Abstract
Objective: The aim of our work was to identify by Western Blot technique (WB) the oncoproteins generated by E6 and E7 genes of high risk Human Papillomavirus (HR-HPV), resulting from integration of viral genomes into cervical cell DNA and their interactions with tumor suppressor human proteins p53 and pRb in premalignant or malignant cervical lesions. Methods: The study was performed on 2,500 women from Caserta Local Health Authority who underwent cervical cytology from June 2010 to September 2011. Informed consent of participants was obtained. The cell samples taken by brushing were stored in liquid based cytology (LBC) and in physiological solution for WB analysis. Cytology diagnoses was based on 2001 Bethesda classification. Proteomic research was performed according to size after extraction and SDS (Sodium Dodecyl Sulfate) electrophoresis. Proteins of interest were detected by primary monoclonal antibodies and WB analysis. Results: Cytology results of 2,500 women were positive for abnormalities of epithelial cells in 3.1%. Atypical squamous cells of undetermined significance (ASC-US) were 1.3%, Low Squamous Intraepithelial Lesion (L-SIL) 1.7% and High Squamous Intraepithelial Lesion (H-SIL) or worse 0.2%. The integration of E6 and E7 genes with their oncoproteins expression were found in 3.1% of ASC-US and in all cases of SIL or worse. On the other hand positive interactions of E6/p53 proteins and E7/pRb were found in 4.8% of L-SIL, in 75.0% of H-SIL or worse and in no ASC-US. Conclusions: Results show that the integration of viral genomes (E6 and E7) is present in all H-SIL or worse and in several L-SIL, but only the most advanced lesions, histologically confirmed, have shown the E6/p53 interaction and E7/pRb. Our protocol allows a selection of women with advanced lesions toward cancer to obtain appropriate management.
Highlights
Human Papillomavirus (HPV) are the primary cause of cervix cancer, involved in 90% of all cases [1,2]
The aim of our work is to identify the protein products of E6 genes and E7 that are detected from integrated phase HPV, and activation between E6 proteins and E7 respectively with the p53 proteins and pRb on cytological diagnoses of Atypical squamous cells of undetermined significance (ASC-US), LSIL and HSIL or worse, using liquid based cytology (LBC) and Western Blot technique (WB) technique
The integration of E6 genes and E7 with their oncoproteins expression were found in 2/32 ASC-US (3.1%); in 42/42 Low Squamous Intraepithelial Lesion (L-SIL) (100%) and in all 4 cases of High Squamous Intraepithelial Lesion (H-SIL) or worse (100%)
Summary
Human Papillomavirus (HPV) are the primary cause of cervix cancer, involved in 90% of all cases [1,2]. Invasive Cervical Cancer (ICC) alters pathways involved in cell cycle control, interacting with and neutralizing the regulatory functions of two important tumor suppresser proteins, p53 and pRb [4]. Two early HPV genes, E6 and E7, are known to play a crucial role in tumorigenesis. Studies both in vitro and in vivo show that the function of E6 proteins and E7, of the “high risk HPV types”, are essential for neoplastic cellular transformation. When low grade lesion occurs HPV DNA penetrates into host nuclei and is present in episomal state [4,5] and E6 expression and E7 is regulated by viral gene and by host cell
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