Elsevier

Gynecologic Oncology

Volume 124, Issue 3, March 2012, Pages 534-541
Gynecologic Oncology

Hypomethylated CpG around the transcription start site enables TERT expression and HPV16 E6 regulates TERT methylation in cervical cancer cells

https://doi.org/10.1016/j.ygyno.2011.11.023Get rights and content

Abstract

Objective

The human papillomavirus (HPV) oncoprotein, E6, activates telomerase reverse transcriptase (TERT) expression and causes cellular immortalization. It remains unclear whether E6 affects TERT transcription by altering DNA methylation profiles. In this study, we explored the methylation status of the TERT promoter in cervical cancer cell lines and its variations after E6 was silenced by RNAi.

Methods

Three kinds of cervical cell lines (HPV16 positive: CaSki and SiHa; HPV18 positive: HeLa), were taken to analyze the methylation status of the TERT promoter by methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing (BS). Stealth RNAi was transiently transfected to these cell lines to silence the expression of HPV16/18 E6, and the subsequent changes of TERT mRNA levels and TERT promoter DNA methylation were examined.

Results

Hypomethylation of the DNA around the TERT transcription start site (− 156 to + 162 bp) was functionally related to its transcription. After transfection with Stealth RNAi, the levels of HPV16/18 E6 and TERT mRNA were greatly decreased. The methylated CpG around the transcription start sites in CaSki and SiHa cells were statistically increased (respectively P = 0.016, P = 0.000). However, there was no significant difference in HeLa cells (P = 0.128).

Conclusion

Hypomethylated CpG around the transcription start site enables the expression of TERT in cervical cancer cells. Our results show for the first time that HPV16 E6 can promote TERT transcription through demethylating the DNA sequence around the TERT transcription start site in cervical squamous cancer cells.

Highlights

► We explore TERT methylation in three HPV+ cervical cells. ► DNA methylation around the transcription start site (Ts) determines TERT expression. ► Decreasing HPV16 E6 will cause denser methylation in TERT promoter. ► HPV16 E6 increases TERT transcription through demethylating TERT promoter around the Ts region.

Introduction

Telomerase stabilizes the length of telomeres by adding hexameric repeats to the ends of linear chromosomes, thus compensating for the continued erosion of telomeres. Maintenance of telomeres is required for cells to escape replicative senescence and proliferate indefinitely [1]. In the past decade, research in the field of telomerases has progressed tremendously, especially in relation to cellular immortality and carcinogenesis [2], [3].

Currently identified components of telomerase consist of the catalytic subunit of human telomerase reverse transcriptase (TERT), the RNA component (TERC) that serves as a template for the repeat sequence, and the telomerase related protein (TEP1) [4]. Among the various components of the human telomerase, TERT and TERC are the essential components [5]. TERC has been shown to be widely expressed in most cell types, and even in telomerase-negative cells, such as differentiated somatic cells. The expression of TERT is tightly regulated during differentiation and is either not expressed or is expressed at very low levels in most somatic cells. The activity of the telomerase holoenzyme is largely governed by the intracellular level of TERT [6], [7]. A positive correlation has been found between the amount of TERT mRNA and telomerase activity, suggesting the key point of telomerase transcription regulation the transcriptional control of the TERT gene [8].

Various transcription factors, such as c-Myc, Sp1, and estrogen, among others, can bind to the 5′-TERT gene regulatory region [1], [9], [10] and this binding correlates with TERT expression. Studies have also found that the TERT promoter contains a cytosine–guanine dinucleotide (CpG) island (CGI) and its transcriptional regulation may therefore involve DNA methylation [11]. Several groups have examined the methylation status of the CGI within this promoter, but contradictory results have been reported. Some researchers found, as expected, that methylation of the TERT promoter was associated with gene silencing [12], [13], [14]. However, other reports have demonstrated no significant correlation between TERT expression and methylation status [15], [16]. Furthermore, other reports indicated the presence of increased DNA methylation levels in the TERT promoter in TERT-positive cancer cells and hypomethylation in normal TERT-negative cells [17]. These varied correlations between DNA methylation and TERT expression may result in part from the varied methods used to study different regions of the TERT promoter and from not using quantitative measures of TERT expression [18].

