- Email: [email protected]
The role of TGF-β signaling pathway in liver regeneration
Poster Sessions
96
immunohistochemistry as well as caspase cleavage after incubation with different TRAIL preparations on freshly isolated primary human hepatocytes of 10 patients. Our data show that some but not all TRAIL preparations independent of their toxicity on tumor cells can induce apoptosis in human liver cells depending on the time point of stimulation. TRAIL resistance correlates with TRAIL-R4 expression. Interestingly at no time chemotherapeutic drugs were not able to sensitise primary human hepatocytes -in contrast to tumor cell lines- for TRAILinduced apoptosis. Our study shows to what extent TRAIL can be used in combination with chemotherapeutics.
Conclusions: Our results indicate that high levels of TGF-b lead to a transient inhibition of hepatocyte proliferation after PH, but not to a significant arrest of the regenerative process. In contrast, the interruption of the TGFb signaling pathway in hepatocytes had no significant effect on the time corn--se of liver regeneration. This implicates that TGF-? is of minor importance in controlling the regenerative process in the liver or that redundant systems, such as the activin system, could compensate for the defective TGF-b pathway.
I I 320
TRAIL-INDUCED
APOPTOSIS
LINES IS REGULATED RECRUITMENT MITOCHONDRIAL
ATTHE
IN HUMAN HEPATOMA
CELL
AND NOT AT THE
LEVEL
TNI-related apoptosis-inducing ligand (TRAIL) exhibits potent anti-tumor activity upon systemic administration, yet non of the deleterious side effects observed with other apoptosis-inducing TNF family members such as TNF and CD95L. About 50% of the investigated tumor cell lines are not sensitive to TRAIL. Thus TRAILbased tumor therapy may be used in the treatment of human cancer in viva. TRAIL induces apoptosis via death domain containing TRAIL-receptor (TRAIL-R) 1 and 2 while TRAIL-R 3 and 4 have been suggested to have an apoptosis-inhibitory function. To evaluate the potential mechanisms of TRAIL resistance we first investigated TRAILreceptor expression on hepatoma cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs such as 5-FU renders the TRAIL resistant hepatoma cell line sensitive to TRAILinduced apoptosis. DISC (Death Inducing Signaling Complex) analysis revealed substantially increased DISC formation after sensitization. The results suggest that TRAIL-mediated apoptosis in this cell lines is controlled through increased caspase 8 recruitment at the DISC level rather than through putative anti-apoptotic receptors or mitochondtial events.
321
THE ROLE OF TGF-j3 SIGNALING
HEPATOCELLULAR
CARCINOMA
TREATED
RADIOFREQUENCY
ABLATION:
EVALUATION
POSITTREATMENT
DISC VIA INCREASED
OF CASPASE-8
T.M. Ganten’, T. Haas2, M. Sprick2, W. Stremmel’, H. Walczak2. ‘Internal Medicine, University Of Heidelberg, Heidelberg, Get-many; 2Apoptosisregulation, DKFZ-Heidelberg, Heidelberg, Get-many
I
322
RESPONSE
CONTRAST-ENHANCED
WITH OF
WITH A NEW
HARMONIC
SONOGRAPHY
F. Giangregorio, M.G. Marinone, M. Di Stasi, F. Fomari. Gastroenterology Department, Piacenza Hospital, Piacenza, Italy A second generation contrast-enhanced ultrasonography (US) was used to evaluate the outcome of radiofrequency thermal ablation (RTA) in a series of hepatocellular carcinoma (HCC), comparing the results with helical CT. 93 cirrhotic patients (54 men, 39 women; mean age 71.69 years; 79 Child A; 14 Child B; 83 HCV positive, 6 HBV positive and 4 post-alcoholic) affected from HCC. Pretreatment US identified 122 lesions with a maximum diameter ranging from 1 to 7 cm (63 nodules: 1-3 cm; 52: 3-5 cm; 7: >5 cm). RTA was performed using an active 15.gauge expandable needle or a 18.gauge internally cooled electrode. The contrast-enhanced harmonic US was performed using a suspension of sulphur hexafluoride in sterile saline (SonoVue, Bracco), as contrast agent, and a machine (Esatune, Esaote) with a new harmonic sonography. In 42 patients (51 lesions) SonoVue-enhanced CnTI was performed before and 24 hours after the treatment: 11 cases (12 nodules) exhibited positive enhancement in the early arterial phase before RTA. The 27 cases with post-treatment partial enhancement at CnTI were: 7 single lesions with diameter >5 cm; 15 with lesions 3-5 cm in diameter and 5 multinodular lesions; Finally SonoVue-enhanced CnTI was used as a guide to target locations for additional RTA SonoVue-enhanced CnTI is an highly accurate technique for assessing the outcome of RTA of HCC nodules, already 24 hours after the treatment; it may be useful for guiding the site of additional treatments; it may allow to reduce the number of helical CT
PATHWAY IN LIVER
REGENERATION
J.F. Garcia-Lazaro, E. Meyer, l? Czochra, S. Lueth, I. Balderas-Renteria, P.R. Galle, A.W. Lohse, S. Kanzler. I. Department Of Medicine, University Of Maim, Maim, Get-many Background: TGF-b is a pleiotropic cytokine with antiproliferative effects on epithelial cells. The aim of this study was to determine the influence of the TGF-b signaling pathway on hepatocyte proliferation and liver regeneration in viva. Methods: Transgenic mice with liver-specific overexpression of TGF-bl (n=80), transgenic mice overexpressing a dominant negative type II TGF-b receptor (dnTGF-R-II; n=30) and wild type mice (Wt; n=90) were subjected to a 213 partial hepatectomy (PH). TGF-bl plasma levels (ELISA), liver TGF-bl mRNA expression, BrdU-labeling, TUNEL-assays and H/Istainings were studied at different time points after pH. Results: Liver TGF-bl mRNA expression and plasma levels increased massively 12 h after PH in TGF-b transgenics compared to WT mice (270 ng/ml vs. 20 ng/ml) and decreased progressively to basal levels at day 5-7. TGF-bl mice showed delayed hepatocyte proliferation and liver growth. This delay was compensated beginning at 3-5 days, when TGF-bl levels decreased. In contrast, Dn-TGF-b-RI1 mice exhibited no difference in hepatocyte proliferation or resconstitution of liver mass after pH. A significant higher mortality was observed in TGF-bl mice compared to the other groups (80% vs. lo%), probably due to an increase in acute phase reaction. Hepatocellular apoptosis was minimal in all three groups.
I 323
P53 AS A PROGNOSTIC CARCINOMA
PREDICTOR
IN HEPATOCELLULAR
(HCC)
S. Gianni’, A. Cecchetto2, G. Altavilla2, M. Michelotto2, M. Rinaldi’, M. De Giorgio’, S. Fagiuoli’, F. Farinati’. ‘Department Of SurgicalAnd Gastroenterological Sciences, Padua, Italy; 2Department of Pathology, Padua, Italy
Background: HCC on cirrhosis is a complex disease in which prognosis depends on tumor stage, phase of the underlying liver disease, type of treatment performed and, possibly, biological factors of the tumor itself. Methods: We prospectively evaluated the survival of 91 consecutive patients with HCC hystologically confirmed on cirrhosis, between 01/98 and 12/99. Clinical features and hystologicaVbiologica1 aspects, including histotype, grading, ~53 overexpression, cytoproliferation and apoptotic markers (Ki67 and BCL2) (monoclonal antibodies), were analyzed. Kaplan Meier Survival Analysis and Cox Regression Analysis were used. Results: From the clinical point of view the single regression analysis of the prognostic factors selected the following parameters: Child-Pugh (p=O, Ol), Okuda (~‘0, OOOl), CLIP staging (~‘0, 0001) and type of treatment (p=O, 0001). In the Cox model CLIP staging was included as single independent predictor of survival at step 1 (~‘0, 0001) with Okuda at step 2 (p=O, 013). Among the biological factors, ~53 overexpression and histotype were significantly related with survival (p=O, 0044 and p=O, 017 respectively). When all the clinical and biological variables were examined
96
immunohistochemistry as well as caspase cleavage after incubation with different TRAIL preparations on freshly isolated primary human hepatocytes of 10 patients. Our data show that some but not all TRAIL preparations independent of their toxicity on tumor cells can induce apoptosis in human liver cells depending on the time point of stimulation. TRAIL resistance correlates with TRAIL-R4 expression. Interestingly at no time chemotherapeutic drugs were not able to sensitise primary human hepatocytes -in contrast to tumor cell lines- for TRAILinduced apoptosis. Our study shows to what extent TRAIL can be used in combination with chemotherapeutics.
