[45] HEPATOCYTE-SPECIFIC DEPLETION OF CASPASE-8 ACCELERATES THE ONSET OF LIVER REGENERATION IN MICE

Parallel Sexxion 5: MOLECULAR BIOLOGY AND CELL SIGNALLING 1 IKK2 HEPATOCYTE SPECIFIC DELETION ALTERS CELL CYCLE PROGRESSION AFTER PARTIAL HEPATECTOMY Y. Malato’, N. Beraza’, M. Pasparakis2, C. Trautwein’ . ‘Drpurtmrnt of Internal Medicine IIL Unioersity Klinikiinz Aachen, Aachen; 2Department of Genetics, linioersiiy of Cologne, Cologne, Gernzany E-mail: [email protected] Background and Aim: NFKB is involved in many cellular responses. In non-stimulated cells, NFKB is sequestred in the cytoplasm by the inhibitory protein TkB. Upon stimulation, TlcB is phosphorylated by the TKK complex, allowing NFKB activation and translocation in the nucleus. The TKK complex consists of two catalytic subunits TKKl and TKK2, and the regulatory subunit NEMO. The liver has the unique ability to restore its mass after liver injury. Since NFKB is involved in early events of hepatocyte proliferation, we studied the impact of hepatocyte-specitic 1KK2 deletion on liver regeneration. Methods: 1KK2 constitutive knock-out mice die embryonically. Thus, we generated a hepatocyte-specific 1KK2 knock-out (lKK2Ahepa) mouse using the Cre/LoxP system. 70% partial hepatectomy (PH) was performed on TKK2fX mice (wild-type) and TKK2Ahepa mice (knockout), and liver regeneration was studied. Results: All mice survived after PH. BrdU analysis showed earlier proliferation in TKK2Ahepa mice compared to TKK2f/f mice. Real Time PCR and Western Blot analysis of cyclin D, E and A further contirmed BrdU analysis. Unexpectedly, no difference in cyclin B expression was observed. In order to understand the earlier induction of hepatocytes proliferation in 1KK2Ahepa mice, we analysed the priming phase. TNF mediated priming of hepatocytes and its ability to induce extracellular matrix remodelling via MMP9, are key events during liver regeneration. TNF and MMP-9 were expressed earlier in 1KK2Ahepa mice compared to 1KK2f/f mice, confirming an earlier priming in 1KK2Ahepa mice and a more rapid proliferative response. Similar RNA and protein levels of cyclin B in both genotypes suggested a G2IM transition lag in TKK2Ahepa mice. The cyclin B-dependent kinase protein CDC2 is a G/2M activator while GADD45 has inhibitory activity. Western blot analysis of these proteins showed reduced CDC2 activity and higher GADD45 expression in 1KK2Ahepa mice, resulting in a slackening of cell cycle progression. Conclusions: Our data demonstrate that hepatocyte-specific TKK2 deletion alters cell cycle progression by triggering an earlier priming phase and proliferative response, while delaying G2/M transition.

HEPATOCYTE-SPECIFIC DEPLETION OF CASPASE-8 ACCELERATES THE ONSET OF LIVER REGENERATION IN MICE

J. Freimuth’, D. Riethmacher2, C. Trautwein’, C. Liedtke’. ’Department of Medicine Ill, University Hospital Aachen, RWTH Aachen; ’Centwfbr Molecular Neurohiology, Hanzburg, Germany E-mail: [email protected] Background and Aims: The cytokine TNF plays a critical role early i n liver regeneration following partial hepatectomy (PH). TNF mediates signals via at least three different pathways involving protective effects via NF-kB, apoptosis signalling through caspase-8 activation and activation of c-Jun N-terminal kinases (JNK). The aim of this study was to evaluate a potential role of caspase-8 in liver regeneration. Methods: Conditional hepatocyte specific knockout (casp8Ahepa) mice for caspase-8 were generated using the cre/loxP recombination system and a cre-transgene under the control of the albumin promoter. Casp8Ahepa and wildtype (WT) mice were subjected to PH and the liver regeneration capacity of both groups was determined. Apoptosis in the regenerating livers was analyzed by TUNEL staining. Hepatocyte proliferation and DNA synthesis was monitored measuring BrdU incorporation. Expression of important cell cycle markers cyclin D (Gl-phase), E (GliS-phase) and A (S-phase) was determined using real-time PCR and immunoblotting.

