Subcellular localization and quantitation of the human papillomavirus type 16 E6 oncoprotein through...

Human papillomavirus (HPV) type 16 E6 is a viral oncoprotein essential for host cell transformation. Due to its role in HPV-induced cancers of the genital, head and neck epithelia, reliable protein-level determination of E6 expression would be an invaluable diagnostic tool. Immunocytochemical detection and subcellular localization of HPV16 E6 has been demonstrated with varying success and a comprehensive review of techniques is lacking. To address these issues, we used established monoclonal antibodies and optimized a standard immunocytochemical method for E6 protein detection inside the HPV16 positive cell lines, SiHa and CaSki. E6 oncoprotein was detected primarily in the nucleus. We also refined quantitative analysis with a software to objectively differentiate between HPV16 positive and negative cells. Our analysis was also able to differentiate expression differences between SiHa and CaSki on par with RT-qPCR. Thus, we provide a long-needed, robust protocol for antibody-mediated detection of the HPV16 E6 oncoprotein inside cultured cells.