Conclusions: Our results indicate that high levels of TGF-b lead to a transient inhibition of hepatocyte proliferation after PH, but not to a significant arrest of the regenerative process. In contrast, the interruption of the TGFb signaling pathway in hepatocytes had no significant effect on the time corn--se of liver regeneration. This implicates that TGF-? is of minor importance in controlling the regenerative process in the liver or that redundant systems, such as the activin system, could compensate for the defective TGF-b pathway.
I I 320
TRAIL-INDUCED
APOPTOSIS
LINES IS REGULATED RECRUITMENT MITOCHONDRIAL
ATTHE
IN HUMAN HEPATOMA
CELL
AND NOT AT THE
LEVEL
TNI-related apoptosis-inducing ligand (TRAIL) exhibits potent anti-tumor activity upon systemic administration, yet non of the deleterious side effects observed with other apoptosis-inducing TNF family members such as TNF and CD95L. About 50% of the investigated tumor cell lines are not sensitive to TRAIL. Thus TRAILbased tumor therapy may be used in the treatment of human cancer in viva. TRAIL induces apoptosis via death domain containing TRAIL-receptor (TRAIL-R) 1 and 2 while TRAIL-R 3 and 4 have been suggested to have an apoptosis-inhibitory function. To evaluate the potential mechanisms of TRAIL resistance we first investigated TRAILreceptor expression on hepatoma cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs such as 5-FU renders the TRAIL resistant hepatoma cell line sensitive to TRAILinduced apoptosis. DISC (Death Inducing Signaling Complex) analysis revealed substantially increased DISC formation after sensitization. The results suggest that TRAIL-mediated apoptosis in this cell lines is controlled through increased caspase 8 recruitment at the DISC level rather than through putative anti-apoptotic receptors or mitochondtial events.
321
THE ROLE OF TGF-j3 SIGNALING
HEPATOCELLULAR
CARCINOMA
TREATED
RADIOFREQUENCY
ABLATION:
EVALUATION
POSITTREATMENT
DISC VIA INCREASED
OF CASPASE-8
T.M. Ganten’, T. Haas2, M. Sprick2, W. Stremmel’, H. Walczak2. ‘Internal Medicine, University Of Heidelberg, Heidelberg, Get-many; 2Apoptosisregulation, DKFZ-Heidelberg, Heidelberg, Get-many
I
322
RESPONSE
CONTRAST-ENHANCED
WITH OF
WITH A NEW
HARMONIC
SONOGRAPHY
F. Giangregorio, M.G. Marinone, M. Di Stasi, F. Fomari. Gastroenterology Department, Piacenza Hospital, Piacenza, Italy A second generation contrast-enhanced ultrasonography (US) was used to evaluate the outcome of radiofrequency thermal ablation (RTA) in a series of hepatocellular carcinoma (HCC), comparing the results with helical CT. 93 cirrhotic patients (54 men, 39 women; mean age 71.69 years; 79 Child A; 14 Child B; 83 HCV positive, 6 HBV positive and 4 post-alcoholic) affected from HCC. Pretreatment US identified 122 lesions with a maximum diameter ranging from 1 to 7 cm (63 nodules: 1-3 cm; 52: 3-5 cm; 7: >5 cm). RTA was performed using an active 15.gauge expandable needle or a 18.gauge internally cooled electrode. The contrast-enhanced harmonic US was performed using a suspension of sulphur hexafluoride in sterile saline (SonoVue, Bracco), as contrast agent, and a machine (Esatune, Esaote) with a new harmonic sonography. In 42 patients (51 lesions) SonoVue-enhanced CnTI was performed before and 24 hours after the treatment: 11 cases (12 nodules) exhibited positive enhancement in the early arterial phase before RTA. The 27 cases with post-treatment partial enhancement at CnTI were: 7 single lesions with diameter >5 cm; 15 with lesions 3-5 cm in diameter and 5 multinodular lesions; Finally SonoVue-enhanced CnTI was used as a guide to target locations for additional RTA SonoVue-enhanced CnTI is an highly accurate technique for assessing the outcome of RTA of HCC nodules, already 24 hours after the treatment; it may be useful for guiding the site of additional treatments; it may allow to reduce the number of helical CT