Human papillomaviruses (HPV) can be classified into high-risk or low-risk groups according to the propensity for malignant progression of their associated cervical lesions. High-risk HPV types, specifically HPV16 and HPV18, are associated with 99.7% of cervical cancers [19]. The activities of the HPV oncogenes, E6 and E7, most clearly linked to carcinogenesis are their ability to inactivate the p53 and retinoblastoma (pRb) tumor suppressors, respectively. Another major function of HPV E6 is its ability to activate TERT expression, [20], [21] which is critical for cell immortalization, thereby maintaining chromosome telomere length and continued cell proliferation.

Data on TERT expression in cervical cancer has revealed that 0–33% of normal cervix exhibited TERT mRNA expression, whereas 80–100% of the cervical cancers showed TERT expression [22]. The activation of TERT is a key point in the progression of the cervical epithelium from cervical intraepithelial neoplasia (CIN) to cervical cancer after infection with high-risk HPV [23]. Some studies showed that methylation of the TERT promoter in cervical cancer was significantly more frequent in comparison to normal cervical tissue, [22] and an increasing frequency of the hypermethylated TERT promoter was found during the progression of cervical oncogenesis [24]. These findings indicate that HPV infection and the progression of cervical oncogenesis should affect TERT methylation status. Whether the activation of TERT is achieved, at least in part, by alterations in the level of TERT promoter methylation remains unknown. The aim of this study was to explore the methylation status of the TERT promoter in cervical cancer cell lines and the effect of the HPV E6 gene on TERT mRNA and the methylation of its promoter.

Section snippets

Cell lines

SiHa and CaSki cervical squamous carcinoma cells (HPV 16 +), and HeLa cervical adenocarcinoma cells (HPV18 +), as well as MCF-7 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Primary human embryonic lung fibroblasts (HELF) were kindly donated by Professor Zebin Mao of the Department of Biochemistry at the Peking University Health Science Center, China, to serve as a normal control. All of the above cells were maintained under 5% CO2 at 37 °C in

Methylation status of TERT promoter

Completely methylated DNA clustered in the upstream and middle-stream of the Ts of the TERT promoter in all three cervical cancer cell lines, whereas the downstream region of the TERT Ts was partially methylated, which was similar to the results of the breast cancer cell line, MCF-7. HELF, a diploid fibroblast, showed partial methylation in the upstream and middle-stream regions and complete methylation in the downstream region (Fig. 1).

In view of the results of MSP, we postulated that the DNA

Discussion

Few researchers have studied the level of DNA methylation in the TERT promoter in cervical cancer cell lines. Using bisulfite genomic sequencing, Devereux et al. [15] studied 72 CpG sites that extended from 500 bases upstream of the transcriptional start site of the TERT gene into the first exon in 37 cell lines, including SiHa. This study detected partially methylated DNA in the TERT promoter in SiHa cells, but no obvious correlation was found between a generalized pattern of site-specific or

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Funding

This research was supported by a grant from the National Natural Science Foundation of China (No. 81001157).

References (33)

  • C.M. Counter et al.

    Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase

    Oncogene

    (1998)
  • S.L. Weinrich et al.

    Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT

    Nat Genet

    (1997)
  • A.L. Ducrest et al.

    Regulation of the human telomerase reverse transcriptase gene

    Oncogene

    (2002)
  • H. Li et al.

    Mechanisms of action of TGF-beta in cancer: evidence for Smad3 as a repressor of the hTERT gene

    Ann N Y Acad Sci

    (2007)
  • S. Kyo et al.

    Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers

    Cancer Sci

    (2008)
  • I. Horikawa et al.

    Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene

    Cancer Res

    (1999)
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