Jia L, Wang X W, Harris C C
Laboratory of Human Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Int J Cancer. 1999 Mar 15;80(6):875-9. doi: 10.1002/(sici)1097-0215(19990315)80:6<875::aid-ijc13>3.0.co;2-z.
Human hepatitis B virus (HBV) is a major risk factor of human hepatocellular carcinoma. Both in vivo and in vitro studies have shown that HBV X protein (HBx) can bind to the p53 tumor-suppressor protein and interfere with the role that p53 plays in the cellular response to DNA damage. Our previous work has shown that HBx protein inhibits p53 sequence-specific transcriptional activation, p53-mediated apoptosis and p53 binding to the TFIIH transcription-nucleotide excision repair (NER) factors, including XPB and XPD. To investigate whether HBx interferes with the NER pathway, we utilized cell-proliferation and colony-formation assays to determine if cells expressing HBx are more sensitive to UVC-induced DNA damage. NER was also measured by a plasmid host cell re-activation assay using a vector containing a luciferase reporter gene. UV-irradiated plasmids were transfected into a human RKO colon carcinoma cell line that contains wild-type (wt) p53 as well as its derivatives, either mutant p53-143ala (RKO-143ala) or human papillomavirus E6 (RKO-E6, a wt p53 protein that is rapidly degraded and non-functional). We found that cells expressing HBx are more sensitive to UVC-induced killing. Moreover, expression of HBx resulted in a reduction of NER efficiency in RKO cells to 52 +/- 2% (compared with control), RKO-143a1a cells to 46 +/- 3% and RKO-E6 cells to 60 +/- 3%. Similar results were also obtained with a HepG2 hepatoblastoma cell line carrying wt p53. In addition, we found that HBx bound directly to either XPB or XPD DNA helicase in vitro. Thus, our data indicate that HBx may interfere with the NER pathway through both p53-dependent and p53-independent mechanisms. Because HBx binds to TFIIH-associated proteins, we propose that HBx may interfere with the NER pathway also through binding to and altering the activities of helicases necessary for NER and, thereby, increase the mutation rate induced by chemical carcinogens, such as aflatoxin B1, during human liver carcinogenesis.
人类乙型肝炎病毒（HBV）是人类肝细胞癌的主要危险因素。体内和体外研究均表明，HBV X蛋白（HBx）可与p53肿瘤抑制蛋白结合，并干扰p53在细胞对DNA损伤反应中所起的作用。我们之前的研究表明，HBx蛋白可抑制p53序列特异性转录激活、p53介导的细胞凋亡以及p53与TFIIH转录-核苷酸切除修复（NER）因子（包括XPB和XPD）的结合。为了研究HBx是否干扰NER途径，我们利用细胞增殖和集落形成试验来确定表达HBx的细胞对紫外线C（UVC）诱导的DNA损伤是否更敏感。还通过使用含有荧光素酶报告基因的载体进行质粒宿主细胞再激活试验来检测NER。将紫外线照射的质粒转染到含有野生型（wt）p53及其衍生物（突变型p53-143ala（RKO-143ala）或人乳头瘤病毒E6（RKO-E6，一种迅速降解且无功能的wt p53蛋白））的人RKO结肠癌细胞系中。我们发现，表达HBx的细胞对UVC诱导的杀伤更敏感。此外，HBx的表达导致RKO细胞中的NER效率降低至52±2%（与对照相比），RKO-143a1a细胞降低至46±3%，RKO-E6细胞降低至60±3%。在携带wt p53的HepG2肝母细胞瘤细胞系中也获得了类似结果。此外，我们发现HBx在体外可直接与XPB或XPD DNA解旋酶结合。因此，我们的数据表明，HBx可能通过p53依赖性和p53非依赖性机制干扰NER途径。由于HBx与TFIIH相关蛋白结合，我们提出，HBx也可能通过结合并改变NER所需解旋酶的活性来干扰NER途径，从而增加黄曲霉毒素B1等化学致癌物在人类肝癌发生过程中诱导的突变率。