PATHWAY IN LIVER
REGENERATION
J.F. Garcia-Lazaro, E. Meyer, l? Czochra, S. Lueth, I. Balderas-Renteria, P.R. Galle, A.W. Lohse, S. Kanzler. I. Department Of Medicine, University Of Maim, Maim, Get-many Background: TGF-b is a pleiotropic cytokine with antiproliferative effects on epithelial cells. The aim of this study was to determine the influence of the TGF-b signaling pathway on hepatocyte proliferation and liver regeneration in viva. Methods: Transgenic mice with liver-specific overexpression of TGF-bl (n=80), transgenic mice overexpressing a dominant negative type II TGF-b receptor (dnTGF-R-II; n=30) and wild type mice (Wt; n=90) were subjected to a 213 partial hepatectomy (PH). TGF-bl plasma levels (ELISA), liver TGF-bl mRNA expression, BrdU-labeling, TUNEL-assays and H/Istainings were studied at different time points after pH. Results: Liver TGF-bl mRNA expression and plasma levels increased massively 12 h after PH in TGF-b transgenics compared to WT mice (270 ng/ml vs. 20 ng/ml) and decreased progressively to basal levels at day 5-7. TGF-bl mice showed delayed hepatocyte proliferation and liver growth. This delay was compensated beginning at 3-5 days, when TGF-bl levels decreased. In contrast, Dn-TGF-b-RI1 mice exhibited no difference in hepatocyte proliferation or resconstitution of liver mass after pH. A significant higher mortality was observed in TGF-bl mice compared to the other groups (80% vs. lo%), probably due to an increase in acute phase reaction. Hepatocellular apoptosis was minimal in all three groups.
I 323
P53 AS A PROGNOSTIC CARCINOMA
PREDICTOR
IN HEPATOCELLULAR
(HCC)
S. Gianni’, A. Cecchetto2, G. Altavilla2, M. Michelotto2, M. Rinaldi’, M. De Giorgio’, S. Fagiuoli’, F. Farinati’. ‘Department Of SurgicalAnd Gastroenterological Sciences, Padua, Italy; 2Department of Pathology, Padua, Italy
Background: HCC on cirrhosis is a complex disease in which prognosis depends on tumor stage, phase of the underlying liver disease, type of treatment performed and, possibly, biological factors of the tumor itself. Methods: We prospectively evaluated the survival of 91 consecutive patients with HCC hystologically confirmed on cirrhosis, between 01/98 and 12/99. Clinical features and hystologicaVbiologica1 aspects, including histotype, grading, ~53 overexpression, cytoproliferation and apoptotic markers (Ki67 and BCL2) (monoclonal antibodies), were analyzed. Kaplan Meier Survival Analysis and Cox Regression Analysis were used. Results: From the clinical point of view the single regression analysis of the prognostic factors selected the following parameters: Child-Pugh (p=O, Ol), Okuda (~‘0, OOOl), CLIP staging (~‘0, 0001) and type of treatment (p=O, 0001). In the Cox model CLIP staging was included as single independent predictor of survival at step 1 (~‘0, 0001) with Okuda at step 2 (p=O, 013). Among the biological factors, ~53 overexpression and histotype were significantly related with survival (p=O, 0044 and p=O, 017 respectively). When all the clinical and biological variables were examined