calmodulin supplementation on NOS catalytic activity in experimental portal hypertension. Methods• Animals were studied 4 weeks after sham surgery or bile duct ligation (BDL). NOS activity was assessed by measuring L-NAME inhibited L-citrulline generation from liver lysates in the presence of a standard concentration ot calmodutin {0.1 /~M) or alternatively lO-fold excess in calmodulin (1.0 /~M). eNOS/iNOS protein levels were assessed by quantitative immunoprecipitation/Western blot analysis. Results, NOS activity was significantly reduced in liver lysates from BDL rat livers in the presence of 0.1 /~M calmodulin, the concentration routinely used in this assay [see Figure (white bars); *P<O.05; sham vs 6DL at 0.1 /~M calmodulin; n = 6]. Western blot analysis demonstrated no reduction in eNOS protein levels or iNOS protein expression in BDL liver lysates. Importantly, supplementation of lO-fold excess calmodulin (1.0/~M) did not affect NOS activity from sham animals, but significantly increased NOS catalytic activity in BDL lysates, thereby correcting deficient hepatic NOS activity (see Figure, **P<O.O5; 0.1 /LM VS. 1.0 ~M calmodulin in BDL; n=6). Conclusion. Provision of excess calmodulin corrects deficient NOS catalytic activity detected from BDL liver lysates in an experimental cell-free assay.

)duhn ~duttn

sham BDL

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Nitric Oxide Modulates Both Pre-Sinusoidal And Sinusoidal Responses To Phenylepbrine In Normal Rat Liver Mauricio R. Loureiro-Silva, Yale Univ Sch of Medicine, New Haven, CT; Gregory Cadelina, VA CT Healthcare System/Hepatic Hemodynamic Lab, West Haven, CT; Roberto J. Groszmann, Yale Univ Sch of MedicineNA CT Healthcare System, New Haven, CT

Background/Aim. We have shown previously that nitric oxide (NO) modulates the intra-hepatic vascular tone (Am J Physiol 1994;267:416-422). Now, using a modified in situ rat liver perfusion as an experimental model, we studied both total and sinusoidal vascular responses to phenylephdne (PE, ~l-adrenergic agonist) as well as the role of NO on these responses in normal livers. Method. Normal rat livers were perfused (40 ml/min) at 37°C with 2% BSA Krebs-Henseleit buffer (pH 7.4) in the absence (n = 5) or the presence (n = 5) of Nw-nitro- L-arginine (NNA, NO-synthase inhibitor). Following a 2O-min incubation, a cumulative dose- response curve to PE (6 doses; 10-6 M to 3x10-4 M) was performed. Pertusion and sinusoidal (wedged hepatic venous pressure) pressures were measured continuously with independent strain-gauge transducers each attached to a side arm connected to the pertosion cannuta or to a catheter wedged into a hepatic vein tributary, respectively. The zero point was identical for both transducers. In 4 preliminary experiments, a cumulative doss-response curve to PE was performed using retrograde perfusion of normal rat livers. In these experiments, the wedge catheter was placed into a portal vein tributary in order to estimate the post-sinusoidal pressure (perfusion pressure minus wedged portal pressure)increase. Dose-response curves were compared using "repeated measures ANOVA". Results• Since perfusion flow is constant, any increase in pressure is equivalent to an increase in vascular resistance. Retrograde perfusions indicated that there is no post-sinusoidal response to PE. The pre-sinusoidal vascular resistance increase induced by PE was higher than the sinusoidal resistance increase both in the absence (P = 0.0164) and the presence of NNA (P<O.OOOf). The presence of NNA enhanced both pre-sinusoidal (P<O.O001) and sinusoidal (P=0.0423) resistance increases induced by PE. In the absence (P=O.0138; t test) and the presence (P<O.OOOl; t test) of NNA, the maximum resistance increase induced by PE was higher in the pre-sinusoidal area than in sinusoidal area (0.125 _+ 0.019 vs. 0.056 _+ 0.012 cmH20.min/ml and 0.390 +_ 0.026 vs. 0.098 +- 0.018 cmH20.min/ml, respectively). Conclusions. The vascular response to ~l- agonists in normal livers is mainly pre-sinusoidal. In normal livers, NO modulates both pre- sinusoidal and sinusoidal vascular tone; however, the main role of NO seems to be pre- sinusoidal. FAPESP 98/14790-1

