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Liver expression of pro-apoptotic and anti-apoptotic genes following hepatectomy in cirrhotic versus normal rats
102
Poster Sessions
HCC. A point to be clarified is wether ischemia during RF has to be transient (by baloon catheter), or long-term (by embolization), and wether the administration of chemotherapic has therapeutic adjunctive value.
I
1044
APOPTIN-INDUCED APOPTOSIS IN HEPATOMA CELLS: OPPORTUNITIES FOR IN VIVO GENE THERAPY OF HCC IN A SYNGENIC RAT MODEL
I.H. Stmatsburg, A.G. van Ginkel, S.A. Rutjes, M. Cooreman, A.M. Pietersen, M.N. Notebom, T.M. van Gulik. Surgical Laboratory, Academic Medical Centel; Amsterdam, The Netherlands Background: Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor. Apoptin, a small protein derived from Chicken Anemia virus, induces apoptosis in transformed cells only, which makes it a promising tool for cancer gene therapy. In nude mice, the size of a subcutaneous tumor of human hepatoma cells (HepG2) was reduced after intratumoral injection of adenoviral Apoptin. To study gene therapy for HCC in the liver of an immunocompetent animal, the syngenic model of rat hepatoma cells (H4IIE) in AC1 rats is used. To study the sensitivity of rat hepatoma cells for Apoptin, H4IIE cells were transfected with Apoptin. Transfection efficiency was compared with transfection in two human hepatoma cell lines (HuH7 and HepG2). Methods: Hepatoma cells were incubated 4-6 hrs with a mixture of liposomes and either Apoptin-, LacZ- or Desmin-DNA. 2-5 days after transfection cells were fixed for immunocytochemistry using monoclonal antibodies against Apoptin, beta galactosidase or desmin and FITC-labeled goat-anti-mouse antibody. DNA was visualized using 2,4-diamino-2-fenylindole. Results: Apoptin was localized in the nucleus and to a lesser degree in the cytoplasm of transfected cells. Nuclear localization is supposedly essential for induction of apoptosis. All cell lines were equally sensitive for Apoptin, reaching 75% apoptosis after 5 days. Transfection with LacZ or desmin DNA resulted in 15-20% apoptosis. Background apoptosis without DNA never exceeded 5%. Conclusion: Our in vitro results show high sensitivity of the rat hepatoma cell line H4IIE for Apoptin. Further studies should be directed towards efficient transduction of Apoptin using retargeted viral vectors for in vivo gene therapy of rat HCC in a syngenic model.
I
1123
LIVER EXPRESSION OF PRO-APOPTOTIC AND ANTI-APOPTOTIC GENES FOLLOWING HEPATECTOMY IN CIRRHOTIC VERSUS NORMAL RATS
M. Scotte, S. Masson, M. Hiron, P. Teniere, J.P. Salier, M. Daveau. General and Digestive Surgery and INSERM U.519, CHU Rouen, France Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven to be activators or inhibitors of hepatocyte proliferation. Following hepatectomy in cirrhotic liver, liver regeneration has been proven to be decreased and delayed as compared to what happens following hepatectomy in normal liver. Apoptosis is defined as a complex mechanism leading to cellular death and seems to play an important role in cellular proliferation and in liver regeneration. Therefore, the aim of this study was to analyse the expression of genes involved in apoptosis following 213 hepatectomy in cirrhotic versus normal rats. Cirrhosis was induced by a weekly intragastric administration of CC14 for 16 weeks. An histological examination of the resected liver was performed and rats were sacrificed 6 h, 12 h, 24 h, or 72 h after liver resection. Using quantitative RT-PCR, we have analysed the expression of proapoptotic (bad, bak, bax) and antiapoptotic (bcl-2, b&XL) genes. In normal rats, we have observed an early increase of antiapoptotic mRNA levels and a delayed increase of proapoptotic mRNA levels following hepatectomy. In cirrhotic liver,
before resection, proapoptotic mRNA levels were significantly higher than those observed in normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis is induced during CC14 induced cirrhosis. As apoptosis seems to play an important role in cellular proliferation, the dysregulation of this mechanism could be involved in the impaired regenerative response observed in cirrhotic liver.
