Akerman G S, Tolleson W H, Brown K L, Zyzak L L, Mourateva E, Engin T S, Basaraba A, Coker A L, Creek K E, Pirisi L
Department of Pathology, University of South Carolina School of Medicine, Columbia 29208, USA.
Cancer Res. 2001 May 1;61(9):3837-43.
Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC:
用全长人乳头瘤病毒16型（HPV16）DNA永生化的人角质形成细胞（HKc）（HKc/HPV16）中，表皮生长因子受体（EGFR）水平显著升高，但EGFR水平的升高实际上先于永生化过程。在一些正常HKc株系中，来自LXSN逆转录病毒载体的HPV16 E6（而非HPV16 E5、HPV16 E7或HPV6 E6）的急性表达使EGFR mRNA水平在感染后24小时即可检测到升高，并在长达10天内保持稳定。然而，我们分析的约一半个体正常HKc株系尽管E6表达强劲且p53降解，但对E6诱导EGFR mRNA无反应。正常HKc株系对E6的反应性与初始EGFR水平呈负相关：虽然基础EGFR水平相对较低的HKc株系生长不佳且难以耐受感染方案，但它们对E6的反应是EGFR mRNA和蛋白增加以及强劲增殖。然而，那些基础EGFR水平高的HKc株系生长良好，但对E6的反应不是EGFR水平升高或增殖。对石蜡包埋的包皮组织进行EGFR免疫染色证实，HKC中EGFR表达存在个体间固有变异性：这些结果促使我们研究EGFR在正常HKC中过表达的影响：用表达全长人EGFR cDNA的LXSN逆转录病毒感染正常HKc导致生长速率显著降低和寿命缩短。虽然单独急性表达（感染后1 - 10天）HPV16 E7不会诱导EGFR，但E6和E7一起急性表达会使单独对E6无反应的正常HKc中的EGFR水平升高。此外，单独感染E7的HKc在延长寿命的早期阶段（感染后第9代）表达的EGFR水平升高，并且单独由HPV16 E7永生化的HKc表达的EGFR水平与E6/E7永生化细胞相当。这些结果支持EGFR在HPV16介导的HKC转化中起关键作用：此外，这些数据表明正常HKc不能耐受过高的EGFR水平/信号传导，为了使HKc被HPV16永生化，必须克服这种不耐受性。我们得出结论，E6和E7都有助于提高EGFR水平，但机制不同：虽然E6可以提高EGFR水平，但它不能克服正常HKc对过高EGFR信号传导的抗性。另一方面，单独不会急性增加EGFR mRNA或蛋白的E7允许在正常HKC中过度表达EGFR。