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The Phosphorylation of Endothelial Nitric Oxide Synthase by Akt Increases Nitric Oxide Production in Early Portal Hypertension Yasuko Iwakiri, Ming-Hung Tsai, William C. Sessa, Yale Univ Sch of Medicine, New Haven, CT; Roberto J. Groszmann, Yale Univ Sch of MedicineNAMC/Hepatic Hemodynamic Lab, New Haven, CT

Akt, also known as protein kinase B, is a serine/threonine kinase, Studies in vitro showed that Akt can directly phosphorylate endothelial nitric oxide sycthase (eNOS) and activate the enzyme, leading to nitric oxide (NO) production. The aim of this study is to test the hypothesis that the phosphorylation of eNOS by Akt plays a role in enhanced NO production observed in early portal hypertension. This is the first study to show that eNOS phosphorylation and activation of enzyme by Akt occur in vivo. Male Sprague-Dawley rats were subjected to either sham or portal vein ligation (PVL). Animals with one day after PVL surgery were used as models for early portal hypertension. Mesenteric bed was used for the ex vivo perfusion study. Superior mesenteric artery (SMA) was used for the Western blot analysis. We showed that mesenteric bed in the PVL group had approximately three- to four-told decrease in response to methoxamine (an 2-1 agonists and vasoconstrictor), compared to the sham group (P< 0.01), When L-NMMA (a NOS inhibitor) was added to the perfusion, the difference in perfusion pressure between two groups was no longer significant, suggesting that increased

NO production in the PVL group blunted the response to the vasoconstrictor. In order to investigate this early mechanism of the increased NO production, we first examined eNOS expression. There was no significant difference in eNOS expression between the sham and PVL groups. The phosphoTytation of eNOS at serine 1179, known as a target by Akt, was significantly increased by two-fold (P<O.O5) in the PVL group. Furthermore, PVL significantly increased Akt phosphorylation (an active form of Akt) by three-fold (P<O.05). These results suggest that the phosphorylation of eNOS by Akt activates the enzyme and may be the first step leading to an initial increase in NO production in portal hypertension. In early portal hypertension, enzyme activation of eNOS by phosphorylation may play a more important role in NO overproduction than eNOS protein expression observed in chronic stages of portal hypertension.

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Gactrln Regulation of H+-K*-ATPase Gene Expression Masashi Matsushima, Andrea Todisco, Najeed Zoubi, Chris J. Dickinson, Univ of Michigan, Ann Arbor, Ul

BACKGROUND: Expression of H+-K+-ATPase in parietal cells plays an important role in the development of the stomach and normal gastric function. Previously, we noted that H +- K +-ATPase gene expression is regulated by gastrin in isolated canine parietal cells. However, the mechanisms that mediate this event remain unknown. Thus, we undertook studies in a human gastric cell line (AGS) stably transfected with the gastrin/CCKB receptor (CCKB-R). METHODS: AGS/CCKB-R cells were transiently transfected with the canine H +-K +-ATPase ~t--subunit promotor of various lengths with a luciferase reporter. Cells were then serum starved overnight, stimulated with various concentrations of amidated gastrin-17, and lucifer- ase activity assayed after an additional 24h. Luciferase activity was corrected by beta-galactosi- dase activity and/or protein concentration. RESULTS: Gastrin induced a dose-dependent (max. 648__.22% at lOSM) increase in luciferase activity using a -619 to +35 promoter-reporter construct (numbered from its known transcription initiation site). This induction was inhibited completely by the CCKB-R antagonists L365260 (lOSM) and D2 (10TM) but not by the CCKA- R antagonist L364718 (IO~M). The MEK inhibitor PD98059 (5O/~M) and the protein kinase C inhibitor GFlO9203X (1/A~1) partially inhibited luciferase activity (36-+7 % and 56-+13 % of maximal, respectively). In contrast, the PI3 kinase inhibitors (LY294002, 5O~M and wortmannin, lOOnM) as well as the P38 kinase inhibitor (S6203580, lOp, M) had no effect on luciferase activity. Deletion reporter constructs indicated that the region from -58 to -6 contained elements necessary for basal as well as gastrin-stimulated expression. This region also contained a putative SP1 binding site. To explore the role of this SP1 site we utilized a thymidine kinase (TK) promotedluciferase construct. The basal TK promoter had a viral SP1 site that induced basel and gestrin-stimulated luciferase activity. However, mutant TK con- structs with SP1 site deletions fully abrogated basal and gastrin-stimulated luciferase activity. Moreover, this SPl-deleted TK promoter had full reconstitution of basal and gastrin-induced luciferase activity with addition of the SP1 binding region of the H +-K+ -ATPase promoter. CONCLUSIONS: Gastrin regulates H +-K + -ATPase ot--subunit gene expression via PKC and MEK pathways that are independent of PI3 and P38 kinases. Both basal and gastrin-stimulated regulation of H +-K+-ATPase expression is dependent on a functioning SP1 binding site.

