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Serum follistatin level as an early detection marker of post-hepatectomy liver failure: Using our novel rat 90% hepatectomy model
the cellular effects resulting from hypothermic exposureappearto overridethose of ischemiainduced, plKB directed NF-KBactivation.Thus, the mechanismof activation of NF-KBappears to be important in cell responseto CH stress.
1788 Human Ductal Pancreatic Adenocarcinoma Cells Can Be Targeted And Destroyed By A Rat Insulin Promoter Thymidine Kinase Construct, In Viva Thomas A. Tirone, Xaioping Wang, Nancy S. Templetone, F Charles Brunicardi, Baylor Coil of Medicine, Houston,IX
1786 Hypoosmotic Stress Stimulates Growth in HepG2 Cells Via PKB-Dependent Activation of AP-1 Robin D. Kim, Timothy P. Ruth, Chad E. Darling, Roceo Ricciardi, Bradley K. Schaffer, Ravi S. Chad, Univ of MA Medical Sch, Worcester, MA Swelling by hypoosmotic stress activates PI-3-K, but its impact on the downstream signal PKB or cell growth is unknown. Although AP-1 activation is in part PI-3-K-dependent, its responseto hypoosmoti¢ stress is also undefined.We hypothesizedthat cell swelling modulates proliferation in HepG2 cells via PKB-dependentactivation of AP-1. Methods: HepG2 incubated for 1 hr with/out 50/~M LY294002 (LY), a PI-3-K inhibitor, were exposed for up to 30 min to hypoosmotic medium (200mOsm/L) to induce swelling. TNF-~ (1.4 nM) or normoosmoticmedium served as positive and negative controls, respectively.Western blots measured cytoplasmic phospho- and total PKB. EMSA measured nuclear AP-1. Methylene blue assays measuredcell proliferation at 24, 48 and 72 hrs after pulse exposureto hypoosmoticstress. T test and ANOVAverified statistical significance.All experimentswere performed in triplicate. Results: [1] Hypoosmotic stress phosphorylated PK6 (P-PKB) by 10 min, and this effect was inhibited by LY. [2] Hypooosmotic stress translocated AP-1 by 30 min, and this effect was inhibited by LY (Fig 1). [3] Hypoosmotic stress potentiatedgrowth by 72 hrs as comparedto both control and LY-inhibitedcells (n = 4 per group, p = 0.009# and p = 0.004*, respectively; p
Fig ] 200 nOsr~. Y
z,p-1
+
+ ÷
Purpose: Preliminary data previously presented suggests that PDA cells can be selectively targeted and killed using the rat insulin promoter (RIP) driving the suicide gene thymidine kinase (tk) in vitro, and that the presenceof the transcription factor PDX-1 in these PDA ceils is vital for insulin promoter activation. The purposeof this study was to investigate if human PDA cells could be targeted and killed in a mouse PDA tumor model and to determine if PDX-1 is present in PDA cells freshly resectedfrom humans. Methods: 5 x 10s PANC-1 cells were injected intraperitoneally (IP) into ICR/scid mice. RIPtk and the reporter gene RIPlacZ were generated. For in vivo gene delivery the DNA was complexed with 20mM extruded DOTAP:cholesterol and delivered IP. Twenty-one days post tumor injection the mice were randomizedto receiveeither RIPtk, RIPlacZ,or nothing. All mice received7 days of ganciclovir (GCV) 40mg/kg IP BID. A subset of mice with tumors that received RIPtacZwere sacrificed at 72 hours and their tissues were fixed and stained with X-gal. Three human PDA tumors were obtained;two hepatic metastasesand one peripancreaticnodule, and western blots were performed with an antibody to PDX-1. Results: PDA cells were targeted in vivo with RIPlacZ; only the tumor cells stained blue. Mice that received RIPtk had a significant reduction in tumor burden; the combination eliminated all microscopic disease in 8/9 mice (See table). All three fresh human PDA specimens contained the transcription factor PDX-1. Conclusion: The data suggest that RIP can be used to target and kill PDA cells in vivo. The presence of PDX-1 in freshly resectedhuman PDA tumors further suggeststhat this therapy may possibly be effective in the treatment of human pancreatic ductal adenosarcinomain the future. TumorSize Vector
Tumor(Gross)
Tumor(Micro)
Size*
RIPtk RIPlacZ None
0/91" 9/9 9/9
1/91` 9/9 9/9
1`p
'°t
