Hepatitis B virus (HBV) X protein activates many viral and cellular genes in trans and functional disruption of the p53 tumor suppressor gene product occurs when X protein is transiently expressed in the cytoplasm of cultured cells. We have carried out investigations to determine the exact location of X protein in X gene transfected cells by using a¯uorescent staining technique as well as by biochemical analyses. Aggregation of mitochondrial structures became evident at the periphery of nucleus in the cytoplasm of X transfected cells. X protein was found associated with the aggregated mitochondrial structures. Furthermore, transiently expressed p53 protein co-localized with X protein in X transfected cells. However, the appearance of aggregated mitochondrial structures at the nuclear periphery was independent of the presence of p53 protein in X transfected cells. X protein expression also caused an appearance of TUNEL positive nucleus, cytochrome c release from mitochondrial, the decrease of mitochondrial membrane potential and the membrane blebbing of X transfected cells, which are characteristic of cell death. Our data suggest that X protein causes an abnormal aggregation of mitochondrial structures in the cell, which may be eventually connected with cell death.
It has been suggested that hepatitis B virus (HBV) X gene activates X gene expression by disrupting the function of p53 tumor suppressor gene (Takada et al., 1996). To ®nd out their connection, eect of X protein expression on the nuclear localization of p53 protein in human hepatoma cells was examined by the immunouorescent double-staining technique. The location of transiently-expressed p53 protein was examined in X gene-transfected cells, where X protein was detected in the cytoplasm. The nuclear location of transientlyexpressed p53 protein was changed to the cytoplasm by X protein co-expression. Endogenous p53 protein was also observed in the cytoplasm by X protein expression. The transcriptional activation domain of X protein and the carboxy-terminal region of p53 protein were found mutually responsible for the cytoplasmic retention of p53 protein in X gene-transfected cells. Therefore, the cytoplasmic retention of p53 protein may be closely correlated to the function of X protein expressed in transfected cells.
Hepatitis B virus (HBV) X gene is known to exhibit a transcriptional activation function and is considered to play a major role in hepatocarcinogenesis. We determined a 20-bp promoter element for the HBV X gene transcription and found a binding protein to this promoter element, designated as an X-PBP. We then examined the effects of HBV X protein and p53 tumor suppressor gene product on X gene transcription from the 20-bp promoter element using the transient expression technique. Activity of the X gene promoter was stimulated by X protein expression, but, in contrast, was repressed by transfected normal p53 gene. On the other hand, mutant p53 gene product exhibited no repression. Moreover, the p53 repression of X gene transcription was canceled by X protein coexpression. Thus, the effects of X protein and normal p53 product appear to be mutually antagonistic in the regulation of X gene expression. However, mutated promoter elements which failed to bind to X-PBP still responded to X protein or p53, indicating that the process of X transactivation or p53 repression may be independent of X-PBP binding to the promoter element. Our data suggest that X protein could disrupt function of normal p53 protein in X gene-transfected cells.
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