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The Intestinal Differentiation-Associated PRL-1 Tyrosine Phosphatase Physically Interacts with a Novel bZIP Transcription Factor Robert H. Diamond, Shuixing Li, Weihong Kong, Charles S. Peters, Univ of Pennsylvania Sch of Medicine, PhiLadelphia, PA

Our laboratory has previously identified the PRL-1 protein tyrosine phosphatase (PTPase) as a 20 kd protein that contains the "signature" amino acid sequence for the active site of PTPases, but otherwise does not contain regions of homology to any previously described protein. PRL-1 is a predominantly nuclear, famesylated PTPase that has been linked to the control of cellular growth and differentiation. PRL-1 is significantly expressed in intestinal epithelia where its expression is associated with cellular differentiation. Specifically, PRL-1 is expressed in villus but not crypt enterocytes, and in differentiated, but not proliferating, Caco-2 cells. Recently, PRL-1 protein was also found to be expressed during development in a number of differentiating epithelial tissues, and it was found to be specifically expressed in the zymogen cells of the adult stomach. Significant insight into PRL-I's specific cellular functions may be derived from identification of PRL-I's cellular partners. To this end, we performed a yeast two-hybrid screen using PRL-1 as bait. We have identified a novel basic leucine zipper (bZIP) protein, designated ATF-7, that physically interacts with PRL-1. ATF-7 is most closely related to members of the ATF/CREB family of bZIP proteins, with highest homology to ATF-4. ATF-7 is expressed in a number of different tissues, and is associated with differentiation in the Caco-2 cell model of intestinal differentiation. We have confirmed the PRL-1/ATF-7 interaction and mapped the regions of ATF-7 and PRL-1 important for interaction to ATF-7's bZIP region and PRL-I's phosphatase domain. We have determined that ATF-7 homodimers bind to but do not transactivata from CRE sites. Finally, we have also shown that PRL-1 is capable of dephosphorylating ATF-7 in vitro. Further insight into ATF- 7's precise cellular roles, transcriptional function and downstream targets are likely be of importance in understanding the mechanisms underlying the complex processes of mainte- nance, differentiation and turnover of digestive epithelia.

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Kinase Suppressor of Ras Is Required for TNF-stimulnted NFKB Activation in Intestinal Epithelial Cells Fang Yan, D. Brent Polk, Yanderbilt Univ, Nashville, TN

BACKGROUND: Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflamma- tory bowel disease by activation of transcription factors, such as nuclear factor (NF) KB. Inhibitor of KB (IKB) kinase (IKK) regulates phosphorylation and subsequent degradation of IKB leading to activation of NFKB. Raf-f regulates NFKB in a number of cell fines by a

