Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.
Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.
复制标题
通过单独引入人乳头瘤病毒 16 型 E6 或 E7 基因，使正常人胚胎成纤维细胞永生化。
DOI：
10.1002/ijc.11219
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Tsutsui,Takeki
中科院分区：
文献类型：
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作者：
Yamamoto,Akito;Kumakura,Shin-ichi;Uchida,Minoru;Barrett,JCarl;Tsutsui,Takeki
检查了人乳头瘤病毒16型（HPV-16）E6或E7基因诱导正常人胚胎成纤维细胞WHE-7细胞永生化的能力。用编码HPV-16 E6或E7单独或E6和E7两者（E6/E7）的逆转录病毒载体感染9个群体倍增（PD）的WHE-7细胞。4个单独携带E6的分离克隆中的一个变得永生，目前处于>445 PD。4个单独携带E7的分离克隆中有4个脱离了危机，目前处于>330 PD。携带E6/E7的5个分离的克隆中的3个也是永生化的，并且目前处于>268 PD。E6基因的克隆体和E6/E7基因的克隆体表达E6蛋白，E7基因的克隆体和E6/E7基因的克隆体表达E7蛋白。表达高水平E6或E7蛋白的永生克隆分别对端粒酶活性或端粒维持的替代机制（称为ALT（端粒替代延长））呈阳性。所有的死亡或危机前克隆都是阴性的两种表型。所有永生克隆在X射线照射DNA损伤后均表现出G1期阻滞的消除。INK 4a蛋白（p16 INK 4a）的表达在E6感染的死亡和永生克隆中检测不到，而Rb蛋白（pRb）仅在永生克隆中过度磷酸化。p16 INK 4a蛋白在所有E7感染的永生克隆和它们的危机前克隆中以及所有E6/E7感染的凡人和永生克隆中过表达，但pRb表达在所有这些克隆中下调。这些结果首次证明，据我们所知，HPV-16 E6或E7单独可以诱导正常人胚胎成纤维细胞的永生化。p16 INK 4a/pRb通路的失活结合端粒维持机制的激活被认为是这些病毒癌基因使正常人胚胎成纤维细胞永生化所必需的。人类细胞对永生化的敏感性可能与细胞的分化状态有关。© 2003 Wiley利斯公司
The ability of the human papillomavirus type 16 (HPV‐16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE‐7 cells was examined. WHE‐7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV‐16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X‐ray irradiation. The expression of INK4a protein (p16INK4a) was undetectable in the E6‐infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16INK4aprotein was overexpressed in all the E7‐infected immortal clones and their clones in the pre‐crisis period as well as all the E6/E7‐infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV‐16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16INK4a/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells. © 2003 Wiley‐Liss, Inc.