Enhanced DNA topoisomerase I inhibitor-induced apoptosis by UDCA in human colon adenocarcinoma cells

of caspase-7, not caspase-3. All three MAPK family members were activated upon paclitaxet treatment. Interestingly, JNK activation and p38MAPK activation were delayedand peakedat 48 hrs, whereas ERK activity was sustained over 72 hrs. In addition, activation of Ras and MEK was concordant with ERK activation. IP/Westem blotting revealedthat neither MEK-1 nor MEK-2 was involved, but did suggest the possibility of a different MEK family member, either known or novel. Although pre-treatment with a general caspase inhibitor, Z-MAD-FMK, rescued apoptosis, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38MAPK, or MEK did not alter the apoptosis induced by paclitaxel.CONCLUSIONS:Paclitaxel induced apoptosis in human esophagealcancer cells through caspase-7activation, a novel finding. Paclitaxelalso inducedprolonged JNK, p38MAPK,and ERKactivation, which occurred independently of the cell death machinery, suggesting that delayed and sustained activation of the MAP kinases may contribute to cellular homeostasis in the face of apoptosis. 3583 Cyciin D1 Mediates the Trophic Properties of Gactdn in Gastric Xdenocarclaoma Basabi Rana, Caroline Choi, Geoffrey M. Crimmins, Boston Univ Sch of Medicine, Boston, MA; Timothy C. Wang, Univ of MA, Worcester, MA; Richard G. Pestell, Chris Albanese, Albert Einstein Coil of Medicine, Bronx, NY; M. Michael Wolfe, Boston Univ Sch of Medicine, Boston, MA Background: Although recognizedprincipally as a stimulant of gastric acid secretion, another biological property attributed to gastrin, as well as to its nonamidatedprecursors, is its potent trophic effects on GI malignancies.Although we havepreviously reportedthat gastdn increases the expression of the cytoplasmic protein/3-catenin, the precise molecular mechanisms by which gastrin exerts its trophic properties have not been elucidated. Purpose: To determine the relationship between gastrin-mediatedgrowth of malignant cells of GI origin and cyclin D1, a major regulator of the cell cycle that is a downstream target of/Y-cateflin and is commonly expressedat high levels in various GI neoplasms. Methods: AGS-Bcells, a human gastric adenocarcinomacell line stably transfected with functional gastrin (CCK-B) receptors, were serum starved for 24 h. Cells were then cultured for various periods of time in the presence or absence of 10 nM gastrin-17 (G-17), after which total RNA or protein was extractedand Northernand Western hybridizationanalysisperformed. In separateexperiments, cyclin D1 promoter activity was measured in AGS-B cells transiently transfected with wild type or mutant 1745-bp cyclin D1/luciferaseconstructs. Cellswere incubatedwith and without gastrin, and luciferase activity was measured using luminometry. Results: Inclusion of G-17 in the culture medium increasedboth/3-catenin and cyclin D1 mRNA levels. ,8-catenin mRNA levels increased by 133.3%, 56.6%, and 66.7%, and cyclin D1 mRNA levels increased by 25.0%, 52.2%, and 103.6% at 1 h, 4.5 h, and 24 h, respectively. These increases were significantly attenuatedby inclusion of the CCK-Breceptorantagonist L365,260 in the medium. Although Western blot analysis did not detect increasesin either total cytoplasmic or nuclear /~-catenin levels, significant increasesin cyclin D1 levels were demonstrated.Moreover, when transfected AGS-B cells were incubated with 10 nM G-17, a 4-5 fold induction of cyclin D1 promoter activity was measured, an effect that was nearly abolished by the inclusion of L365,260 in the culture medium. Mutation of the cAMP responseelement (CRE) of the cyclin D1 promoter demonstrated similar induction as measured with the wild type construct, suggesting that the effects of gastrin on the cyclin D1 promoter are not mediated through the CREelementin the promoter. Conclusion:The results of these studiesindicatethat oastrininduced tumor growth appears to be mediated, at least in part, via modulation of cyclin D1 expression. 3584 Enhanced DNA Topoisomerose I inhibitor-induced Apoptosis By UOCA in Human Colon Adsnocarcinoma Ceils Tadashi Ikegami, Joe Cotruvo, Susan Ceryak, George Washington Univ, Washington, DC; Yasushi Matuszaki,Tsukuba Univ, Tsukuba Japan; Bernard Bouscarel, George Washington Univ, Washington,DC Backgroundand Hypothesis:Theoral administration of ursodeoxycholicacid (UDCA)has been proposedto prevent diarrheaassociatedwith administration of CPT-11, a DNAtopoisomerase I inhibitor. Since UDCA has been reported to have an anti-apoptotic effect in hepatocyteand non-hepatic tumor cell lines, we hypothesizedthat UDCAalso has an anti-apoptntic effect on CPT-11-induced intestinal cellular injury. Methods:Cytotoxicityand growth-inhibition by SN38, the most potent metabolite of CPT-11, were determined by MTI assay, and colony formation assay, respectivelyin human colon adenocarcinomaHT-29 cells. For the detection and quantification of SN-38-induced apoptosis, determination of internucleosomal DNA fragmentation (IDF) in agarose-gelelectrophoresisand determination of phosphatidylserine(PS) externalization by flow cytometry using GFP-labeledAnnexin-V were employed. Cell cycle determination by flow cytometry was performed using propidium iodide to probe DNAcontent. [~4C]SN-38 was used to determine intrasellular accumulation. Results: The result of MTi" assay did not show any significant difference in SN-38-induced cytotoxicity when tested in either the presenceor absenceof UDCA.However,the IDFinducedby increasingconcentrations of SN-38(0-2 #M)was enhancedby 50/~M UDCAat every concentrationtested. Furthermore, UDCA potentiated the PS externalization induced by 0.5 p,M SN-38 in a dose-dependent fashion to a maximum of over 50 % observed with 50/~M UDCA (P