1789 Protective Effect of a Selective Endothelin A Receptor Antagonist (BSF 268075) on Graft Pancroatitis in a Pig Transplant Model Helmut Witziomann, Stefan Ludwig, Evelin Escher, Gabor Gaebel, BaateArmaun, D Teupser, Andrea Tannapfel, Dirk Uhlmann, Johann Hauss, Univ of Leipzig, Leipzig Germany
1787
Objective: To evaluate the effect of a new Endothelin A receptor antagonist (ET-RA) on pancreatic microcirculation in a pig transplant model. Background: Impaired microcirculation is a major determinant of graft pancreatitis post transplant in humans. In animal models Endothelin 1 has been shown to reduce local blood flow in the pancreas and to aggravate pancreatitis. Methods: Heterotopicpancreastransplantationwas performed in GOttingerminipigs (n = 16 donors, n = 16 recipients,female 20-25 kg). The grsf~ were stored for 6 hours in University of Wisconsin solution at 4°C. CyclosporineA and Prednisolonwere administered for immunosuppression. The animals were randomizedto a control (n = 8) and treatment group (n = 8) that received 10 mg/kg of the selective endothelin A receptor antagonist (BSF 208075) i.v. during reperfusion and after 6 h. Target parameter measured before and after reperfusion included quantification of the morphological damage by a score, tissue oxygenation, laser doppler flowmetry, serum amylase, serum lipase, and trypsinogen-activation peptide (TAP) Results:A rapid onset of morphologicalalterationsimmediatelyafter reperfusion was noted. The tissue damagescore was significant lower in the treatment group (p
Serum Follistatin Level as an Early Detection Marker of Poct-hepatectomyLiver Failure: Using Our Novel Rat 90% Hepatectomy Model Kazuaki Takabe,The Salk Institute, La Julia, CA',Yasumi Shintani, Tokushima Univ, Tokushima Japan; Toru Kubota, Hiroshi Shimada, Yokohama City Univ, YokohamaJapan; Wylie Vale, The Balk Institute, La Julia, CA Background: Extensivehepatectomyis inevitableto somepatients with serious liver diseases in order to obtain cure. However,excessivesurgical removal of liver tissue can lead to posthepatectomyliver failure. Therefore,a marker to predict whether the remaining liver is capable to regenerate,or is prone to develop liver failure is an immediate need.Activins are members of the TGF-fl superfamily, which are an autocrine inhibitory growth factor of hepatocytas.We have shown that follistatin, an activin binding protein, blocks the anti-proliferative effect of endogenous activin, and thereby stimulate hepatocyte growth (Endoorino/ogy 1999; 140: 3125-3132). This was also the case in normal adult rat liver, which was demonstrated to increase in size by adenovirus-mediatedoverexpressionof follistatin (Hepato/ow32: (4), Pt.2 Supp/; 321A). Moreover, serums of patients with liver diseasewere shown to have elevated follistatin levels. Method: Male Wistar rats (180-200g) were used for rat study, and human samples were obtained from patients who admitted YokohamaCity University Hospital with their consent. Serum follistatin was measured by ELISA assay. Results: We have developed a novel technique by which we can perform a 90% hepatectomy(9O%Hx) in rats that results in 100% survival for more than 2 weeks. This model is an excellenttool to study differences betweensurvival case (90%Hx) and mortal liver failure case (95%Hx) after extensivehepatectomy. Total and direct bilirubin levelswere elevatedafter both 90%Hx and 95%Hx. In contrast to the 90%Hx, in which their levels of bilirubin decreaseddramaticalty in 48 hours, the levels remainedsignificantly elevatedin the 95%Hx rats. Therewere no significant difference between 90%Hx and 95%Hx in AST, ALT, Albumin, and ALP levels. However, serum free follistatin levels remained significantly elevated only in the 95%Hx group 24 hours after surgery, in contrast to the 30, 70, and 90%Hx groups. Therefore, serum free follistatin levels represents a novel early detection marker for liver failure. Furthermore, we found that in clinical samples of two patients who suffered severe liver failure following hepatectomies had elevation of their serum follistatin levels, when compared with patients who were dischargedwithout any unusual event after either hepatectomy or extensive gaetrectomy. Conclusion: Our results suggest that serum follistatin level can be used as an early predictor for post-hepatectomy liver failure.
