Abstract
Problem: High-risk human papillomaviruses (HPV) types 16 and 18 have been associated with the development of cervical cancer and more recently with head and neck carcinomas. HPV produces two early proteins, E6 and E7, which can disrupt the cell cycle and immortalize cells. In order to study the potential effects of other viruses on the regulation of E6 and E7 transcription within HPV-transformed cell lines, a real-time quantitative RT-PCR method was needed.
Methods: An assay for the real-time quantitative detection of HPV16-E6 and -E7 RNA using the ABR Prism 7700 (TaqMan) sequence detector was developed. In order for a single-tube multiplex comparative assay, separate fluorogenic probes were designed for the E6 and E7 regions. The RNA samples were normalized to glyceraldehyde-3-phosphate dehydrogenase, allowing for relative quantitation of HPV16-E6 and -E7 using FAM- and VIC-labeled probes, respectively. HPV-transformed SiHa cervical cells were transfected with a human herpesvirus-8 (HHV-8) expression plasmid, and RNA extractions were prepared and analyzed using the assay.
Results: This method produces reproducible quantitative results for the detection of HPV16-E6 and -E7 RNA sequences in HHV-8-transfected SiHa cells. The data suggest upregulation of HPV16-E6 and -E7 transcription 24 hours after transfection.
Conclusion: This newly developed multiplex real-time RT-PCR method allows for rapid quantitation of specific HPV16-E6 and -E7 sequences within the same RNA sample. This becomes invaluable when multiple HPV16-transformed cell lines are analyzed for the upregulation of E6 and/or E7 transcription after manipulation with HHV-8 genes.
Significance: Rapidly detecting specific E6 and E7 genomic sequences within HPV16-transformed cells may help delineate the cause for cell cycle dysregulation in some HPV16-positive oral squamous cell carcinomas. This is an important in vitro method for rapidly determining E6/E7 concentrations within HPV-transformed cells. Future studies can analyze tissue samples from patients at risk for head and neck cancer.
Support: None reported.