Subcellular localization and quantitation of the human papillomavirus type 16 E6 oncoprotein through immunocytochemistry detection.
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.
Thunder Bay Regional Research Institute, Probe Development & Biomarker Exploration, 980 Oliver Road, Thunder Bay, Canada ON P7B 6V4. rjackso1@lakeheadu.ca
Robert
R
Jackson
Melissa
M
Togtema
Ingeborg
I
Zehbe
D000911:Antibodies, Monoclonal / Q000276:immunology; D002277:Carcinoma / Q000378:metabolism / Q000821:virology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000821:virology
D000911:Antibodies, Monoclonal; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins