Detection of Human Papillomavirus Type 18 E7 Oncoprotein in Cervical Smears: a Feasibility Study

Abstract

Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6-to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears. P ersistent infections by human papillomaviruses (HPVs) are the main etiologic factor for cervical cancer (41). Approximately 85% of cervical cancers are squamous cell carcinomas (SCCs) which arise from dividing keratinocytes in the squamous epithelium of the ectocervix and 15% are adenocarcinomas (ACs) which arise from glandular cells located in the endocervix (3, 25). About 40 HPV genotypes that can infect epithelial squamous and glandular cells in the cervical mucosa have been described. On the basis of epidemiological and biochemical data, only a subgroup of HPV types, referred to as high-risk HPVs, is associated with intraepithelial lesions that have a high potential for progression to invasive carcinoma. Infections by high-risk HPV genotypes have been detected in virtually all cervical cancers (37). At least 15 highrisk HPV types have been associated with these cancers. HPV type 16 (HPV16) and HPV18 are the most prevalent genotypes worldwide in SCCs as well as in ACs (23).A persistent infection with oncogenic HPVs is necessary for the development of cervical precancer and cancer (3, 37, 40, 41). Initial events of cervical carcinogenesis after viral infection by highrisk HPV types are specific changes that overcome the transcriptional control of viral gene expression in the infected keratinocytes (40). Inactivation of these cellular control functions permits deregulated transcription of the early viral genes E6 and E7. This event triggers reprogramming of cell proliferation, apoptosis, differentiation, metabolism, epigenetic reorganization, and genomic instability (21). These changes can support the integration ...

Depletion of the cdk Inhibitor p16 INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells

“…HPV-18 E7 oncoprotein expression was determined by Western blotting, using monoclonal rabbit anti-HPV-18 E7 antibodies (clone 143-7) (19). These experiments revealed that depletion of p16…”

“…Equal amounts of protein were subjected to SDS-PAGE (12.5% acrylamide) and blotted onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% skim milk in PBS-0.1% Tween 20 and incubated overnight with the following primary antibodies: rabbit polyclonal antiserum to HPV-16 E7 (5), monoclonal rabbit anti-HPV-18E7/anti-HPV-45E7 antibodies (clone 143-7 [19]), anti-human p16…”

A New Sandwich ELISA Test Simultaneously Detecting E7 Proteins of HPV-16, 18 and 45 in Cervical Smears

“…New technologies for cervical cancer screening are urgently needed. Previous studies suggest that high-risk HPV E7 proteins are regularly expressed in cervical carcinoma and in their high-grade precursor lesions, suggesting that high-risk E7 oncoproteins are necessary for this cancer and may serve as new tumor biomarkers [10,[17][18][19][20]. In the present study, we developed and validated a new ELISA test capable for the simultaneous detection of the E7 proteins of HPV-16, HPV-18, and HPV-45.…”

“…The generation of rabbit monoclonal antibodies (RabMAbs) 42-3 and 143-7, as well as polyclonal goat antisera to HPV-16 E7 (Goat 1) and HPV-18 E7 (Goat 2), was described previously [17,20]. Increasing amounts of the E7 proteins encoded by HPV-16, HPV-18, HPV-45 and HPV-11 (low risk) were added in 100 µl coating buffer (0.1 M NaHCO 3 , pH 9.6) to each well of a 96-well plate (Maxisorp F, Nunc, Vienna, Austria) and incubated overnight at 4°C.…”

“…ELISA plates were coated with increasing concentrations of recombinant E7 proteins, and incubated with increasing amounts of purified RabMAbs 42-3 [17] and 143-7 [20], respectively. The bound rabbit monoclonal antibodies were detected by incubation with HRP-conjugated anti-rabbit-antibodies, incubated with substrate and quantitated by a Plate reader.…”