| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 92 === Hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC). Hep3B is a well-differentiated human HCC cell line with one to two HBV genome equivalents per cell. One end of the HBV integration site is at chromosome 4q21 and the other end is at chromosome 13q31, both regions have been shown to have high frequency of allele loss.
To address the question of whether HBV integration was accompanied by a change in cellular chromatin, DNase I hypersensitive site mapping analyses were performed. In addition to two hypersensitive sites in HBV preS/S promoter and HBV enhancer I, we demonstrated the existence of a hypersensitive site in chromosome 4q, located approximately 10.3 kb downstream of the HBV integration site.
In Hep3B cells, because of integration, the C-terminal portion of HBx protein, i.e., amino acids 141-145, is replaced by a host sequence of 61 amino acids. To study functional difference between proteins of wild type HBx and fusion HBxt-3B, we compared their intracellular distribution. We found that wild type HBx accumulated as granula in the cytosol. In contrast, HBxt-3B fusion protein showed an overexpression and aggregation pattern in the cytosol. Furthermore, colony formation assay indicated HBx, but not of HBxt-3B, caused a substantial reduction in the number of colony in HuH-7 cell line. Moreover, the result of cell cycle analysis suggests that the effect of HBx on growth suppression was probably not due to the induction of cell cycle arrest or cell apoptosis. Thus, the HBxt-3B fusion protein has lost the growth suppressive activity. On the other hand, NF-кB, p105, a member of NF-кB inhibitor proteins, is shown to locate in chromosome 4q24, a region proposed to harbor tumor suppressor genes. Our result shows that HBxt-3B fusion protein, mediates a reduction in exogeneous p105 protein level. However, in Hep3B cells, no HBxt-3B protein could be detected by western blot using X specific antibody. We propose that at the early stage of tumor development, the undesired p105 was inactivated by HBxt-3B protein. As tumor progressing, the gene of HBxt-3B was silenced to allow p105, protein functions in regulating many important biological processes, to activate again.
|
|---|