1790 Cytokine Modulation of Pancreatic Cancer Cell Growth, Invasion and Metastasis Richard Essner, Masayuki Kojima, Young Hunyh, Mark C. Kelley, John Wayne Cancer Institute, Santa Monica, CA Background: The natural history of pancreatic carcinoma is cbaractedzedby rapid growth, local invasionand metastases.Theseprocessesare in part modulatedthrough the equalibrium between matrix metalloproteinases (MMP) and their physiologic inhibitor (TIMP1) while another cytokine, hepatocyte growth factor (HGF) has been shown to be important in the generation of hepatic metastases in colorectal carcinoma. We have recently demonstrated the presenceof the HGF receptor (HGF-R or c-met) and the cytokine receptors of interieukin4 (IL-4R) and interleukin-2 receptor .y (IL-2R~/c) on human pancreatic carcinoma cell lines. The hypothesis was that the growth and tissue invasion of these cells may be modulated through common signal pathways induced by the MMP and cytokine receptors IL-4R and IL2R~e. Methods: Eight human pancreatic cell lines (CRL 1420, CRL 1469, CRL 1682, CRL 1687, CRL 2000, CRL 2110, HTB 79, and HTB 134 obtained from American Type Culture
A-347
1788 Human Ductal Pancreatic Adenocarcinoma Cells Can Be Targeted And Destroyed By A Rat Insulin Promoter Thymidine Kinase Construct, In Viva Thomas A. Tirone, Xaioping Wang, Nancy S. Templetone, F Charles Brunicardi, Baylor Coil of Medicine, Houston,IX
1786 Hypoosmotic Stress Stimulates Growth in HepG2 Cells Via PKB-Dependent Activation of AP-1 Robin D. Kim, Timothy P. Ruth, Chad E. Darling, Roceo Ricciardi, Bradley K. Schaffer, Ravi S. Chad, Univ of MA Medical Sch, Worcester, MA Swelling by hypoosmotic stress activates PI-3-K, but its impact on the downstream signal PKB or cell growth is unknown. Although AP-1 activation is in part PI-3-K-dependent, its responseto hypoosmoti¢ stress is also undefined.We hypothesizedthat cell swelling modulates proliferation in HepG2 cells via PKB-dependentactivation of AP-1. Methods: HepG2 incubated for 1 hr with/out 50/~M LY294002 (LY), a PI-3-K inhibitor, were exposed for up to 30 min to hypoosmotic medium (200mOsm/L) to induce swelling. TNF-~ (1.4 nM) or normoosmoticmedium served as positive and negative controls, respectively.Western blots measured cytoplasmic phospho- and total PKB. EMSA measured nuclear AP-1. Methylene blue assays measuredcell proliferation at 24, 48 and 72 hrs after pulse exposureto hypoosmoticstress. T test and ANOVAverified statistical significance.All experimentswere performed in triplicate. Results: [1] Hypoosmotic stress phosphorylated PK6 (P-PKB) by 10 min, and this effect was inhibited by LY. [2] Hypooosmotic stress translocated AP-1 by 30 min, and this effect was inhibited by LY (Fig 1). [3] Hypoosmotic stress potentiatedgrowth by 72 hrs as comparedto both control and LY-inhibitedcells (n = 4 per group, p = 0.009# and p = 0.004*, respectively; p
Fig ] 200 nOsr~. Y
z,p-1
+
+ ÷
Purpose: Preliminary data previously presented suggests that PDA cells can be selectively targeted and killed using the rat insulin promoter (RIP) driving the suicide gene thymidine kinase (tk) in vitro, and that the presenceof the transcription factor PDX-1 in these PDA ceils is vital for insulin promoter activation. The purposeof this study was to investigate if human PDA cells could be targeted and killed in a mouse PDA tumor model and to determine if PDX-1 is present in PDA cells freshly resectedfrom humans. Methods: 