- Email: [email protected]
[42] DEPLETION OF CYCLIN E IN MICE INCREASES CELL CYCLE ACTIVITY AND REGENERATION EFFICIENCY OF THE LIVER AFTER PARTIAL HEPATECTOMY
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Thur~xduy,I 2 April
responsible for the different outcome observed after TNF challenge following mGSH depletion. Methods: Cultured hepatocytes from wild type and ASMase-’- mice (C57BL/6), obtained by collagenase perfusion, were depleted of mGSH by (R, S)-3-hydroxy-4-pentenoate(HE 0.5mM). Survival and chromatin morphology were assessed by GST release and Hoechst/PI staining. Bax, tBid and pBim translocation to mitochondria, JNK phosphorylation, and caspase-3 activation were determined by western blot. Mitochondria1 membrane permeabilization (MMP) was assessed by the distribution of calceidTMRM. Cardiolipin levels were analyzed by HPLC. ROS generation was analyzed fluorimetrically. Results: The signaling events upstream of mitochondria such as mitochondrial Bax translocation and oligomerization, Bid truncation, NFkB activation and transient JNK phosphorylation, were preserved in ASMase-’- hepatocytes regardless of mGSH depletion. Only wild type mGSH-depleted cells underwent MMP with lower intact cardiolipin levels (40-50%) due to enhanced cardiolipin hydroperoxides, cyt c release into the cytosol and caspase 3 activation followed by cell death. None of these features were observed in mGSH-depleted ASMase’- cells. Interestingly, an early burst of ROS (beginning 15-30 min after TNF addition) was also only observed in mGSH-depleted wild type hepatocytes, and not in TNF-alone treated hepatocytes, nor in ASMase-’- cells despite mGSH depletion. Furthermore, ganglioside GD3 colocalized with mitochondria (as soon as 30 min after TNF addition) in wild type but not in ASMase’-, suggesting that ASMase is necessary for this event. Conclusion: These suggest that mGSH modulates hepatocellular sensitivity to TNF through control of mitochondrial ROS generation targeting mitochondrial cardiolipin status, and that ASMase links TNF signaling to mitochondrial ROS generation.
DEPLETION OF CYCLIN E2 IN MICE INCREASES CELL CYCLE ACTIVITY AND REGENERATION EFFICIENCY OF THE LIVER AFTER PARTIAL HEPATECTOMY Y.A. Nevzorova’ , D.F. Tschaharganeh’ , P. Sicinski’, C. Trautwein’, C. Liedtke’ . ‘Depurtnzent of Medicine Ill, Universiiy Hospital Aachen,
RWTH Auchen lJniwrsity, Aachen, Germany; ’Dana Furber Cancer Institute, Boston, MA, 7JSA E-mail: [email protected] Background: The current understanding of cell cycle regulation in mammals has completely changed as recent studies demonstrate that the E-type cyclins E l and E2 are not essential for cell proliferation per se, but for transition of quiescent cells into the cell cycle. Therefore the aim of this study was to investigate the role of E-type cyclins during liver regeneration. Methods: Partial hepatectomy (PH) was performed in cyclin El and E2 knockout (KO) mice and wildtype (WT) controls. Regeneration was monitored measuring the liveribody weight ratio after PH and cell cycle markers for GliS-Phase (PCNA, cyclin E l , E2) and S-phase (BrdU, Cyclin A). Cell cycle progression was detected by FACS analysis. Activity of cyclin Eicdk2 complexes were analysed through histone H1 kinase assays. Results: Following PH both E l KO and E2KO animals displayed different proliferation kinetics compared to WT controls. ElKO revealed a slight delay of GliS phase transition but stronger hepatocyte proliferation at later time points (48-7211 post PH) and a prolonged cdk2 kinase activity compared to WT animals. In E2KO mice we found an unexpected effect. Hepatocyte proliferation started earlier and was significantly higher in course of regeneration. Cdk2 kinase activity was observed from 36 to 96 hours post PH whereas in WT mice cdk2 kinase activity was only found from 40 to 48 hours after PH. Accordingly, E2KO mice showed a 40% higher liveribody weight ratio in comparison to control animals 7 days after PH. During liver regeneration we found an identical gene expression profile of both cyclins in WT animals. However, the mRNA expression level of cyclin E2 was approximately 2 magnitudes higher compared to cyclin E l .