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mechanism that is not fully understood. We have recently shown that kinase suppressor of Ras (KSR) directly phosphorylates Rat-1 in response to TNF, enhancing its kinase activity towards MEK1. The present study focused on determining the role of KSR in TNF-induced NFKB activation. METHODS: We studied young adult mouse colon (YAMC) cells stably expressing dominant negative, kinase inactive (ki) KSR, or wild-type (wt) KSR, or transiently expressing two different dominant negative Rat-1 constructs. Ceils were treated with TNF (100 ng/ml) or cell-permeable Cs-ceramide (100 riM) and lysates prepared for Western blot analysis, to determine protein expression and phosphorylation state, or immunoprecipitates prepared for in vitro kinase assays. Immunostaining with anti-p65/RelA antibody was performed to detect NFKB nuclear translocation. RESULTS: TNF stimulates NFKB nuclear translocation and activa- tion in cells expressing endogenous KSR, wtKSR, or vector. Expression of the dominant- negative kiKSR blocks both NFKB nuclear translosation and activation. Likewise, TNF or Ca- ceramide-stimulated IKB phosphorylation and degradation are inhibited by kiKSR expression. TNF stimulates IKKa serine phosphorylation, and IKKa in vitro kinase activity towards IKB~, which are both inhibited only in the cells expressing kiKSR. Furthermore, TNF-stimolated IKB~ phosphorylation and degradation are both inhibited by transient expression of dominant negative Raf-1. However, Raf-1 activated by KSR phosphorylation in vitro is unable to directly serine phosphorylate IKK~, or to enhance its in vitro kiuase activity towards IKB. CONCLU- SIONS: KSR kinase activity is required for TNF-stimulated NFKB activation in intestinal epithelial cells upstream of IKK~ serine phosphorylation. Rat-l, a KSR kinase substrate, is also necessary in this pathway, but not by direct phosphorylation of IKK~. These novel findings indicate KSR regulates TNF-induced NFKB activation in intestinal epithelial cells through Raf-1.

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Increased Posttranscription Of The JunD Geoe And Growth InhibHion Following Polyamine Depletion Li Li, Rao N. Jaladanki, Xin GoD, Lan Liu, Univ of Maryland and VA Medical Ctr, Baltimore, MD; Eric D. Strauch, Univ ot Maryland, Baltimore, MD; Barbara L. Bass, Jian-Ying Wang, Univ of Maryland and VA Medical Ctr, Baltimore, MD

The maintenance of intestinal integrity requires cellular polyamines that regulate expression of the genes involved in proliferation, growth arrest, and apoptosis. JunD is one of three Jun proteins that contribute to the AP-1 transcription factor complex and the activation of the JunD gene slows cell growth in some cell types. We recently demonstrate that polyamine depletion activates JunD/AP-1 activity in normal intestinal epithelial cells, which is associated with the growth arrest. This study was to investigate the mechanisms of regulation of JunD gene expression by potyamines and to further determine the role of JunD/AP-1 activity in the process of growth inhibition following polyamine depletion. Methods:. Studies were conducted in IEC-6 cells, derived from rat small intestinal crypt cells. Cellular polyamines were depleted by treatment with DFMO, a specific inhibitor for polyamine synthesis; JunD gene transcription and the mRNA stability were measured at 4 and 6 days after plating. Results.. Depletion of cellular potyamines by DFMO induced JunD mRNA and protein levels, increased sequence- specific JunD/AP-1 binding activity, and inhibited cell growth. PolyamJne depletion did not induce the rate of Juno gene transcription as measured by nuclear run-on assays. In contrast, the stability of mRNA for JunD increased dramatically following polyamine depletion. The half-life of JunD mRNA in normal cells was -45 min and increased to >8 h in polyamine- deficient cells. Exogenous polyamine spermidine, when given together with DFMO, prevented the increased half-life of Juno mRNA in IEC-6 cells. To determine the function of JunO/AP- 1 activity, antisense JunD was added into the cultures containing DFMO. Inhibition of the JunD expression by the antisense significantly promoted cell growth in polyamine-deticlent cells. Conclusion~ 1) polyamine depletion induces the activation of the JunD geue through the posttranscriptional regulation and 2) increased JunD/AP-1 activity plays a critical role in the process of growth inhibition following polyamine depletion.