5 x 10s PANC-1 cells were injected intraperitoneally (IP) into ICR/scid mice. RIPtk and the reporter gene RIPlacZ were generated. For in vivo gene delivery the DNA was complexed with 20mM extruded DOTAP:cholesterol and delivered IP. Twenty-one days post tumor injection the mice were randomizedto receiveeither RIPtk, RIPlacZ,or nothing. All mice received7 days of ganciclovir (GCV) 40mg/kg IP BID. A subset of mice with tumors that received RIPtacZwere sacrificed at 72 hours and their tissues were fixed and stained with X-gal. Three human PDA tumors were obtained;two hepatic metastasesand one peripancreaticnodule, and western blots were performed with an antibody to PDX-1. Results: PDA cells were targeted in vivo with RIPlacZ; only the tumor cells stained blue. Mice that received RIPtk had a significant reduction in tumor burden; the combination eliminated all microscopic disease in 8/9 mice (See table). All three fresh human PDA specimens contained the transcription factor PDX-1. Conclusion: The data suggest that RIP can be used to target and kill PDA cells in vivo. The presence of PDX-1 in freshly resectedhuman PDA tumors further suggeststhat this therapy may possibly be effective in the treatment of human pancreatic ductal adenosarcinomain the future. TumorSize Vector
Tumor(Gross)
Tumor(Micro)
Size*
RIPtk RIPlacZ None
0/91" 9/9 9/9
1/91` 9/9 9/9
1`p
'°t
1789 Protective Effect of a Selective Endothelin A Receptor Antagonist (BSF 268075) on Graft Pancroatitis in a Pig Transplant Model Helmut Witziomann, Stefan Ludwig, Evelin Escher, Gabor Gaebel, BaateArmaun, D Teupser, Andrea Tannapfel, Dirk Uhlmann, Johann Hauss, Univ of Leipzig, Leipzig Germany
1787
Objective: To evaluate the effect of a new Endothelin A receptor antagonist (ET-RA) on pancreatic microcirculation in a pig transplant model. Background: Impaired microcirculation is a major determinant of graft pancreatitis post transplant in humans. In animal models Endothelin 1 has been shown to reduce local blood flow in the pancreas and to aggravate pancreatitis. Methods: Heterotopicpancreastransplantationwas performed in GOttingerminipigs (n = 16 donors, n = 16 recipients,female 20-25 kg). The grsf~ were stored for 6 hours in University of Wisconsin solution at 4°C. CyclosporineA and Prednisolonwere administered for immunosuppression. The animals were randomizedto a control (n = 8) and treatment group (n = 8) that received 10 mg/kg of the selective endothelin A receptor antagonist (BSF 208075) i.v. during reperfusion and after 6 h. Target parameter measured before and after reperfusion included quantification of the morphological damage by a score, tissue oxygenation, laser doppler flowmetry, serum amylase, serum lipase, and trypsinogen-activation peptide (TAP) Results:A rapid onset of morphologicalalterationsimmediatelyafter reperfusion was noted. The tissue damagescore was significant lower in the treatment group (p
Serum Follistatin Level as an Early Detection Marker of Poct-hepatectomyLiver Failure: Using Our Novel Rat 90% Hepatectomy Model Kazuaki Takabe,The Salk Institute, La Julia, CA',Yasumi Shintani, Tokushima Univ, Tokushima Japan; Toru Kubota, Hiroshi Shimada, Yokohama City Univ, YokohamaJapan; Wylie Vale, The Balk Institute, La Julia, CA Background: Extensivehepatectomyis inevitableto somepatients with serious liver diseases in order to obtain cure. However,excessivesurgical removal of liver tissue can lead to