Toni Urbanik

Knockout of myeloid cell leukemia‐1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes

Myeloid cell leukemia‐1 (Mcl‐1) is an antiapoptotic member of the Bcl‐2 protein family. It interacts with proapoptotic Bcl‐2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl‐1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl‐1 by growth factors rescues primary human hepatocytes from CD95‐mediated apoptosis. This prompted us to further analyze the relevance of Mcl‐1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte‐specific Mcl‐1 knockout mouse (Mcl‐1flox/flox‐AlbCre). Deletion of Mcl‐1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl‐1flox/flox‐AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl‐1. Importantly, hepatic pericellular fibrosis occurs in Mcl‐1 negative livers in response to chronic liver damage. Furthermore, Mcl‐1flox/flox‐AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti‐CD95 antibodies or concanavalin A. Conclusion: The present study provides in vivo evidence that Mcl‐1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction. (HEPATOLOGY 2009.)

Hepatocyte‐specific deletion of the antiapoptotic protein myeloid cell leukemia‐1 triggers proliferation and hepatocarcinogenesis in mice

Regulation of hepatocellular apoptosis is crucial for liver homeostasis. Increased sensitivity of hepatocytes toward apoptosis results in chronic liver injury, whereas apoptosis resistance is linked to hepatocarcinogenesis and nonresponsiveness to therapy‐induced cell death. Recently, we have demonstrated an essential role of the antiapoptotic Bcl‐2 family member Myeloid cell leukemia‐1 (Mcl‐1) in hepatocyte survival. In mice lacking Mcl‐1 specifically in hepatocytes (Mcl‐1Δhep), spontaneous apoptosis caused severe liver damage. Here, we demonstrate that chronically increased apoptosis of hepatocytes coincides with strong hepatocyte proliferation resulting in hepatocellular carcinoma (HCC). Liver cell tumor formation was observed in >50% of Mcl‐1Δhep mice already by the age of 8 months, whereas 12‐month‐old wild‐type (wt) and heterozygous Mcl‐1flox/wt mice lacked tumors. Tumors revealed a heterogenous spectrum ranging from small dysplastic nodules to HCC. The neoplastic nature of the tumors was confirmed by histology, expression of the HCC marker glutamine synthetase and chromosomal aberrations. Liver carcinogenesis in Mcl‐1Δhep mice was paralleled by markedly increased levels of Survivin, an important regulator of mitosis which is selectively overexpressed in common human cancers. Conclusion: This study provides in vivo evidence that increased apoptosis of hepatocytes not only impairs liver homeostasis but is also accompanied by hepatocyte proliferation and hepatocarcinogenesis. Our findings might have implications for understanding apoptosis‐related human liver diseases. (HEPATOLOGY 2010.)

Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice.

BACKGROUND & AIMS CYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. Reduced CYLD expression has been observed in other tumor entities, including hepatocellular carcinoma. In the present study, we analyzed the role of CYLD in liver homeostasis and hepatocarcinogenesis in vivo. METHODS Mice with liver-specific deletion of CYLDexon7/8 (CYLD(FF)xAlbCre) were generated. Liver tissues were histologically analyzed and oval cell activation was investigated. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB). Microarray expression profiling of livers was performed in untreated as well as DEN/PB-treated mice. NF-κB signaling was assessed by ELISA, quantitative real-time PCR, and Western blotting. RESULTS CYLD(FF)xAlbCre hepatocytes and cholangiocytes did not express full-length CYLD (FL-CYLD) protein but showed increased expression of the naturally occurring short-CYLD splice variant (s-CYLD). CYLD(FF)xAlbCre mice exhibited a prominent biliary phenotype with ductular reaction and biliary-type fibrosis. In addition, CYLD(FF)xAlbCre mice showed a significantly increased sensitivity towards DEN/PB-induced hepatocarcinogenesis. Moreover, we could observe the development of cholangiocellular carcinoma, in line with enhanced oval cell activity. NF-κB-signaling was increased in livers of CYLD(FF)xAlbCre mice and likely contributed to the inflammatory and fibrotic response. CONCLUSIONS The deletion of exon7/8 of the CYLD gene activates oval cells, leads to a biliary phenotype, and increases the susceptibility towards carcinogenesis in the liver. Thus, our study presents a novel model of biliary damage and liver fibrosis, followed by cancer development.

Pan-Bcl-2 Inhibitor Obatoclax Delays Cell Cycle Progression and Blocks Migration of Colorectal Cancer Cells

Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC), prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes

AIM To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models. METHODS Hepatocyte cell death in CYLD knockout mice (CYLD(-/-) ) was analyzed by application of liver injury models for CD95- (Jo2) and tumor necrosis factor (TNF)-α- [D-GalN/lipopolysaccharide (LPS)] induced apoptosis. Liver injury was assessed by measurement of serum transaminases and histological analysis. Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases. Nuclear factor (NF)-κB, ERK, Akt and jun amino-terminal kinases signaling were assessed. Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-α and with the CD95-ligand Jo2. Cell viability was analyzed by MTT-assay. RESULTS Livers of CYLD(-/-) mice showed increased anti-apoptotic NF-κB signaling. In both applied liver injury models CYLD(-/-) mice showed a significantly reduced apoptosis sensitivity. After D-GalN/LPS treatment CYLD(-/-) mice exhibited significantly lower levels of alanine aminotransferase (ALT) (295 U/L vs 859 U/L, P < 0.05) and aspartate aminotransferase (AST) (560 U/L vs 1025 U/L, P < 0.01). After Jo injection CYLD(-/-) mice showed 2-fold lower ALT (50 U/L vs 110 U/L, P < 0.01) and lower AST (250 U/L vs 435 U/L, P < 0.01) serum-levels compared to WT mice. In addition, isolated CYLD(-/-) primary murine hepatocytes (PMH) were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2, XIAP, cIAP1/2, survivin and c-FLIP expression upon TNF- and CD95-receptor triggering, respectively. Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of anti-apoptotic proteins and re-sensitized CYLD(-/-) PMH towards TNF- and CD95-receptor mediated cell death. CONCLUSION CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.