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EP2 Receptor Null Mice Have an Impaired Intestinal Response to Radiation Injury Courtney W. Houcben, Washington Univ Sch of Medicine, St Louis, MO; Brian K. Dieckgraefe, Mark A. Sturmoski, William F. Stenson, Washington Univ, St Louis, MO

Prostaglandins (PGs), such as PGE2 are critical mediators of epithelial integrity in the gastroin- testinal tract and play a major role in the regulation of apoptosis, cell differentiation and oncogenesis in many tissues. We have shown that PGE2 synthesized by Cox-1 are important regulators of intestinal epithelial stem cell survival and apoptosis in response to radiation injury. The biological activities of PGE 2 are mediated through plasma membrane G-protein coupled receptors termed EP receptors (EP1-4). We have shown that EP2 receptor mRNA and protein are upregulated after radiation injury in adult mice and in the human epithelial cell line 1407. We have also shown that epithelial cells express increased levels of the EP2 receptor in the context of human inflammatory bowel disease(IBD) induced mucosa. To determine whether PGE2 signal transduction through the EP2 receptor plays a role in the regulation of epithelial injury, we evaluated epithelial stem cell survival and crypt epithelial apoptosis in response to radiation injury in EP2 null mice and their wild type littermates. Methods: Adult EP2 - / - mice and their wild type littermates received 13 Gy "y- irradiation and intestinal stem cell survival was determined using the microcolony assay. In separate experiments adult mice received 12 Gy ~t- irradiation and crypt epithelial apoptosis was scored 6hr after irradiation using the TUNEL assay, and confirmed by morphologic criteria. Immunohistochemical analysis was used to determine cell specific expression of the EP2 receptor. EP2 mRNA in IBD was determined in human resection specimens by RNase protection analysis. Results : Crypt survival was diminished in EP2- / - mice (5.1 surviving crypts / cross section) compared with wild-type littermates (8.8 surviving crypts/cross section). This 41% reduction in crypt survival is similar to the 50 % reduction previously observed in Cox- 1 - / - mice. Apoptosis induced by radiation injury was significantly increased in EP2 - / - mice compared with wild type littermates. Spontaneous apoptosis occurred infrequently (0.38 apoptotic cells/crypt) in wild type mice. Radiation injury induced a large increase in apoptosis in both wild type and EP2 - / - mice. The number of apoptotic cells per crypt was 1.6 told higher in EP2 - / - mice (5.9 apoptotic cells/crypt) than in wild type mice (3.5 apoptotic cells/crypt). Conclusion: Taken together these data suggest that the effects of PGE2

on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP2 receptor.

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Overaxpression of COX-2 in Intestinal Cells Inhibits Heat Shock Factor-1 Binding by Decreasing Phosphatase Activity Richard T. Ethridge, Mark B. Hellmich, B Mark Evers, University of Texas Medical Branch, Galveston, TX

6ACKGROUND. Cyclooxygenase (COX) catalyzes the rate-limiting step in the conversion of arachidonic acid to prostaglandins; the inducible isoform, COX-2, plays an important rule in the inflammatory response of various tissues. Heat shock proteins (HSPs) are stress-respon- sive genes important for cell survival; regulation of HSP expression is dependent upon activation of the heat shock transcription factors (HSFs). We have shown that the overexpres- sion of COX-2 in a rat intestinal epithelial (RIE) cell line results in the inhibition of HSP70 protein and gene induction and HSE-1 binding activity. The purpose of this study was to delineate the mechanisms responsible for decreased HSF-1 binding activity associated with COX-2 overexpression. METHODS. RIE cells stably transfected with the empty vector (RIE- P) or the COX-2 gene in either the sense (RIE-S) or antisense (RIE-AS) direction were used in these studies. Cells were heated to 43°C and harvested for protein over a time course. Western immunoblots were performed to assess HSP70 and HSF-1 protein expression. Electrophoretic mobility shift assays (EMSAs) were performed to determine HSF-1 binding activity. Endogenous phosphatase activity was assessed by a commercially available kit. RESULTS. Increased HSP70 expression and HSE-1 binding activity was noted in the RIE-P and RE-AS cells after heating to 43°C; in contrast, RIE-S cells demonstrated no induction of either HSP70 expression or HSF-1 binding. Phosphatase activity was increased in the RIE- P and RE-AS cells following heating, but no significant induction of phosphatase activity was noted in RIE-S cells. Moreover, basal levels of phosphatase activity in RIE-S cells were significantly lower than RIE-P and RIE-AS cells. To further confirm the role of phosphatase in HSP70 expression, RIE-P cells were treated with the phosphatase inhibitor, catyculin A (20 nM), which resulted in decreased HSF-1 binding activity and suppression of HSP70 expression. CONCLUSIONS. Our findings demonstrate a functional link between the COX/prostaglandin pathway and the highly conserved heat shock pathway in intestinal cells. We show that the inhibition of HSF-1 binding and subsequent suppression ot HSP70 gene and protein expression is the direct result of decreased phosphatase activity in intestinal cells that overexpress COX- 2. Inhibition of HSP expression by COX-2 may play an important role in the overall inflammatory response in cells.

