Mi Y, Borger D R, Fernandes P R, Pirisi L, Creek K E
Children's Cancer Research Laboratory, University of South Carolina, Columbia, SC 29208, USA.
Virology. 2000 May 10;270(2):408-16. doi: 10.1006/viro.2000.0283.
Human keratinocytes (HKc) immortalized by human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through growth factor-independent (HKc/GFI) and differentiation-resistant stages (HKc/DR). This progression is associated with a loss of sensitivity to growth inhibition by both all-trans-retinoic acid (RA) and transforming growth factor-beta (TGF-beta). In the accompanying article (Borger et al., 2000, Virology 270, 397-407), we demonstrate that RA resistance in HKc/HPV16 arises despite functional nuclear retinoid receptors and that TGF-beta mediates growth inhibition by RA. To investigate the basis for the loss of TGF-beta sensitivity during in vitro progression of HKc/HPV16, we explored the expression of TGF-beta receptors type I and type II in independently derived HKc/HPV16 lines and their corresponding HKc/GFI and HKc/DR derivatives. While TGF-beta receptor type II mRNA levels were unchanged during progression, mRNA levels for TGF-beta receptor type I decreased dramatically as the cells became TGF-beta resistant. At the HKc/DR stage, loss of TGF-beta receptor type I mRNA, compared to low-passage cells, ranged from 55 to 87% in four HKc/HPV16 lines examined. Immunohistochemistry, using anti-TGF-beta receptor type I antibodies, confirmed a loss of TGF-beta receptor type I expression in HKc/DR. Reintroduction of the TGF-beta-receptor type I into TGF-beta-resistant HKc/DR completely restored growth inhibition by TGF-beta. Southern blot analysis of DNA extracted from normal HKc, HKc/HPV16, and HKc/DR ruled out any gross changes in the TGF-beta receptor type I gene. The activity of the TGF-beta receptor type I promoter, cloned upstream of a luciferase reporter gene, was decreased in HKc/DR, to an extent comparable to the decrease in mRNA levels for the TGF-beta receptor type I. Thus, TGF-beta resistance at late stages of HPV16-mediated transformation of HKc is the result of a loss of expression of TGF-beta receptor type I.
由人乳头瘤病毒16型DNA永生化的人角质形成细胞（HKc/HPV16）通过不依赖生长因子（HKc/GFI）和抗分化阶段（HKc/DR）向恶性转化发展。这种进展与对全反式维甲酸（RA）和转化生长因子-β（TGF-β）介导的生长抑制敏感性丧失有关。在随附文章（Borger等人，2000年，《病毒学》270卷，397 - 407页）中，我们证明尽管存在功能性核视黄酸受体，HKc/HPV16中仍出现RA抗性，并且TGF-β介导RA对生长的抑制作用。为了研究HKc/HPV16体外进展过程中TGF-β敏感性丧失的基础，我们探讨了独立衍生的HKc/HPV16细胞系及其相应的HKc/GFI和HKc/DR衍生物中I型和II型TGF-β受体的表达。虽然在进展过程中II型TGF-β受体mRNA水平未发生变化，但随着细胞对TGF-β产生抗性，I型TGF-β受体的mRNA水平急剧下降。在HKc/DR阶段，与低代细胞相比，在所检测的四个HKc/HPV16细胞系中，I型TGF-β受体mRNA的缺失范围为55%至87%。使用抗I型TGF-β受体抗体的免疫组织化学证实了HKc/DR中I型TGF-β受体表达的缺失。将I型TGF-β受体重新导入对TGF-β耐药的HKc/DR中，完全恢复了TGF-β对生长的抑制作用。对从正常HKc、HKc/HPV16和HKc/DR中提取的DNA进行Southern印迹分析，排除了I型TGF-β受体基因的任何重大变化。克隆到荧光素酶报告基因上游的I型TGF-β受体启动子的活性在HKc/DR中降低，其程度与I型TGF-β受体mRNA水平的降低程度相当。因此，HKc在HPV16介导的转化后期对TGF-β的抗性是I型TGF-β受体表达缺失的结果。