posthepatectomyliver failure. Therefore,a marker to predict whether the remaining liver is capable to regenerate,or is prone to develop liver failure is an immediate need.Activins are members of the TGF-fl superfamily, which are an autocrine inhibitory growth factor of hepatocytas.We have shown that follistatin, an activin binding protein, blocks the anti-proliferative effect of endogenous activin, and thereby stimulate hepatocyte growth (Endoorino/ogy 1999; 140: 3125-3132). This was also the case in normal adult rat liver, which was demonstrated to increase in size by adenovirus-mediatedoverexpressionof follistatin (Hepato/ow32: (4), Pt.2 Supp/; 321A). Moreover, serums of patients with liver diseasewere shown to have elevated follistatin levels. Method: Male Wistar rats (180-200g) were used for rat study, and human samples were obtained from patients who admitted YokohamaCity University Hospital with their consent. Serum follistatin was measured by ELISA assay. Results: We have developed a novel technique by which we can perform a 90% hepatectomy(9O%Hx) in rats that results in 100% survival for more than 2 weeks. This model is an excellenttool to study differences betweensurvival case (90%Hx) and mortal liver failure case (95%Hx) after extensivehepatectomy. Total and direct bilirubin levelswere elevatedafter both 90%Hx and 95%Hx. In contrast to the 90%Hx, in which their levels of bilirubin decreaseddramaticalty in 48 hours, the levels remainedsignificantly elevatedin the 95%Hx rats. Therewere no significant difference between 90%Hx and 95%Hx in AST, ALT, Albumin, and ALP levels. However, serum free follistatin levels remained significantly elevated only in the 95%Hx group 24 hours after surgery, in contrast to the 30, 70, and 90%Hx groups. Therefore, serum free follistatin levels represents a novel early detection marker for liver failure. Furthermore, we found that in clinical samples of two patients who suffered severe liver failure following hepatectomies had elevation of their serum follistatin levels, when compared with patients who were dischargedwithout any unusual event after either hepatectomy or extensive gaetrectomy. Conclusion: Our results suggest that serum follistatin level can be used as an early predictor for post-hepatectomy liver failure.
1790 Cytokine Modulation of Pancreatic Cancer Cell Growth, Invasion and Metastasis Richard Essner, Masayuki Kojima, Young Hunyh, Mark C. Kelley, John Wayne Cancer Institute, Santa Monica, CA Background: The natural history of pancreatic carcinoma is cbaractedzedby rapid growth, local invasionand metastases.Theseprocessesare in part modulatedthrough the equalibrium between matrix metalloproteinases (MMP) and their physiologic inhibitor (TIMP1) while another cytokine, hepatocyte growth factor (HGF) has been shown to be important in the generation of hepatic metastases in colorectal carcinoma. We have recently demonstrated the presenceof the HGF receptor (HGF-R or c-met) and the cytokine receptors of interieukin4 (IL-4R) and interleukin-2 receptor .y (IL-2R~/c) on human pancreatic carcinoma cell lines. The hypothesis was that the growth and tissue invasion of these cells may be modulated through common signal pathways induced by the MMP and cytokine receptors IL-4R and IL2R~e. Methods: Eight human pancreatic cell lines (CRL 1420, CRL 1469, CRL 1682, CRL 1687, CRL 2000, CRL 2110, HTB 79, and HTB 134 obtained from American Type Culture
A-347