Chesters P M, McCance D J
Department of Microbiology, United Medical School, Guy's Hospital, Guy's Campus, London, U.K.
J Gen Virol. 1989 Feb;70 ( Pt 2):353-65. doi: 10.1099/0022-1317-70-2-353.
Using a focus assay we have shown that the entire human papillomavirus type 16 (HPV-16) genome is capable of cooperating with an activated ras oncogene to transform secondary rat embryo fibroblast (REF) cells as indicated by focus formation of unselected cells. However, this assay failed to detect any similar activity with either the whole HPV-6 genome or with subgenomic fragments. In contrast, transformed colonies appeared when G418 (geneticin)-resistant colonies were selected after cotransfection with activated ras DNA and either the entire HPV-6 genome or subgenomic fragments containing the E6/E7 open reading frames (ORFs) of HPV-6 or HPV-16. The transformation assessment was based on the development of a ras-transformed appearance in G418-resistant colonies. The appearance of this morphology did not imply the ability of transformed cells to produce colonies in semi-solid agarose (anchorage-independent growth), and extended culture for about 10 to 20 population doublings was necessary before transfected cells exhibited anchorage-independent growth. Transformation of REF cells was not observed with the E5 ORF of HPV-16 under the control of an exogenous promoter (the long terminal repeat of Rous sarcoma virus) in cooperation with activated ras DNA. No transformation was observed using an activated myc oncogene with either HPV-6 or HPV-16 DNA.
我们使用焦点分析表明，整个人乳头瘤病毒16型（HPV-16）基因组能够与激活的ras癌基因协同作用，使原代大鼠胚胎成纤维细胞（REF）发生转化，未筛选细胞的焦点形成即表明了这一点。然而，该分析未能检测到整个HPV-6基因组或亚基因组片段具有任何类似活性。相反，在用激活的ras DNA与整个HPV-6基因组或含有HPV-6或HPV-16的E6/E7开放阅读框（ORF）的亚基因组片段共转染后，选择对G418（遗传霉素）有抗性的菌落时，出现了转化菌落。转化评估基于对G418抗性菌落中ras转化外观的观察。这种形态的出现并不意味着转化细胞能够在半固体琼脂糖中形成菌落（不依赖贴壁生长），并且在转染细胞表现出不依赖贴壁生长之前，需要进行约10至20次群体倍增时间的延长培养。在 Rous肉瘤病毒的长末端重复序列这一外源性启动子的控制下，HPV-16的E5 ORF与激活的ras DNA协同作用时，未观察到REF细胞发生转化。使用激活的myc癌基因与HPV-6或HPV-16 DNA时，也未观察到转化现象。
