Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade
Introduction
Specific genotypes of the human papillomavirus (HPV) are known to be the principal cause of cervical squamous intraepithelial lesions (SILs) and cervical cancer [1]. To date, more than 100 different HPV types have been characterized and, based on their potential to induce cervical epithelial malignant transformation, have been classified as low-risk (LR) and high-risk (HR) viruses [2], [3]. The entry of HPV into cervical host cells may be followed by three events: (1) viral DNA is maintained as an episome, thereby establishing a latent infection; (2) conversion of latent into productive infection is associated with the assembly of complete infective virions; (3) viral DNA is integrated into the host genome. It is now generally accepted that the integration of HPV DNA into the host genome and subsequent constitutive expression of the oncoproteins E6 and E7 represent two activation mechanisms for the progression of preinvasive lesions to cervical carcinoma [4], [5]. Integration of viral genomic sequences can cause deletions and/or a disruption of the E2 gene and result in a loss of its function as a regulator of viral gene expression [5]. This event is often followed by up-regulation of E6/E7 gene transcription. E6 and E7 proteins deregulate cell-cycle control through interaction with different cell proteins, for example, tumor suppressor gene products such as p53 and retinoblastoma protein (Rb), thereby initiating the transformation and immortalization of HPV-infected cells [6], [7].
Various studies have demonstrated the presence of integrated HPV16 and 18 genomes in the vast majority of cancers and in cell lines isolated from cervical malignancies [8], [9], [10], [11], [12], [13]. However, the role of the integration of viral DNA into the host genome as a major event leading to malignant transformation of dysplastic cervical epithelium is still discussed, as there have been several studies reporting only the presence of episomal forms of HPV16 DNA in preneoplastic and neoplastic cervical lesions. For example, Matsukura et al. [14] detected exclusively episomal forms of HPV16 DNA in infected cervical epithelium, albeit this form was present in at least 70% of investigated cervical malignancies. Similarly, Fuchs et al. [15] and Das et al. [16] reported the presence of viral HPV16 DNA in up to 30% of cervical carcinomas, and again, viral DNA was exclusively present in the episomal form.
To elucidate the role of HPV6, 11, 16, 18, 31, 33, 52b, 58 DNA integration and mRNA expression patterns in the transformational process of cervical epithelium in benign and dysplastic/preinvasive lesions of the uterine cervix, we used polymerase chain reaction (PCR) and bi-dimensional (2D)-gel electrophoresis to analyze the type-specific integration patterns of viral DNA. In addition, mRNA expression of E6/E7 genes was evaluated by a reverse transcriptase (RT)-PCR and correlated with HPV DNA integration.
We established an RT-PCR test (amplification of papillomavirus oncogene transcripts) that allows discrimination of HPV E6/E7 mRNAs derived from integrated and/or episomal viral genomes. In combination with virus load and Northern blot in certain cases, we defined and confirmed the prevalence of integrate-derived HR-HPV16, 18, 31, 33, 52b and 58 transcripts in cervical swabs from patients with CIN lesions in various stages of progression.
Section snippets
Specimens and patient population
Clinical samples obtained from 275 patients attending our clinic between February 1997 and November 2001 were considered suitable for the study. The ethics committee of the University of Vienna approved this study. Only women who had signed the informed consent form were enrolled. The study population was restricted to women of Caucasian origin undergoing their biennial or triennial routine cytological analysis and/or patients referred to our clinic for colposcopy because of an abnormal
Presence and typing of HPV DNA
To determine the physical state and expression pattern of HPV DNA in preneoplastic and neoplastic cervical lesions, clinical specimens were collected from 275 patients enrolled in the study. Swab specimens were tested for the presence of HPV infection by the HC II test. As shown in Table 1, among the total HPV DNA, the LR- and HR-HPV types were found in 148 patients (53.8%). The HPV detection rate ranged from 27.5% (28 of 102 patients) in women with normal Pap smears (within normal limits, or
Discussion
In the present study, we established a method to detect the physical state and the mRNA expression pattern of viral genomes of several LR- and HR-HPV types in benign and premalignant/preinvasive clinical samples obtained from 78 out of 275 patients. The persistence and progression of dysplastic cervical lesions are influenced by several factors such as lesion severity [21], type of HPV present in the epithelial cells [22], [23], the prevalence and amount of E6/E7 transcripts detected in
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2021, Experimental and Molecular PathologyCitation Excerpt :As the HPV E2 protein functions as a repressor for the expression of the E6 and E7 oncogenes, a disruption of the E2 gene during viral integration further elevates the E6 and E7 gene expression, resulting in a change in the metabolism of the host cell favoring neoplastic development (Ault, 2006). Previous studies indicate that integration of HPV into the host genome is correlated with an increased risk of developing cervical cancer, suggesting that viral integration may be a potential marker for preneoplastic disease progression (Hudelist et al., 2004; Cricca et al., 2007). Several studies have assessed the viral integration status by calculating an E2/E6 ratio to determine whether the viral genome is integrated (ratio < 1) or exists as an episome (ratio ~ 1)(Cricca et al., 2007; Boulet et al., 2009).