Abstract
The hepatitis B virus-encoded HBx protein coactivates transcription of viral and cellular genes, and it is believed to play an important role in hepatitis B virus-related liver cancer. HBx has been shown to alter the coordinated balance between proliferation and programmed cell death, being able to either induce or block apoptosis. Here, we demonstrate for the first time that the HBx is a potent caspase 3 inhibitor. Rat fibroblasts (REV2) and hepatoma cells (Hep) synthesizing the HBx protein were resistant to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor alpha, or anti-Fas antibodies administration. In these cells, HBx prevented DNA fragmentation and cell death in the absence of de novo protein synthesis, with a similar efficiency as the competitive caspase 3 substrates inhibitors VAD-FMK and DEVD-FMK. Protein extracts obtained from the HBx positive cells contained a very low caspase activity, and addition of anti-HBx antibody restored the endogenous caspase activity. To obtain a functional map of the anti-caspase activity of HBx, various cell lines were established that synthesized either N-terminally or C-terminally truncated HBx molecules. These gene dissection experiments revealed that the regions required for the anti-caspase activity overlap with the two known transactivation domains of HBx.
Highlights
Caspase 3 (CPP32) is a member of the cysteine protease family, activating downstream substrates of the apoptosis pathway by proteolytic cleavage (e.g.: poly(ADP)-polymerase (PARP)),1 which leads to DNA fragmentation and to cell death [1]
HBx has been reported to contain two Kunitz-type serine protease inhibitor consensus sequences that overlap with its two transcription activation domains [11], implying that the HBx may prevent cell death via caspase inhibition. To test this hypothesis we established several cell lines expressing either wild type HBx or different deletion/insertion mutants and analyzed the apoptotic response of these cells upon application of different apoptotic stimuli. These experiments revealed that HBx efficiently blocks caspase 3 (CPP32) activity and apoptosis in both rat fibroblasts and hepatoma cells
REV2 cells were grown in DulbeccoЈs modified EagleЈs medium (DMEM) with 5% fetal calf serum (FCS), hepatoma cells were grown in DMEM with 10% FCS, 1 g/ml insulin, and 1 g/ml dexamethasone
Summary
Caspase 3 (CPP32) is a member of the cysteine protease family, activating downstream substrates of the apoptosis pathway by proteolytic cleavage (e.g.: poly(ADP)-polymerase (PARP)),1 which leads to DNA fragmentation and to cell death [1]. HBx prevented DNA fragmentation and cell death in the absence of de novo protein synthesis, with a similar efficiency as the competitive caspase 3 substrates inhibitors VAD-FMK and DEVD-FMK.
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