Activation of tumor necrosis factor receptor 1 or Fas leads to the generation of reactive oxygen species, which are important to the cytotoxic effects of tumor necrosis factor ␣ (TNF-␣) or Fas ligand. However, how these radicals are generated following receptor ligation is not clear. Using primary hepatocytes, we found that TNF-␣ or anti-Fas antibodyinduced burst of oxygen radicals was mainly derived from the mitochondria. We discovered that Bid-a pro-death Bcl-2 family protein activated by ligated death receptors-was the main intracellular molecule signaling the generation of the radicals by targeting to the mitochondria and that the majority of oxygen radical production was dependent on Bid. Reactive oxygen species contributed to cell death and caspase activation by promoting FLICE-inhibitory protein degradation and mitochondrial release of cytochrome c. For the latter part, the oxygen radicals did not affect Bak oligomerization but instead promoted mitochondrial cristae reorganization and membrane lipid peroxidation. Antioxidants could reverse these changes and therefore protect against TNF-␣ or anti-Fas-induced apoptosis. In conclusion, our studies established the signaling pathway from death receptor engagement to oxygen radical generation and determined the mechanism by which reactive oxygen species contributed to hepatocyte apoptosis following death receptor activation. (HEPATOLOGY 2004;40:403-413.)

Mechanisms that control the proliferation capability of the initiated cells during hepatocarcinogenesis are still largely unclear. We investigated the role of a pro-death Bcl-2 family protein, Bid, in liver tumor development using a neonatal diethylnitrosamine model. Diethylnitrosamine was administrated to 15-day-old wild-type and bid-null mice. The development of microfoci at the early stage and of gross tumors at the later stage was compared between the two groups of mice. Both microfoci and gross tumor development were significantly retarded in the bid-null mice, despite reduced cell death as measured by TUNEL staining. Further studies indicated that there were significantly less proliferating cells in diethylnitrosamine-treated bid-null livers. The regulation of cell proliferation by Bid was confirmed in two other systems not involving carcinogenesis. Hepatocyte proliferation following partial hepatectomy and T lymphocyte proliferation following anti-CD3 stimulation were both retarded in bid-null mice. Thus, these studies revealed a previously undisclosed function of Bid in regulating cell proliferation, which can be important to tumor development. Furthermore, the role of Bid in promoting hepatocarcinogenesis is in contrast to its reported role in suppressing myeloid leukemia and thus suggests an organ-and/or etiology-specific role of the Bcl-2 family proteins in regulating oncogenesis. Hepatocellular carcinoma is a worldwide health concern with diverse etiology. Viral hepatitis, alcohol liver disease, and carcinogen exposures are the major causes.1 Rodents have been traditionally used to study carcinogen-induced liver cancers and to screen potential carcinogens. The mouse models have become increasingly important with the availability of various transgenic/knockout mice, which offer a definite way to dissect genetic pathways in the carcinogenesis. Diethylnitrosamine (DEN) is one of the most extensively studied carcinogens for the liver.2,3 It is a complete genotoxic carcinogen, and it can induce liver tumors at a proper dosage without any promoting agents, particularly in neonatal mice, whose proliferating hepatocytes are particularly susceptible to it.3 The molecular mechanism of how DEN induces liver cancer is not completely understood. It is likely that the mutations accumulated in the genome eventually lead to oncogenic transformation and tumor development. Histologically, numerous microfoci appear in the first several months after a single DEN injection at the neonatal stage, followed by the development of adenomas and carcinomas by 8 to 12 months. 2Genetic elements that control and regulate DEN-induced carcinogenesis are still in large part unknown, although it seems that many different genes can affect the process. Mice overexpressing c-myc or TGF-␣ in the liver developed spontaneous hepatocellular carcinoma and were also more susceptible to DEN-induced hepatocarcinogenesis, although their actual contributions under non-overexpression conditions are less clear. 4 On the other hand, deletion of cycli...

The death receptor pathway is coupled to the mitochondria apoptosis pathway. However, mitochondrial participation, which is stimulated by Bid but suppressed by Bcl-2/Bcl-x L , is required in certain cells (Type II), but not in others (Type I). While these differences were originally characterized in the lymphoid cell lines, the typical Type II cells are represented by hepatocytes in vivo. The molecular mechanisms that distinguish Type II from Type I cells and the regulation are not fully understood. Fas can be sequestered by the HGF receptor c-Met and high doses of HGF can promote cell death by freeing Fas from c-Met complex. We thus reasoned that treatment of the Type II cells with high doses of HGF could enhance Fas-mediated apoptosis and spare the mitochondria amplification. Indeed, such treatment led to increased apoptosis in Type II lymphoid cells, which could not be blocked by Bcl-x L . Moreover, significant hepatocyte apoptosis was induced by this scheme in the absence of Bid with increased dissociation of Fas from c-Met. These findings indicate that high doses of HGF could be used to promote apoptosis in Type II cells bypassing the requirement for mitochondria activation.

How a central apoptosis mechanism could be modulated during a specific developmental or homeostatic process to comply with the specific needs of a particular tissue is poorly understood. Bcl-2 is a key anti-apoptosis regulator and its deletion resulted in multiple defects in mice, indicating its broad involvement in development and homeostasis of various tissues. We found that the severity and extensiveness of the defects could be greatly influenced by the genetic background of the mice. Hence, Bcl-2-deficient mice predominantly on C57BL/6 background had the most severe presentation with increased embryonic lethality, whereas Bcl-2-deficient mice predominantly on 129/SvJ background had a significantly minor phenotype. In particular, the 129/SvJ background could almost completely rescue the polycystic kidney disease phenotype of the Bcl-2 deficiency, resulting in normal renal functions. These observations would be consistent with the assumption that the C57BL/6 background is more pro-death while the 129/SvJ background is more pro-survival. Concurrent deletion of Bid, a BH3-only molecule, in either genetic background, could significantly increase the birth rate of the Bcl-2 deficient progenies and lessen lymphocytopenia, although the double knockout mice still developed the polycystic kidney diseases. Overall, our work indicates that the phenotype of Bcl-2 deficiency can be affected by multiple genetic elements, resulting in tissue-specific modulations of the cell death program during development and cellular homeostasis.