Pirisi L, Batova A, Jenkins G R, Hodam J R, Creek K E
Department of Pathology, University of South Carolina School of Medicine, Columbia 29208.
Cancer Res. 1992 Jan 1;52(1):187-93.
Human papillomavirus (HPV) type 16 (HPV16) is associated with a large percentage of cervical malignancies, and HPV16 DNA can immortalize human keratinocytes in vitro. The transforming ability of the virus resides primarily in the open reading frames E6 and E7. Retinoids are potent modulators of growth and differentiation of keratinocytes and have been shown to reverse cervical lesions resulting from HPV infection. We compared the sensitivity of normal human foreskin keratinocytes (HKc) and four immortalized HKc lines, independently obtained by transfection of different normal HKc strains with HPV16 DNA (HKc/HPV16), to growth control by retinoic acid (RA). All the HKc/HPV16 lines were 10- to 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc, while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. In addition, HKc/HPV16 lines were more sensitive than normal HKc to modulation of keratin expression by RA and retinol. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Dot blot analysis of RNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 open reading frames E6 and E7 is reduced 2- to 4-fold by RA. In addition, Northern blot analysis demonstrated that RA inhibition of E6 and E7 expression was both dose and time dependent. Overall, these results suggest that the increased sensitivity of the HKc/HPV16 lines to growth control by RA may be mediated by an inhibition of the expression of HPV16 gene products which are required for the maintenance of continuous growth.
16型人乳头瘤病毒（HPV16）与很大比例的宫颈恶性肿瘤相关，并且HPV16 DNA可在体外使人类角质形成细胞永生化。该病毒的转化能力主要存在于开放阅读框E6和E7中。维甲酸是角质形成细胞生长和分化的有效调节剂，并且已显示可逆转由HPV感染引起的宫颈病变。我们比较了正常人包皮角质形成细胞（HKc）和通过用HPV16 DNA转染不同正常HKc菌株独立获得的四个永生化HKc系（HKc/HPV16）对维甲酸（RA）生长控制的敏感性。在克隆和大规模培养生长试验中，所有HKc/HPV16系对RA生长抑制的敏感性均比正常HKc高10至100倍。RA的前体视黄醇也被发现是比正常HKc更有效的HKc/HPV16生长抑制剂，而β-胡萝卜素既不抑制正常HKc也不抑制HKc/HPV16的生长。此外，HKc/HPV16系比正常HKc对RA和视黄醇对角蛋白表达的调节更敏感。在正常HKc和HKc/HPV16之间未观察到[3H]RA或[3H]视黄醇摄取率的差异。对在不存在或存在10（-7）M RA的情况下培养的HKc/HPV16提取的RNA进行斑点印迹分析表明，RA使HPV16开放阅读框E6和E7的表达降低2至4倍。此外，Northern印迹分析表明RA对E6和E7表达的抑制是剂量和时间依赖性的。总体而言，这些结果表明HKc/HPV16系对RA生长控制的敏感性增加可能是由对维持持续生长所需的HPV16基因产物表达的抑制介导的。