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Results: The efficiency ofthe caspase-8 knockout was analyzed in primary hepatocytes on DNA, RNA and protein levels revealing a deletion efficiency of >90%. Following hepatectomy, we observed liver regeneration in both WT and casp8Ahepa mice within 96 hours. In the regenerating livers we could not detect significant levels of apoptosis in control groups or casp8Ahepa mice. Following hepatectomy, in casp8Ahepa mice expression of S-phase marker cyclin A started 6h earlier and was Sfold increased 36h post PH compared to controls. Consistently, casp8Ahepa livers revealed approximately 30% more BrdU positive hepatocytes 36h and 48h after surgery implicating prolonged S-phase. At later time points (60-96h) no difference in BrdU incorporation was observed between both groups. In agreement with these data, cyclin D and E peaked 4h earlier (36h post PH) in casp8Ahepa mice also showing higher expression levels than the controls. Conclusions: Our data implicates that caspase-8 inactivation strongly enhances early proliferative effects without afecting the outcome of liver regeneration. However, caspase-8 mediated apoptotic mechanisms seem to be dispensable for the termination of liver regeneration.

C-FOS OVEREXPRESSION INCREASES THE PROLIFERATION OF IMMORTALIZED HUMAN HEPATOCYTES BY STABILIZING CYCLIN D1 M.C. Guller’, A. Legrand2, D. Bernuau’, F. Daniel2. ‘hsernzlJ~!,7/H~/)ital Saint-Louis, Paris; ’lnsernz lJ773 centre de rrchrrchr hiomkliculr Bichut-Beuujon (CRB3)/H@itul Bichut, Puris, France E-mail: [email protected]

Background and Aims: The proto-oncogene c-fos is an important member of the Activating Protein I (AP-I) transcription factor involved in cell proliferation and transformation. c-Fos overexpression might be an important step towards the development of liver cancer but its precise role remains ill-defined. We therefore aimed to clarify c-Fos implication in hepatocarcinogenesis. Methods: Immortalized human hepatocytes (THH) were stably transfected with a c-Fos expression vector. Cell proliferation was assessed by [’HI thymidine incorporation and growth curve assays. Anchorageindependent growth was studied in soft agar. The cell cycle was analyzed by flow cytometry and cell cycle regulatory proteins were analyzed by western blot. Protein stability was determined by cycloheximide treatment. c-Fos expression was silenced by transient small interfering RNA (SiRNA) transfections. Results: c-Fos overexpression induced in vitro transformation as shown in soft agar assays with an increase in the number and size of colonies. c-Fos also increased cell proliferation under low serum conditions and resulted in a two-fold increase in [’Hlthymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation of quiescent cells, Cyclin DI was more abundantly expressed in c-Fos overexpressing cells. Cyclin D I accumulation did not result from increased transcriptional activation. Cycloheximide experiments showed that Cyclin D1 was stabilized in the nucleus ofc-Fos overexpressing cells, which explains the rapid nuclear accumulation ofthis protein after serum stimulation. This stabilization was abolished when c-Fos gene expression was silenced via siRNA. Conclusions: c-Fos overexpression leads to anchorage-independent growth, increased DNA synthesis following serum addition, accelerated cell cycle and increased proliferation of immortalized human hepatocytes under low serum conditions. c-Fos also increases the stability of Cyclin D1 in the nucleus following serum stimulation of quiescent cells. Cyclin D1 retention in the nucleus during the cell cycle is known to be a cancer promoting or predisposing event. Our results therefore strongly suggest that c-Fos may contribute to hepatocarcinogenesis by increasing the nuclear stability of Cyclin Dl ,