Consistent with the higher regeneration capacity of cyclin E2KO mice, we measured much higher levels of cyclin El in these animals compared to control group. In El KO cyclin E2 expression had overall lower level compared to WT. Conclusions: Cyclin El and E2 alone are not essential for liver regeneration. However, depletion of cyclin E2 leads to higher proliferation rate, prolonged cdk2 kinase activity and enhanced liver mass implicating that cyclin E2 might be a negative regulator of cyclin E l .
SUBTYPE CLASSIFICATION OF HEPATOCELLULAR ADENOMAS BY MOLECULAR MARKERS AND IMMUNOHISTOCHEMISTRY P. Bioulac-Sage’ , S. Rebouissou’, C. Thomas’, J.F. Blanc3, J. Saric4, E. Jeannot’, A. Sa Cunha4, G. Couchy2, S. Imbeaud5, C. Balabaud3, J. Zucman-Rossi’. ‘Depurtment of Puthology, INSERM E362, CHU Bordeuux, Bordeuux; ’ I N S E M U6 74, Puris; ’De~~urtment of Heputology, CHU Bordeuux, Bordeuux; 4De~~urtment of Surgery, CHU Bordeuux, CNRS, villejuif,~France E-mail: [email protected] Background and Aims: Hepatocellular adenomas (HCA) are rare benign liver tumors, most frequently occurring in women using oral contraception. Recently, 4 HCA subtypes defined by HNFla inactivating mutations, (i-catenin activating mutations and inflammatory infiltrates were defined. Considering the high risk of malignant transformation related to [hatenin activated HCA and to simplify the use of this classification, we searched for new markers useful in a routine diagnosis. Methods: We tested for the expression of candidate genes using quantitative RT-PCR in 42 classified HCA. The diagnostic value of markers was assessed by AUC. We analyzed the protein expression level of these markers using immunohistochemistry to test their specificity and sensibility in 72 classified HCA. Finally, four markers were validated using IHC on paraffin sections in a whole monocentric series of 89 HCA. Results: We validated 6 markers using quantitative RT-PCR for their diagnostic value. LFABP and UGT2B7 were down regulated in HNFIa inactivated HCA (P<0.0002; AUC (SE; 95%CI) = 0.9 (0.1; 0.7-1)); GLUL and GPR49 over-expression were correlated with (i-catenin activating mutations (P<0.0005; AUC (SE; 95%CT) = 0.86 (0.08; 0.7-1)); SAA2 and CRP were significantly up-regulated in inflammatory HCA (P=O.OOOl; AUC (SE; 95%CT) 3 0.86 (0.07; 0.74-0.97)). We validated using immunohistochemistry the absence of LFABP to predict HNFla mutation (sensitivity loo%, specificity 94%), GLUL overexpression and nuclear (i-catenin staining to predict (i-catenin activating mutation (sensitivity 85%, specificity IOO’XO) and SAA2 hepatocytic staining to predict inflammatory HCA (sensitivity and specificity 9 1%). Finally, the overall series of 89 HCA were classified using the four validated immunohistochemical markers and correlations were searched with clinical and phenotypic features. In summary, (i-catenin HCA (20%) were associated with HCC (PiO.0Ol) at diagnosis (4 cases) or in the follow-up (2 case); male was also more frequent in (i-catenin subtype (P=O.O02). HNFIa subtype (33%) was associated with steatosis (P < 0.001), adenomatosis defined by more than 10 nodules (P=O.OOl) and a normal serum level of GGT (P=0.002). All adenomas with a telangiectatic phenotype andor inflammatory phenotype (38%) were immunostained for SAA2. Conclusion: These results provided a valuable tool to classify hepatocellular adenomas in a routine test using quantitative RT-PCR or IHC.
