E6 and E7 Gene Silencing and Transformed Phenotype of Human Papillomavirus 16-Positive Oropharyngeal Cancer Cells

Abstract

The E6 and E7 genes of human papillomavirus type 16 (HPV16) encode oncoproteins that bind and degrade p53 and retinoblastoma (pRb) tumor suppressors, respectively. We examined the effects of repressing E6 and E7 oncogene expression on the transformed phenotype of HPV16-positive oropharyngeal cancer cell lines. Human oropharyngeal squamous cell cancer 147T and 090 (harboring integrated HPV16 DNA) and 040T (HPV DNA-negative) cells were infected with retroviruses that expressed a short hairpin RNA (shRNA) targeting the HPV16 E6 and E7 genes or a scrambled-sequence control shRNA. Flow cytometry, terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay, and immunoblotting for annexin V were used to assess apoptosis in shRNA-infected cell lines. Biochemical analysis involved quantitative real-time polymerase chain reaction analysis of p53- and pRb-target gene expression and immunoblotting for p53 and pRb protein expression. In 147T and 090 cells, shRNA-mediated inhibition of HPV16 E6 and E7 expression reduced the E6 and E7 mRNA levels by more than 85% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in restoration of p53 and pRB protein expression, increased expression of p53-target genes (p21 and FAS), decreased expression of genes whose expression is increased in the absence of functional pRb (DEK and B-MYB), and induced substantial apoptosis in 147T and 090 cells compared with the control shRNA-infected cells (from 13.4% in uninfected to 84.3% in infected 147T cells and from 3.3% in uninfected to 71.2% in infected 090 cells). Repression of E6 and E7 oncogenes results in restoration of p53 and pRb suppressor pathways and induced apoptosis in HPV16-positive oropharyngeal squamous cell cancer cell lines.