HPV16 E2 gene disruption and polymorphisms of E2 and LCR: Some significant associations with cervical cancer in Indian women
Introduction
Human papillomavirus (HPV) infection has been considered to be the major etiological agent for the development of cervical carcinoma (CaCx). HPV DNA is found in 99.7% of CaCx cases, of which 50% are HPV16 DNA-positive [1], [2], [3]. The viral genes that contribute to transformation of cells are E6 and E7. The E2 viral protein regulates transcription of these genes through its binding to sites adjacent to the promoter responsible for this transcription. Thus, an increase in expression of E2 results in repression of E6/E7 expression [4], while loss of E2 function most commonly by integration into the viral genome confirms an upregulation of E6/E7 proteins [5], [6].
The viral genome contains a region spanning 1024 base pair termed as the long control region (LCR) that regulates replication and transcription. The entire LCR is divided into three functionally distinct segments, viz., the 5′, the central and the 3′ segment. The 5′ region of the LCR has a high A + T content (up to 85%) and contains a negative regulatory element acting at the level of late mRNA stability [7] and a nuclear matrix attachment region that represses viral oncoprotein expression [8], [9]. The central segment (400 base pair) functions as an epithelial-specific transcriptional enhancer [10], [11], [12]. Multiple transcription factors have been found to bind in vitro to more than 20 sites within the HPV16 enhancer sequence which include nuclear factor-1 (NF1, NF-IL6), Jun–Fos heterodimers (AP-1, AP-2), transcriptional enhancer factor-1 (TEF-1), Octamer-1 (Oct-1), glucocorticoid responsive element (GRE), Yin Yang 1 (YY1) and papillomavirus silencing motifs [13], [14], [15], [16], [17], [18]. The 3′ segment contains the origin of replication and the E6 and E7 promoter, P97 [19], [20]. Thus, it is a complex network of events that guide either to cause positive or negative effect.
A number of factors could be involved in modifying processes that disrupt the natural progression of HPV infection towards CaCx development. One such is the disruption of the E2 gene, often observed in CaCx but not in premalignant lesions [21], [22], [23]. As a result of disruption, the E2 protein in its truncated form is unable to displace the cellular transcription factors, Sp1 or TFIID, from their cognate binding sites next to the P97 promoter, resulting in the increased expression of the E6 and E7 genes [5], [22]. This phenomenon is often attributed to disease progression from CIN to invasive CaCx [10], [11], [24] and to poor disease-free survival.
HPV16, harboring intact E2 in the episomal form, are often found to coexist with disrupted forms in CaCx [25], but in relatively low frequencies. In fact, a few studies have also reported an increased frequency of intact episomal HPV16 DNA in CaCx [26], reflecting a lack of repression of E6 and E7 genes by intact E2. Taken together, such findings indicate that E2 gene disruption may not be a prerequisite for CaCx development. Among alternative mechanisms of enhanced viral oncogene expression, altered E2 functions, as a result of variations in the E2 ORF, have been reported by a few studies [27], [28], [29], [30], including one from India [24]. Some studies have also observed elevated levels of E6 and E7 in primary or metastasizing tumors carrying intact E2, due to deletions or point mutations affecting one or more binding sites of the transcription factor YY1 [31], [32] in the viral LCR.
HPV16 has been found to be the most prevalent high risk HPV type in CaCx in India [33] as well as in HPV-infected individuals of the normal population [33], [34]. In this study, we evaluated the status of the E2 gene (disrupted or intact) in a group of CaCx cases (invasive squamous cell carcinomas) and population controls (cytologically normal) harboring HPV16 DNA. Furthermore, we also determined the nucleotide sequence alterations within intact E2 genes and the corresponding LCR of the HPV16 isolates. Our objective was to determine the viral factors that were significantly associated with HPV16-related CaCx in Indian women.
Section snippets
Sample collection and DNA isolation
The malignant cervical biopsy tissues used for this study were all histopathologically confirmed as invasive squamous cell carcinomas. These were derived from married subjects aged 27–80 years (median age: 50 years), attending a cancer referral hospital (Cancer Centre Welfare Home and Research Institute, South 24 Parganas, West Bengal, India). All of these malignant subjects were clinically diagnosed to have tumors of stage III and above, as per FIGO classification. The control samples were
HPV16 E2 gene disruption
Thirty of the eighty-one cases (37.04%) and four of the twenty-seven controls (14.81%) showed E2 disruption. Each sample was amplified twice, using the W1/W2 primers, to confirm the results. Thus, E2 disruption was significantly higher among the cases [P = 0.018; OR (95% CI) = 3.38 (1.07–10.72)]. Subsequently, each portion of E2 gene was amplified individually using the three sets of overlapping primers indicated in Table 1 to map the exact region that got most frequently disrupted. The
Discussion
In this study, we analyzed E2 disruption as well as E2 and LCR sequence variations in HPV16 isolates from CaCx cases and cytologically normal subjects to identify key viral factors associated with disease development. Our data indicate that 37.04% of the HPV16-positive CaCx against 14.81% of controls harbor disrupted E2, which showed significant association with disease development. Such disruption was most prominent in the region encoding the DNA-binding domain of E2 protein (nucleotides
Acknowledgments
We thank Dr. Chandralekha Duttagupta (formerly of Biochemistry Unit, Indian Statistical Institute, Kolkata, India) for her constant inspiration and untiring support through this work; Professor Partha P Majumder and Dr. Sourabh Ghosh of Human Genetics Unit, Indian Statistical Institute, Kolkata, India, for their comments on the manuscript and for providing guidelines for statistical analysis of the data; Cancer Centre Welfare Home and Research Institute (Thakurpukur, South 24 Parganas, West
References (47)
- et al.
Effect of glucocorticoid hormones on viral gene expression, growth, and dysplastic differentiation in HPV16-immortalized ectocervical cells
Exp. Cell Res.
(1997) - et al.
Oct-1 activates the epithelial-specific enhancer of human papillomavirus type 16 via a synergistic interaction with NF1 at a conserved composite regulatory element
Virology
(1995) - et al.
Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization
Virology
(1992) - et al.
Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers
Virology
(1989) - et al.
Physical status and expression of HPV genes in cervical cancers
Gynecol. Oncol.
(1997) - et al.
Intratype HPV 16 sequence variation within LCR of isolates from asymptomatic carriers and cervical cancers
J. Clin. Virol.
(2001) - et al.
Prevalence of human papillomavirus in cervical cancer: a world wide perspective. International biological study on cervical cancer (IBSCC) Study Group
J. Natl. Cancer Inst.
(1995) - et al.
Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 in cervical preneoplastic and neoplastic lesions
J. Gen. Virol.
(1992) - et al.
Human papillomavirus genotype as a predictor of persistence and development of high-grade lesions in women with minor cervical abnormalities
Int. J. Cancer
(1996) - et al.
The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID
J. Virol.
(1994)