Gene expression profiling to identify markers associated with deregulated hTERT in HPV‐transformed keratinocytes and cervical cancer

Abstract

Although high-risk human papillomavirus (HPV) infection plays a major role in the development of cervical cancer, additive oncogenic events are involved as well. One key event involves increased activity of telomerase resulting from a deregulated expression of its catalytic subunit hTERT. Our previous microcell-mediated chromosome transfer studies revealed that introduction of human chromosome 6 in the HPV16-immortalized keratinocyte cell line FK16A and in the HPV16-containing cervical cancer cell line SiHa induced growth arrest, resulting from a repression of hTERT mRNA expression and telomerase activity. Here, this model was used to analyze expression profiles associated with hTERT deregulation in HPV-transformed cells. Microarray expression analysis of 12 FK16A/chromosome 6 hybrids, 4 of which were negative for endogenous hTERT and 8 of which were positive for endogenous hTERT, resulted in the identification of 164 differentially expressed genes. Differential expression of a selection of 5 genes was verified by real-time RT-PCR. Of these 164 genes, 32 were also differentially expressed in other HPV transformed cells with deregulated hTERT. For 2 of these genes, encoding AQP3 and MGP, altered expression in hTERT positive cervical carcinomas was confirmed by real-time RT-PCR and immunohistochemistry, respectively. Moreover, increased MGP protein expression was significantly more frequent in high-grade cervical premalignant lesions with elevated hTERT mRNA expression compared to those without. In summary, we identified 32 candidate biomarkers for deregulated hTERT mRNA expression, which may enable the identification of cervical premalignant lesions that are at highest risk to progress to invasive cancer. ' 2007 Wiley-Liss, Inc.Key words: HPV; hTERT; telomerase; cervical carcinogenesis; microarray expression analysis The main risk factor for the development of cervical cancer is infection with high-risk human papillomavirus (hrHPV), 1 which is supported by the detection of hrHPV in virtually all cervical carcinomas. 2 Cervical cancer evolves from preexisting noninvasive premalignant lesions referred to as cervical intraepithelial neoplasia (CIN), graded CIN1 to CIN3. Progression of an hrHPV infected CIN lesion to invasive cancer is a result of further specific (epi)genetic alterations within the host cell genome. 3,4 One of the consequences that is crucial for tumor development is an increased activity of telomerase. 5,6 Telomerase is a ribonucleoprotein that can elongate the chromosomal ends or telomeres, thereby compensating for telomere shortening that is inherent to DNA replication. 7 Whereas most cancer cells display elevated telomerase activity and stabilized telomeres, resulting in an unlimited lifespan, most primary somatic cells exhibit absence of detectable telomerase activity and continuous telomere erosion during cell division. The latter ultimately leads to the induction of senescence. 7 Telomerase consists of several subunits, including a structural RNA component (hTR) that serves as a template dur...

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

“…Gene alterations previously detected in our in vitro model system, such as GATA-3, hTERT, MGP and CADM1, were also found in clinical samples (Steenbergen et al, 2001(Steenbergen et al, , 2002(Steenbergen et al, , 2004de Wilde et al, 2007). Therefore, we believe that the methylation events identified in the in vitro model system provide potential markers for cervical cancer detection as well, which is underlined by their large overlap with methylated gene promoters found in cervical carcinomas.…”

Differential In Vitro Immortalization Capacity of Eleven, Probable High-Risk Human Papillomavirus Types

“…For hTERT mRNA expression analysis, 500 ng of total RNA was treated with RQ1 RNase-free DNase (Promega), and qRT-PCR was performed as described previously (35).…”

“…Infections with the hrHPV types 16,18,31,33,35,45,52, and 58 comprise the highest long-term (Ͼ5-year) risk of CIN3 or higher lesions, whereas most of the other hrHPV types present an almost negligible long-term risk (8)(9)(10). Furthermore, a large, randomized, controlled prospective trial conducted in the Netherlands has revealed that among hrHPV-positive women with normal cytology or borderline to moderate dyskaryosis, those infected with hrHPV types 16, 18, 31, and 33 have an increased 18-month risk of CIN3 or cancer (8).…”

Identification of differential expressed transcripts in cervical cancer of Mexican patients

“…After statistical analysis, we were able to validate 28 upregulated and seven downregulated genes. Our findings represent a collection of cancer-related genes and a variety of genes associated with cell development and cell cycle mechanisms that have not been reported by other authors [8,[25][26][27][28].…”