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C y s t o o x y ~ 2 Promoter Can MHigata the Lethal Toxicity in HSV-TK Based Adenoviral Suicide Gene Therapy of Gastrointestinal Cancers Masato Yomamoto, Ramon Alemany, Yasuo Adachi, Willium E Grizzle, David T. Curiel, Univ of Alabama at Birmingham, 6irmingharn, AL

Background: Adenoviral vectors have been widely employed for suicide gene tharapy, Le. herpes simplex virus thymidine kinase (TK) in combination with gancyclovir (GCV). However, the application of this method to the disseminated or intrahepatic lesion of gastrointestinal (GI) cancers has not currently been feasible due to the severe hepatotoxicity caused by transgene expression in the liver after systemic administration of adenoviral vectors. Cyclooxy- genase-2 (cox-2) promoter incorporated into adenoviral vector is "ON" in the various GI cancer cells, in vitro and in vivo, and "OFF" in the liver. We apply this promoter to TK-GCV gene therapy to mitigate the lethal liver toxicity on suicide gene therapy of GI cancers. Methods: Cox-2 M (-883/+ 59) and cox-2 L (-1432/+ 59) promoters were incorporated into adenovirus vectors to regulate TK expression (Adcox-2M TK and Adcox-2L TK, respectively). In vitro, cell lines were infected with the TK expression vectors and the viability of the cells was analyzed alter 5 days of cultivation with GCV. To analyze anti-tumor effect, 10 g pfu of the vectors were injected into the tumor xenograft on nude mice and GCV was administrated intraperitoneally. To investigate in vivo toxicity, 109 pfu of the vectors were injected via tail vein and GCV administered intraperitoneally. Results: 6oth of Adcox-2M TK and Adcox-2L TK showed specific cytocidal effect on cox-2 positive cells, in contrast to the non-specific effect with the CMV driven vector. Intratumorai administration of Adcox-2L TK in combination with GCV showed anti-tumor effect on MKN45 (cox-2 positive gastric cancer cell) without any animal death, in contrast, 4/5 of the mice received CMV driven vector died after 14 days of GCV administration. In the analysis of toxicity after systemic administration, the CMV promoter driven TK expression vector caused lethal toxicity in 2/5 of the mice on the 5th days of GCV administration, and surviving mice showed severe hepatotoxicity with microvesi- cular fatty change and elevation of the total bilirubin level. In the group with Adcox-2L TK and GCV, this lethal hepatotoxicity was not observed. Conclusion: Cox-2 promoter successfully mitigated the lethal hepatotoxicity of TK suicide gene therapy by means of selective expression of TK in the tumor but not in the liver. This may allow suicide gene therapy on variety of cox-2 positive tumors by systemic administration.

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Eradication of Experimental Colon Cancer Liver Metastasis and Survival Prolongation Using IL-12 and Trimerized 4-1BB Ligand Gene Therapy Bernhard V. Sauter, Olivier Martinet, Mei Gan, Yun-Juan Zang, Savio Lc Woo, Shu-Hsia Chen, Institute for Gene Therapy, Mount Sinai Sch of Medicine, New York, NY

Background: Previously, we showed that in vivo adenovirus-mediated IL-12 gene transfer to established hepatic metastases of murine colon cancer cells induced tumor shrinkage and prolonged survival. The long-term survivors (20-30%) exhibited a powerful natural killer cell response (innate immunity). To enhance this anti-tumor effect, we studied 4-1BB ligand (4- 1BBL), a co-stimulatory molecule ot the TNF~uperfamily that can induce a sustained cytotoxic T-cell response (adaptive immunity). Aim: To determine whether monomeric or trimeric 4- 1BBL when used in combination with IL-12 would produce a better anti-tumor effect than

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