Kim C. J., Um S. J., Kim T. Y., Kim E. J., Park T. C., Kim S. J., Namkoong S. E., Park J. S.
Division of Gynecologic Oncology, Department of Obstetrics & Gynecology, Catholic University Medical College, Catholic Cancer Center, Seoul, Korea.
Int J Gynecol Cancer. 2000 Mar;10(2):157-164. doi: 10.1046/j.1525-1438.2000.00016.x.
Human papillomavirus (HPV) infection is known as the major cause of the development of cervical cancer. The E6 and E7 proteins of oncogenic HPV can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, it has been determined that long-term use of oral contraceptives may be a risk factor for cervical cancer. Investigation of the estrogenic and antiestrogenic effects on the proliferation of cervical cancer cells and the gene expression of HPV would help to explain the role of estrogen in the HPV-associated pathogenesis of cervical cancer. In this study, cervical cancer cells (HeLa, CaSki, and C33A) were cultured in vitro in the presence of 17beta-estradiol or tamoxifen to observe their regulatory growth effect and HPV E6/E7 gene expression. The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient transfection assay, which was conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. The supplemental effect of estrogen receptors on URR promoter activity was also evaluated. The proliferation of HeLa and CaSki cells was stimulated by estradiol at physiologic concentration levels (</=1 x 10-6 M). At a low concentration (0.1 x 10-6 M), tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, the proliferation of C33A was not influenced by exogenous estradiol or tamoxifen, indicating that HPV might play a role in the hormonal stimulation of cell growth. Interestingly, the proliferation of HeLa was markedly suppressed at high concentrations of estradiol and tamoxifen (5 and 10 x 10-6 M). The levels of HPV-18 E6 and E7 mRNA were significantly increased by estradiol at a concentration of 0.5 x 10-6 M. Transient transfection experiments using the HPV URR-CAT reporter plasmid in C33A cells indicated that the expression of HPV E6/E7 genes was increased by the treatment of estradiol and tamoxifen. Co-transfection of estrogen receptors (ER) and URR-CAT leads to a fourfold increase in CAT activity by estradiol or tamoxifen at physiologic concentrations. When estradiol or tamoxifen was administered at high concentrations (5 x 10-6 M), a DNA ladder, typically indicative of apoptosis, was observed in HeLa cells. In conclusion, estradiol stimulated the growth of HPV-positive cervical cancer cells, as did tamoxifen at low concentrations (0.1 x 10-6 M). The growth stimulation of HPV-positive cervical cancer cells by estrogen appeared to be related to the increased expression of HPV E6/E7. Growth suppression observed at high concentrations of estradiol and tamoxifen in HeLa cells might be a result of apoptosis. Taken together, these data suggested that exogenous estradiol might be a risk factor in HPV-mediated cervical carcinogenesis.
人乳头瘤病毒（HPV）感染是宫颈癌发生发展的主要原因。致癌性HPV的E6和E7蛋白在宫颈上皮细胞的永生化和恶性转化中起关键作用。根据以往的流行病学数据，已确定长期使用口服避孕药可能是宫颈癌的一个危险因素。研究雌激素和抗雌激素对宫颈癌细胞增殖及HPV基因表达的影响，将有助于解释雌激素在HPV相关宫颈癌发病机制中的作用。在本研究中，将宫颈癌细胞（HeLa、CaSki和C33A）在17β-雌二醇或他莫昔芬存在的情况下进行体外培养，以观察它们对细胞生长的调节作用以及HPV E6/E7基因的表达。使用HPV-18 URR-CAT报告质粒在C33A细胞中进行瞬时转染试验，进一步证实了雌激素对HPV上游调节区（URR）启动子活性的影响。同时评估了雌激素受体对URR启动子活性的补充作用。生理浓度水平（≤1×10-6 M）的雌二醇刺激HeLa和CaSki细胞的增殖。低浓度（0.1×10-6 M）时，他莫昔芬也刺激HeLa和CaSki细胞的增殖。与HPV阳性宫颈细胞不同，C33A细胞的增殖不受外源性雌二醇或他莫昔芬的影响，这表明HPV可能在激素刺激细胞生长中起作用。有趣的是，高浓度（5和10×10-6 M）的雌二醇和他莫昔芬显著抑制HeLa细胞的增殖。浓度为0.5×10-6 M的雌二醇可使HPV-18 E6和E7 mRNA水平显著升高。在C33A细胞中使用HPV URR-CAT报告质粒进行的瞬时转染实验表明，雌二醇和他莫昔芬处理可增加HPV E6/E7基因的表达。雌激素受体（ER）与URR-CAT共转染导致生理浓度的雌二醇或他莫昔芬使CAT活性增加四倍。当高浓度（5×10-6 M）给予雌二醇或他莫昔芬时，在HeLa细胞中观察到典型的凋亡DNA梯带。总之，雌二醇刺激HPV阳性宫颈癌细胞的生长，低浓度（0.1×10-6 M）的他莫昔芬也有同样作用。雌激素对HPV阳性宫颈癌细胞生长的刺激作用似乎与HPV E6/E7表达增加有关。在HeLa细胞中高浓度雌二醇和他莫昔芬观察到的生长抑制可能是凋亡的结果。综上所述，这些数据表明外源性雌二醇可能是HPV介导的宫颈癌发生的一个危险因素。
