The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to downregulate TNF-␣-induced activation of NF-B. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-␣, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-␣, but nottoetoposide,indicatingthatthephysiologicalantiapoptotic role of this protein is specific to death receptormediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BIDinduced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein.Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (Po2.2eÀ16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

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This is the first study of the role of BRE in embryonic development using early chick embryos. BRE is expressed in the developing neural tube, neural crest cells, and somites. BRE thus plays an important role in regulating neurogenesis and indirectly somitogenesis during early chick embryo development.