Thur~xduy,I 2 April
responsible for the different outcome observed after TNF challenge following mGSH depletion. Methods: Cultured hepatocytes from wild type and ASMase-’- mice (C57BL/6), obtained by collagenase perfusion, were depleted of mGSH by (R, S)-3-hydroxy-4-pentenoate(HE 0.5mM). Survival and chromatin morphology were assessed by GST release and Hoechst/PI staining. Bax, tBid and pBim translocation to mitochondria, JNK phosphorylation, and caspase-3 activation were determined by western blot. Mitochondria1 membrane permeabilization (MMP) was assessed by the distribution of calceidTMRM. Cardiolipin levels were analyzed by HPLC. ROS generation was analyzed fluorimetrically. Results: The signaling events upstream of mitochondria such as mitochondrial Bax translocation and oligomerization, Bid truncation, NFkB activation and transient JNK phosphorylation, were preserved in ASMase-’- hepatocytes regardless of mGSH depletion. Only wild type mGSH-depleted cells underwent MMP with lower intact cardiolipin levels (40-50%) due to enhanced cardiolipin hydroperoxides, cyt c release into the cytosol and caspase 3 activation followed by cell death. None of these features were observed in mGSH-depleted ASMase’- cells. Interestingly, an early burst of ROS (beginning 15-30 min after TNF addition) was also only observed in mGSH-depleted wild type hepatocytes, and not in TNF-alone treated hepatocytes, nor in ASMase-’- cells despite mGSH depletion. Furthermore, ganglioside GD3 colocalized with mitochondria (as soon as 30 min after TNF addition) in wild type but not in ASMase’-, suggesting that ASMase is necessary for this event. Conclusion: These suggest that mGSH modulates hepatocellular sensitivity to TNF through control of mitochondrial ROS generation targeting mitochondrial cardiolipin status, and that ASMase links TNF signaling to mitochondrial ROS generation.
DEPLETION OF CYCLIN E2 IN MICE INCREASES CELL CYCLE ACTIVITY AND REGENERATION EFFICIENCY OF THE LIVER AFTER PARTIAL HEPATECTOMY Y.A. Nevzorova’ , D.F. Tschaharganeh’ , P. Sicinski’, C. Trautwein’, C. Liedtke’ . ‘Depurtnzent of Medicine Ill, Universiiy Hospital Aachen,
RWTH Auchen lJniwrsity, Aachen, Germany; ’Dana Furber Cancer Institute, Boston, MA, 7JSA E-mail: [email protected] Background: The current understanding of cell cycle regulation in mammals has completely changed as recent studies demonstrate that the E-type cyclins E l and E2 are not essential for cell proliferation per se, but for transition of quiescent cells into the cell cycle. Therefore the aim of this study was to investigate the role of E-type cyclins during liver regeneration. Methods: Partial hepatectomy (PH) was performed in cyclin El and E2 knockout (KO) mice and wildtype (WT) controls. Regeneration was monitored measuring the liveribody weight ratio after PH and cell cycle markers for GliS-Phase (PCNA, cyclin E l , E2) and S-phase (BrdU, Cyclin A). Cell cycle progression was detected by FACS analysis. Activity of cyclin Eicdk2 complexes were analysed through histone H1 kinase assays. Results: Following PH both E l KO and E2KO animals displayed different proliferation kinetics compared to WT controls. ElKO revealed a slight delay of GliS phase transition but stronger hepatocyte proliferation at later time points (48-7211 post PH) and a prolonged cdk2 kinase activity compared to WT animals. In E2KO mice we found an unexpected effect. Hepatocyte proliferation started earlier and was significantly higher in course of regeneration. Cdk2 kinase activity was observed from 36 to 96 hours post PH whereas in WT mice cdk2 kinase activity was only found from 40 to 48 hours after PH. Accordingly, E2KO mice showed a 40% higher liveribody weight ratio in comparison to control animals 7 days after PH. During liver regeneration we found an identical gene expression profile of both cyclins in WT animals. However, the mRNA expression level of cyclin E2 was approximately 2 magnitudes higher compared to cyclin E l .