0 1139
CYCLINS (A, B, Dl, E) AND CYCLIN DEPENDENT KINASE INHlBlTORS (P16, PPl) EXPRESSION IN THE LIVER OF THIOACETAMIDE INTOXICATED RATS
SE. Theocharis, H. Kanelli, A. Margeli, A. Koutselinis. Forensic Medicine and Toxicology, University of Athens, Medical School, Greece Objective-Purpose: Loss of hepatic mass due to toxin-induced injury, induces quiesent hepatocytes to enter the cell cycle. Cell cycle progression requires activation of cyclin dependent kinases (Cdks) regulated by cyclins and Cdk inhibitors. In the present study we investigated the expression of the cyclins A, B, Dl, E and Cdk inhibitors p16 and p21 in a rat model of injury and regeneration induced by thioacetamide (TAA) administration. Material and Methods: Adult male Wtstar rats were administered intraperitoneally with 300 mg TAA/Kg b.w. Toxicological end points and markers of hepatocellular regeneration were assessed at various time points (0, 12,24, 36,48, 60,72 h) after toxin injection. Serum biochemical markers and liver histology were used to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocytes’ mitotic index were used as indices of regeneration. The expression of cyclins A, B, Dl and E and Cdk inhibitors p16 and p21 was examined immunohistochemical-ly in paraffin embedded liver tissue sections, using appropriate antibodies. Results and Conclusion: TAA administration caused severe hepatic injury, followed by hepatocellular proliferation. Marked alterations of cyclins Dl, E and p16 expression were observed in hepatoceytes throughout the experimental timein relation to liver proliferating capacity, while cyclins A, B and p21 expression was found in isolated hepatocytes. Our data for first time describe the immunohistochemical distribution of cyclins and Cdk inhibitors in rat liver post TAA-induced injury, suggesting reliable markers of cell cycle progression during the repair mechanism process.
I 1146
DETERMINATION OF OPTIMAL RADIATION DOSE IN EXTERNAL RADIOTHERAPY FOR HEPATOCELLULAR CARCINOMA
J. Seong, H.C. Park, K.H. Han, C.Y. Chon, Y.M. Moon, C.O. Suh. Radiation Oncology, Yonsei University Medical College, South Korea Recent reports have shown that external radiotherapy could achieve a substantial tumor response in hepatocellular carcinoma (HCC). In this study, we investigated optimal radiotherapy dose with regard to both tumor control and normal tissue toxicity. From January 1992 to March 2000, 158 patients, treated with external radiotherapy for unresectable HCC, were included. UICC stages were III in 67 and IVa in 91. Mean tumor size was 8.95 f 3.4 cm. Radiotherapy was given by 10 MV X-ray with a mean tumor dose of 48.2 f 7.9 Gy in daily 11.8 Gy fractions. As a result, an objective response was seen in lO6/158 (67.1%). Radiation dose was the most significant factor for tumor response rate with 29.2% in less than 40 Gy, 68.6% in 4O-50 Gy, and 77.1% in more than 50 Gy (p < 0.05). However, increase of radiation dose resulted in increased toxicity; gastroduodenal toxicity occurred 4.2% in less than 40 Gy, 9.9% in 4O-50 Gy, and 13.2% in more than 50 Gy (p < 0.05). This complication was shown preferentially in case of tumors in or proximity to the left lobe. Radiation-induced liver disease was 4.2% in less than
Poster Sessions
HCC. A point to be clarified is wether ischemia during RF has to be transient (by baloon catheter), or long-term (by embolization), and wether the administration of chemotherapic has therapeutic adjunctive value.
I
1044
APOPTIN-INDUCED APOPTOSIS IN HEPATOMA CELLS: OPPORTUNITIES FOR IN VIVO GENE THERAPY OF HCC IN A SYNGENIC RAT MODEL
I.H. Stmatsburg, A.G. van Ginkel, S.A. Rutjes, M. Cooreman, A.M. Pietersen, M.N. Notebom, T.M. van Gulik. Surgical Laboratory, Academic Medical Centel; Amsterdam, The Netherlands Background: Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor. Apoptin, a small protein derived from Chicken Anemia virus, induces apoptosis in transformed cells only, which makes it a promising tool for cancer gene therapy. In nude mice, the size of a subcutaneous tumor of human hepatoma cells (HepG2) was reduced after intratumoral injection of adenoviral Apoptin. To study gene therapy for HCC in the liver of an immunocompetent animal, the syngenic model of rat hepatoma cells (H4IIE) in AC1 rats is used. To study the sensitivity of rat hepatoma cells for Apoptin, H4IIE cells were transfected with Apoptin. Transfection efficiency was compared with transfection in two human hepatoma cell lines (HuH7 and HepG2). Methods: Hepatoma cells were incubated 4-6 hrs with a mixture of liposomes and either Apoptin-, LacZ- or Desmin-DNA. 2-5 days after transfection cells were fixed for immunocytochemistry using monoclonal antibodies against Apoptin, beta galactosidase or desmin and FITC-labeled goat-anti-mouse antibody. DNA was visualized using 2,4-diamino-2-fenylindole. Results: Apoptin was localized in the nucleus and to a lesser degree in the cytoplasm of transfected cells. Nuclear localization is supposedly essential for induction of apoptosis. All cell lines were equally sensitive for Apoptin, reaching 75% apoptosis after 5 days. Transfection with LacZ or desmin DNA resulted in 15-20% apoptosis. Background apoptosis without DNA never exceeded 5%. Conclusion: Our in vitro results show high sensitivity of the rat hepatoma cell line H4IIE for Apoptin. Further studies should be directed towards efficient transduction of Apoptin using retargeted viral vectors for in vivo gene therapy of rat HCC in a syngenic model.
