Sizemore N, Choo C K, Eckert R L, Rorke E A
Department of Environmental Health Sciences, Case Western Reserve University, Cleveland, Ohio, 44106, USA.
Exp Cell Res. 1998 Oct 10;244(1):349-56. doi: 10.1006/excr.1998.4179.
We have previously demonstrated that human papillomavirus 16 (HPV16)-immortalized human ectocervical epithelial cells and cells derived from tumors which express HPV16 oncogenes express high levels of epidermal growth factor receptor (EGFR) compared to normal cervical cells. We have also shown that proliferation of these cells is inhibited by retinoid treatment. We have hypothesized that the retinoid inhibition of cell proliferation may be due to the retinoid-dependent reduction in EGFR level. In this study we examine the regulation of EGFR expression in cervical cells with emphasis on two aspects: (1) the mechanism of retinoid-dependent suppression of EGFR levels in HPV16-positive cells and (2) the mechanism of EGFR upregulation by HPV16. EGFR levels were found to be elevated 5-, 3. 7-, and 1.25-fold in the HPV16-immortalized ECE16-1, ECE16-D1, and ECE16-D2 cells, respectively, compared to normal cervical cells. Treatment of ECE16-1 and ECE16-D1 cells with retinoic acid suppresses proliferation, EGFR level, EGFR mRNA level, and EGFR promoter activity. The reduction in EGFR promoter activity appears to account for the reduction in EGFR protein and mRNA levels. In contrast, retinoic acid does not affect cell growth or EGFR level in ECE16-D2 cells or normal cervical cells. To study the mechanisms regulating EGFR expression in HPV16-positive cells, normal ECE cells were cotransfected with an EGFR promoter reporter plasmid and an expression plasmid encoding the HPV16 E6/E7 open reading frames. In the presence of E6/E7, EGFR promoter activity was increased by 2- to 3-fold, suggesting that the E6/E7 proteins are directly or indirectly responsible for the increased EGFR level and that the EGFR promoter contains the DNA elements necessary to mediate this response. Nevertheless expression of E6/E7 proteins did not confer retinoic acid regulation, as EGFR promoter activity remained elevated in normal cells cotransfected with pHPVE6/E7 and treated with retinoic acid. These results suggest that human papillomavirus and retinoic acid regulate EGFR levels by independent effects on the EGFR promoter.
我们之前已经证明，与正常宫颈细胞相比，人乳头瘤病毒16型（HPV16）永生化的人宫颈外膜上皮细胞以及源自表达HPV16癌基因的肿瘤的细胞，表达高水平的表皮生长因子受体（EGFR）。我们还表明，这些细胞的增殖受到类视黄醇处理的抑制。我们推测类视黄醇对细胞增殖的抑制作用可能是由于类视黄醇依赖性的EGFR水平降低。在本研究中，我们重点从两个方面研究宫颈细胞中EGFR表达的调控：（1）HPV16阳性细胞中类视黄醇依赖性抑制EGFR水平的机制，以及（2）HPV16上调EGFR的机制。与正常宫颈细胞相比，在HPV16永生化的ECE16 - 1、ECE16 - D1和ECE16 - D2细胞中，EGFR水平分别升高了5倍、3.7倍和1.25倍。用视黄酸处理ECE16 - 1和ECE16 - D1细胞可抑制增殖、EGFR水平、EGFR mRNA水平以及EGFR启动子活性。EGFR启动子活性的降低似乎是EGFR蛋白和mRNA水平降低的原因。相比之下，视黄酸不影响ECEI6 - D2细胞或正常宫颈细胞的细胞生长或EGFR水平。为了研究HPV16阳性细胞中调控EGFR表达的机制，将正常ECE细胞与EGFR启动子报告质粒和编码HPV16 E6/E7开放阅读框的表达质粒共转染。在存在E6/E7的情况下，EGFR启动子活性增加了2至3倍，这表明E6/E7蛋白直接或间接导致了EGFR水平的升高，并且EGFR启动子包含介导这种反应所需的DNA元件。然而，E6/E7蛋白的表达并未赋予视黄酸调控作用，因为在用视黄酸处理的同时与pHPVE6/E7共转染的正常细胞中，EGFR启动子活性仍然升高。这些结果表明，人乳头瘤病毒和视黄酸通过对EGFR启动子的独立作用来调控EGFR水平。