Consistent with the higher regeneration capacity of cyclin E2KO mice, we measured much higher levels of cyclin El in these animals compared to control group. In El KO cyclin E2 expression had overall lower level compared to WT. Conclusions: Cyclin El and E2 alone are not essential for liver regeneration. However, depletion of cyclin E2 leads to higher proliferation rate, prolonged cdk2 kinase activity and enhanced liver mass implicating that cyclin E2 might be a negative regulator of cyclin E l .
SUBTYPE CLASSIFICATION OF HEPATOCELLULAR ADENOMAS BY MOLECULAR MARKERS AND IMMUNOHISTOCHEMISTRY P. Bioulac-Sage’ , S. Rebouissou’, C. Thomas’, J.F. Blanc3, J. Saric4, E. Jeannot’, A. Sa Cunha4, G. Couchy2, S. Imbeaud5, C. Balabaud3, J. Zucman-Rossi’. ‘Depurtment of Puthology, INSERM E362, CHU Bordeuux, Bordeuux; ’ I N S E M U6 74, Puris; ’De~~urtment of Heputology, CHU Bordeuux, Bordeuux; 4De~~urtment of Surgery, CHU Bordeuux, CNRS, villejuif,~France E-mail: [email protected] Background and Aims: Hepatocellular adenomas (HCA) are rare benign liver tumors, most frequently occurring in women using oral contraception. Recently, 4 HCA subtypes defined by HNFla inactivating mutations, (i-catenin activating mutations and inflammatory infiltrates were defined. Considering the high risk of malignant transformation related to [hatenin activated HCA and to simplify the use of this classification, we searched for new markers useful in a routine diagnosis. Methods: We tested for the expression of candidate genes using quantitative RT-PCR in 42 classified HCA. The diagnostic value of markers was assessed by AUC. We analyzed the protein expression level of these markers using immunohistochemistry to test their specificity and sensibility in 72 classified HCA. Finally, four markers were validated using IHC on paraffin sections in a whole monocentric series of 89 HCA. Results: We validated 6 markers using quantitative RT-PCR for their diagnostic value. LFABP and UGT2B7 were down regulated in HNFIa inactivated HCA (P<0.0002; AUC (SE; 95%CI) = 0.9 (0.1; 0.7-1)); GLUL and GPR49 over-expression were correlated with (i-catenin activating mutations (P<0.0005; AUC (SE; 95%CT) = 0.86 (0.08; 0.7-1)); SAA2 and CRP were significantly up-regulated in inflammatory HCA (P=O.OOOl; AUC (SE; 95%CT) 3 0.86 (0.07; 0.74-0.97)). We validated using immunohistochemistry the absence of LFABP to predict HNFla mutation (sensitivity loo%, specificity 94%), GLUL overexpression and nuclear (i-catenin staining to predict (i-catenin activating mutation (sensitivity 85%, specificity IOO’XO) and SAA2 hepatocytic staining to predict inflammatory HCA (sensitivity and specificity 9 1%). Finally, the overall series of 89 HCA were classified using the four validated immunohistochemical markers and correlations were searched with clinical and phenotypic features. In summary, (i-catenin HCA (20%) were associated with HCC (PiO.0Ol) at diagnosis (4 cases) or in the follow-up (2 case); male was also more frequent in (i-catenin subtype (P=O.O02). HNFIa subtype (33%) was associated with steatosis (P < 0.001), adenomatosis defined by more than 10 nodules (P=O.OOl) and a normal serum level of GGT (P=0.002). All adenomas with a telangiectatic phenotype andor inflammatory phenotype (38%) were immunostained for SAA2. Conclusion: These results provided a valuable tool to classify hepatocellular adenomas in a routine test using quantitative RT-PCR or IHC.