Hawley-Nelson P, Vousden K H, Hubbert N L, Lowy D R, Schiller J T
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD 20892.
EMBO J. 1989 Dec 1;8(12):3905-10. doi: 10.1002/j.1460-2075.1989.tb08570.x.
The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
最常与宫颈癌相关的人乳头瘤病毒类型（HPV），如HPV16，此前已有报道称其能在体外使正常人包皮角质形成细胞永生化，而主要与宫颈良性病变相关的类型则不能。在本研究中，我们确定了HPV16中负责病毒基因组永生化活性的基因。用一种质粒转染，其中E6和E7是仅有的完整开放阅读框（ORF），可诱导角质形成细胞产生无限寿命，其效率与病毒DNA的整个早期区域相似。E6E7克隆中E6或E7有失活损伤的突变体未能诱导永生化。单独转染时，E7可诱导细胞过度增殖，但这些细胞最终会衰老。E6自身无活性，将一个带有完整E6 ORF的质粒与另一个带有完整E7 ORF的质粒共转染，可产生具有无限生长潜能的角质形成细胞系。在由E6E7 DNA诱导的细胞系以及E6和E7质粒共转染产生的细胞系中均检测到了E6和E7蛋白。因此，我们得出结论，HPV16 E6和E7协同作用可在体外使人类角质形成细胞永生化。由于所有检测的角质形成细胞系均为非整倍体，因此细胞基因表达的变化可能也是永生化所必需的。从E6E7诱导的细胞系中分离出了血清和钙抗性亚系，表明其他HPV基因在产生分化抗性方面并非起必需作用。（摘要截短于250词）