I
1123
LIVER EXPRESSION OF PRO-APOPTOTIC AND ANTI-APOPTOTIC GENES FOLLOWING HEPATECTOMY IN CIRRHOTIC VERSUS NORMAL RATS
M. Scotte, S. Masson, M. Hiron, P. Teniere, J.P. Salier, M. Daveau. General and Digestive Surgery and INSERM U.519, CHU Rouen, France Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven to be activators or inhibitors of hepatocyte proliferation. Following hepatectomy in cirrhotic liver, liver regeneration has been proven to be decreased and delayed as compared to what happens following hepatectomy in normal liver. Apoptosis is defined as a complex mechanism leading to cellular death and seems to play an important role in cellular proliferation and in liver regeneration. Therefore, the aim of this study was to analyse the expression of genes involved in apoptosis following 213 hepatectomy in cirrhotic versus normal rats. Cirrhosis was induced by a weekly intragastric administration of CC14 for 16 weeks. An histological examination of the resected liver was performed and rats were sacrificed 6 h, 12 h, 24 h, or 72 h after liver resection. Using quantitative RT-PCR, we have analysed the expression of proapoptotic (bad, bak, bax) and antiapoptotic (bcl-2, b&XL) genes. In normal rats, we have observed an early increase of antiapoptotic mRNA levels and a delayed increase of proapoptotic mRNA levels following hepatectomy. In cirrhotic liver,
before resection, proapoptotic mRNA levels were significantly higher than those observed in normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis is induced during CC14 induced cirrhosis. As apoptosis seems to play an important role in cellular proliferation, the dysregulation of this mechanism could be involved in the impaired regenerative response observed in cirrhotic liver.
0 1139
CYCLINS (A, B, Dl, E) AND CYCLIN DEPENDENT KINASE INHlBlTORS (P16, PPl) EXPRESSION IN THE LIVER OF THIOACETAMIDE INTOXICATED RATS
SE. Theocharis, H. Kanelli, A. Margeli, A. Koutselinis. Forensic Medicine and Toxicology, University of Athens, Medical School, Greece Objective-Purpose: Loss of hepatic mass due to toxin-induced injury, induces quiesent hepatocytes to enter the cell cycle. Cell cycle progression requires activation of cyclin dependent kinases (Cdks) regulated by cyclins and Cdk inhibitors. In the present study we investigated the expression of the cyclins A, B, Dl, E and Cdk inhibitors p16 and p21 in a rat model of injury and regeneration induced by thioacetamide (TAA) administration. Material and Methods: Adult male Wtstar rats were administered intraperitoneally with 300 mg TAA/Kg b.w. Toxicological end points and markers of hepatocellular regeneration were assessed at various time points (0, 12,24, 36,48, 60,72 h) after toxin injection. Serum biochemical markers and liver histology were used to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocytes’ mitotic index were used as indices of regeneration. The expression of cyclins A, B, Dl and E and Cdk inhibitors p16 and p21 was examined immunohistochemical-ly in paraffin embedded liver tissue sections, using appropriate antibodies. Results and Conclusion: TAA administration caused severe hepatic injury, followed by hepatocellular proliferation. Marked alterations of cyclins Dl, E and p16 expression were observed in hepatoceytes throughout the experimental timein relation to liver proliferating capacity, while cyclins A, B and p21 expression was found in isolated hepatocytes. Our data for first time describe the immunohistochemical distribution of cyclins and Cdk inhibitors in rat liver post TAA-induced injury, suggesting reliable markers of cell cycle progression during the repair mechanism process.
I 1146
DETERMINATION OF OPTIMAL RADIATION DOSE IN EXTERNAL RADIOTHERAPY FOR HEPATOCELLULAR CARCINOMA
J. Seong, H.C. Park, K.H. Han, C.Y. Chon, Y.M. Moon, C.O. Suh. Radiation Oncology, Yonsei University Medical College, South Korea Recent reports have shown that external radiotherapy could achieve a substantial tumor response in hepatocellular carcinoma (HCC). In this study, we investigated optimal radiotherapy dose with regard to both tumor control and normal tissue toxicity. From January 1992 to March 2000, 158 patients, treated with external radiotherapy for unresectable HCC, were included. UICC stages were III in 67 and IVa in 91. Mean tumor size was 8.95 f 3.4 cm. Radiotherapy was given by 10 MV X-ray with a mean tumor dose of 48.2 f 7.9 Gy in daily 11.8 Gy fractions. As a result, an objective response was seen in lO6/158 (67.1%). Radiation dose was the most significant factor for tumor response rate with 29.2% in less than 40 Gy, 68.6% in 4O-50 Gy, and 77.1% in more than 50 Gy (p < 0.05). However, increase of radiation dose resulted in increased toxicity; gastroduodenal toxicity occurred 4.2% in less than 40 Gy, 9.9% in 4O-50 Gy, and 13.2% in more than 50 Gy (p < 0.05). This complication was shown preferentially in case of tumors in or proximity to the left lobe. Radiation-induced liver disease was 4.2% in less than