The hepatitis B virus X protein (HBx) is thought to be implicated in the development of hepatocellular carcinoma, but its exact function remains controversial. Transgenic mice from PEX7 and AX16 lineages that express HBx in the liver under control of di erent viral regulatory elements develop no liver pathology (Billet et al., 1995). We have crossed these two mouse lineages with WHV/c-myc oncomice in which liver-speci®c expression of c-myc driven by woodchuck hepatitis virus (WHV) regulatory sequences causes liver cancer in all animals. The average tumor latency was shortened by 2 to 3 months in bitransgenic animals from all populations compared with simple c-myc transgenic littermates. At preneoplastic stages, adult bitransgenic mice showed four to ®vefold enhanced expression of the c-myc transgene, increased hepatocyte proliferation and more extensive liver lesions compared with simple WHV/c-myc transgenics. Thus in this model, HBx alone has no direct pathological e ect but it is shown to accelerate tumor development induced by c-myc. The data presented here ®rmly establish the oncogenic potential of HBx, apparently acting as a tumor promoter. This model o ers unique opportunities to investigate the mechanisms by which HBx trans-activates the expression of target genes and deregulates the hepatocyte growth control in vivo.

The hepatitis B virus protein HBx is a promiscuous transactivator implicated in both cell growth and death and in the development of hepatocellular carcinoma. We recently reported that HBx can potentiate c-myc-induced liver oncogenesis in a transgenic model where low level expression of HBx induces no pathology. To assess if HBx could a ect the hepatocyte turnover, we investigated the HBx-elicited apoptotic responses in transgenic livers and in primary hepatocyte cultures. Here we show that transgenic expression of HBx is associated with a twofold increase of spontaneous cell death in the mouse liver. The ®nding that apoptosis was enhanced to similar extents in HBx mice carrying homozygous p53 null mutations implied that functionally intact p53 was not required to transduce the death signal. A direct, dosedependent apoptotic function of HBx was demonstrated in transient transfections of liver-derived cell lines. We further show that stable expression of HBx at low, presumably physiological levels in primary hepatocytes, induced cellular susceptibility to diverse apoptotic insults, including growth factor deprivation, treatment with antiFas antibodies or doxorubicine and oxidative stress. HBx expression, but not p53 status profoundly a ected the commitment of cells to die upon apoptotic stimuli. These data strengthen the notion that HBX may contribute to HBV pathogenesis by enhancing apoptotic death in the chronically infected liver.

There is compelling evidence that Bax channel activity stimulates cytochrome c release leading ultimately to cell death, which is a key event in ischemic injuries and neurodegenerative diseases. Here 3,6-dibromocarbazole piperazine derivatives of 2-propanol are described as the first small and potent modulators of the cytochrome c release triggered by Bid-induced Bax activation in a mitochondrial assay. Furthermore, a mechanism of action is proposed, and fluorescent derivatives allowing the localization of such inhibitors are reported.

The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial. Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53. In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo. HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes. In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2. Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal. Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory e ect of Bcl-2 against Fas cytotoxicity. This e ect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X L or Bax could not be evidenced in yeast two-hybrid assays. We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation. Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.

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We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHVinfected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus-and host-specific determinants to liver pathology and tumorigenesis.

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Pro-apoptotic members of the Bcl-2 family can be subdivided in two classes according to their structure: a group including Bax, Bak, and Bok that display Bcl-2 homology (BH) 1, BH2 and BH3 domains and a second group including Bid (BH3 interacting domain death agonist), Bad, Bim (Bcl-2 interacting mediator of cell death) and several others that contain only a BH3 domain, the BH3-only proteins. The BH3-only proteins have been proposed to activate pro-apoptotic members of the Bax subfamily to trigger a mitochondrial pathway that leads to the release of cytochrome c and other apoptogenic factors. Here we report that the mechanism of action of Bim is di¡erent from that of Bid. Although overexpression of Bid or Bim in cells leads to cytochrome c release, only Bid is able to trigger the release of cytochrome c through Bax activation when added directly to isolated mitochondria. Bim L , although unable to activate Bax, can directly inhibit Bcl-2 or Bcl-x L . Our data suggest two functional classes of BH3-only proteins: those such as Bid which directly activate Bax-like proteins leading to mitochondrial membrane permeability and apoptosis and those such as Bim which inhibit anti-apoptotic proteins and render the cells more susceptible to apoptogenic stimuli. ß