title: Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry.
abstract: The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.
authors: Department of Pathology, Georgetown University Medical School, Washington, DC, USA.
authors: Ziad
authors: Z
authors: Sahab
authors: Sawali R
authors: SR
authors: Sudarshan
authors: Xuefeng
authors: X
authors: Liu
authors: YiYu
authors: Y
authors: Zhang
authors: Alexander
authors: A
authors: Kirilyuk
authors: Christopher M
authors: CM
authors: Kamonjoh
authors: Vera
authors: V
authors: Simic
authors: Yuhai
authors: Y
authors: Dai
authors: Stephen W
authors: SW
authors: Byers
authors: John
authors: J
authors: Doorbar
authors: Frank A
authors: FA
authors: Suprynowicz
authors: Richard
authors: R
authors: Schlegel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D052162:Human papillomavirus 16 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D002583:Uterine Cervical Neoplasms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000821:virology; D000086082:Viroporin Proteins
chemical_list: D009856:Oncogene Proteins, Viral; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins