keratin polypeptides8 and 18 (K8/18) are the major IF proteins. K8/18 undergo dramatic hyperphosphorylationduring mitosis and a variety of stress conditions, including virus infection and heat, at K8 phospho-(p)-sarine (S)73/pS431 and K18 pS33/pS52. Aims: Examinethe effect of hypotonic stress on cell growth and keratin phosphorylation,given that several phosphatasesand kinasesare activatedduring hypo-osmotic conditions and the highly dynamic regulation of keratins by phosphoryletion. Methods: Three colon-caminema cell lines HT29, HRT18 and CaCo2 were used. Hypotonic treatmentwas carried out by 1:1 dilution of the culture medium with distilled water. Phosphoryiation of K8/18 was assessed by Western blotting using site-specific anti-phoslY~H~n antibodiesor by 2-dimensionalgel analysis.Cellcycle analysiswas doneafter propidium iodide staining, and keratin distribution was examinedby immunofluorescence staining. Results:All three colonic cell lines were arrested at G1/S upon a 6 hour exposure to hypotonic stress (average baseline®hypotoniccell cycle changes: %GO/G1, 38.67; %S, 40.25; and %G2/ M 22,8). This effect was unique to the colonic cell lines since two tested pancreatic cell lines (AsPG1 and PaCa-2)did not manifest a change in their cell cycle profile. Upon hypotonic stress, K8 $73 phosphoryiationincreasedin all three colonic cell lines, and K8 $431 phosphoryiation increased in HRT18 and CaCo2 cells, while surprisingly K8 5431 phospho~tation decreaseddramatically and reversibly in HT29 cells. No significant K18 $33/$52 phoephoryiation effects were noted. Upon exposure to okadaic acid, a protein phosphetaseinhibitor, K8 $431 phosphoryiation increased dramatically in HT29 cells after hypotonic stress, but was unalteredin HRT18cells therebysuggestingphosphataseactivation in HT29cells. Conclusions: We describe, for the first time, a hypotonic-inducedG1/S block in cultured tumor cell lines. This effect occurred in colonic but not in two tested pancreatic cell lines. H l ~ cells appear to manifest a unique K8 pS431 hypotonic stress-selective phosphataseactivity, independent of cell cycle arrest.

Inmmmmmbalitatim Of Endogenous Glucegon-Like Peptide-2 Reduces The Adaptive Intestinal Growth in Diabetic Rats. Boletts Hartmann, Jesper Thulesen, Krlstine J. Hare, HannelouiseKissow, Cathrine Orskov, Steen S. Ponlsen, ,lens J. Hoist, Univ of Copenhagen,CopenhagenDenmark Background:Treatmentwith suprephysiologicaldoses of glueagon-likepeptide-2 (GLP-2) has been shown to induce intestinal growth by increasing villus height and crypt depth and decreasing apoptosis, but a physiological effect of GLP-2 has not yet been demonstrated. Elevatedlevels of endogenousGLP-2 in plasma havebeenreported in untreatedstreptozotocin diabetic rats, associatedwith marked intestinal growth. The aim of this study was, therefore, to investigate the role of endogenous GLP-2 for this adaptive growth response. Methods: Experiments were performed on adult female Wistar rats (weighing approximately 180 g), housed individually in wira-bettomed cages in a temperature- (21 C) and humidify-controlled (55%) environment with a 12:12 h light:dark cycle. Thirty rats were used of which twentyfour were made diabetic by streptozutocin injection (60 mg/kg). The animals were allocated into the following groups of six: 1) diabetic rats treated with NaCI, 2) diabetic rats treated with non-specific antiserum, 3) diabetic rats treated with polyclonal GLP-2 antiserum, and 4) control rats treated with NaCI. All animals were treated for two weeks with intraperitoneal injections of either antiserum or NaCI once daily and killed on day 15. At death, the weight

A-502