Suppression ofp53 expression by RNAi induces EGF receptor levels and facilitates immortalization by HPV16 E7 in human keratinocytes.

摘要

We have previously shown that human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins cooperate to increase epidermal growth factor receptor (EGFR) levels in HPV16-transformed human keratinocytes (HKc). Experiments conducted using LXSN retroviral vectors expressing E6, E7, or E6/E7 demonstrated that while E6 expression results in an increase in EGFR levels in normal HKc, detectable as early as 24 h after infection, expression of E7 alone does not modify, acutely, EGFR levels. However, E7 is required in order for normal HKc to escape early senescence induced by EGFR overexpression. E6 interacts with many cellular components, but one of the major functions of E6 is the degradation of p53. In order to investigate the role of p53 in the EGFR mRNA and protein induction by E6, we used a p53 RNAi (pSuper.p53) vector to selectively suppress p53 expression in normal HKc in the absence of E6. Transfection with p53 RNAi alone only slightly extended the life span of normal HKc above that observed in LSXH (vector) transfected cells and no immortalized clones were obtained. p53i efficiently cooperated with E7 to induce life span extension of HKc, and immortalization (defined as continuous growth past crisis) in 7 out of 12 normal HKc strains transfected. Transfection with LXSHE7 resulted in extended life span in 2 of those individuals. EGFR protein levels increased significantly in cells with extended life span obtained with p53 RNAi alone, and to a greater extent in those obtained with p53 RNAi and E7. EGFR protein levels were found to continue to rise throughout passaging in cells co-transfected with p53 RNAi and E7. EGFR mRNA levels were also found to be significantly increased in cells with p53 RNAi and E7, with levels reaching about 2.5-fold higher than untransfected controls by passage 11. These results indicate that p53 RNAi efficiently replaces E6 to cooperate with E7 in immortalization of HKc.