Journal of the National Cancer Institute Advance Access originally published online on March 10, 2009
JNCI Journal of the National Cancer Institute 2009 101(6):412-423; doi:10.1093/jnci/djp017
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© The Author 2009. Published by Oxford University Press.
ARTICLES |
E6 and E7 Gene Silencing and Transformed Phenotype of Human Papillomavirus 16–Positive Oropharyngeal Cancer Cells
Affiliations of authors: Department of Medical Oncology (TR, PW, AP) and Department of Otolaryngology (CS, PW), Yale University School of Medicine, New Haven, CT
Correspondence to: Amanda Psyrri, MD, PhD, Yale University School of Medicine, New Haven, CT 06520 (dp237{at}email.med.yale.edu or psyrri237{at}yahoo.com).
Background: 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.
Methods: 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.
Results: 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).
Conclusion: 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.
| CONTEXT AND CAVEATS Prior knowledge Epidemiological and molecular pathology data have suggested that human papillomavirus type 16 (HPV16) is associated with a subset of oropharyngeal cancers, but experimental data showing a causal association between transcriptionally active HPV16 and HPV16-positive oropharyngeal squamous cell carcinomas are lacking. Study design Retrovirus-mediated delivery of short hairpin RNAs (shRNAs) targeting the HPV16 E6 and E7 oncogenes was used to examine the effect of decreased E6 and E7 oncogene expression on cell survival and the p53 and Rb tumor suppressor pathways in HPV16-positive and HPV16-negative human oropharyngeal cancer cell lines. Contribution shRNA-mediated repression of HPV16 E6 and E7 oncogene expression resulted in the activation of the p53 and pRb tumor suppressor pathways and in the induction of apoptosis in HPV16-positive oropharyngeal cancer cell lines. Implications Continuous expression of HPV E6 and E7 is required to maintain the proliferative state and to prevent apoptosis in HPV16-positive human oropharyngeal cancer cells. Limitations The study was limited to in vitro experimental data. Experimental evidence in an animal model will ultimately be needed to prove a causal association. From the Editors
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Manuscript received July 7, 2008; revised December 19, 2008; accepted January 13, 2009.
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J Natl Cancer Inst 2009 101: 361.
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  C. H. Chung and M. L. Gillison Human Papillomavirus in Head and Neck Cancer: Its Role in Pathogenesis and Clinical Implications Clin. Cancer Res., November 15, 2009; 15(22): 6758 - 6762. [Abstract] [Full Text] [PDF]
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