Telomerase Suppression by Chromosome 6 in a Human Papillomavirus Type 16-Immortalized Keratinocyte Cell Line and in a Cervical Cancer Cell Line

Steenbergen, Renske D.M.; Kramer, Debbie; Meijer, Chris J.L.M.; Walboomers, Jan M. M.; Trott, Deborah A.; Cuthbert, Andrew; Newbold, Robert F.; Overkamp, Wilhelmina J. I.; Zdzienicka, Małgorzata Z.; Snijders, Peter J.F.

doi:10.1093/jnci/93.11.865

Cited by 66 publications

(81 citation statements)

References 26 publications

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“…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.…”

Section: Discussionsupporting

confidence: 62%

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

et al. 2007

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We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n ¼ 11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARb and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

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Section: Discussionsupporting

confidence: 62%

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

et al. 2007

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“…4,7 Moreover, activation of telomerase, by up-regulation of its catalytic subunit human telomerase reverse transcriptase gene, seemed to be necessary and sufficient for immortalization of HPV-transfected keratinocytes. 6,8,9 In addition, expression of the viral oncogene HPV 16 E6 from a heterologous promoter has been shown to activate telomerase in primary keratinocytes. 10 However, expression of HPV 16 and HPV 18 E6 from their native promoter in the context of the fulllength virus genome did not result in telomerase activation, 4 indicating that additive events are required.…”

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confidence: 99%

“…6,9 To identify host cell genes that may be involved in telomerase regulation and HPV-mediated immortalization we performed mRNA differential display analysis 11 on RNAs isolated from a panel of HPV-transformed keratinocytes at pre-immortal and subsequent immortal stages to idenSupported by grant 28-28360 from Zorg Onderzoek Nederland and the Royal Netherlands Academy of Arts and Sciences (fellowship to R. D. M. S.).…”

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confidence: 99%

“…[2][3][4] Studies involving somatic cell fusions and microcell-mediated chromosome transfers have indicated that particularly recessive gene alterations are crucial for immortalization of HPV-containing epithelial cells. 5,6 HPV-mediated immortalization is associated with an arrest of telomeric shortening and activation of the telomere lengthening enzyme telomerase. 4,7 Moreover, activation of telomerase, by up-regulation of its catalytic subunit human telomerase reverse transcriptase gene, seemed to be necessary and sufficient for immortalization of HPV-transfected keratinocytes.…”

mentioning

confidence: 99%

“…6,9 To identify host cell genes that may be involved in telomerase regulation and HPV-mediated immortalization we performed mRNA differential display analysis 11 on RNAs isolated from a panel of HPV-transformed keratinocytes at pre-immortal and subsequent immortal stages to iden-tify genes showing differential expression. Two clonally derived HPV-16 transformed cell lines, designated FK16A and FK16B, as well as two clonally derived HPV 18-transformed cell lines, designated FK18A and FK18B, were analyzed.…”

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confidence: 99%

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Down-Regulation of GATA-3 Expression during Human Papillomavirus-Mediated Immortalization and Cervical Carcinogenesis

Steenbergen

OudeEngberink

Kramer

et al. 2002

The American Journal of Pathology

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To identify cellular genes that may be involved in human papillomavirus (HPV)-mediated immortalization mRNA differential display analysis was performed on preimmortal and subsequent immortal stages of four human keratinocyte cell lines transformed by HPV type 16 or 18 DNA. This yielded a cDNA fragment encoding the transcription factor GATA-3 that was strongly reduced in intensity in all immortal stages of the four cell lines. A marked reduction in both GATA-3 mRNA and protein expression in HPV-immortalized cell lines was confirmed by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry and was also shown to be apparent in cervical carcinoma cell lines. Immunohistochemical analysis of cervical tissue specimens showed a clear nuclear staining for GATA-3 in normal cervical squamous epithelium (n ‫؍‬ 14) and all cervical intraepithelial neoplasia (CIN) I (n ‫؍‬ 6) and CIN II lesions (n ‫؍‬ 2). In contrast, 11% (1 of 9) of CIN III lesions and 67% (8 of 12) of cervical squamous cell carcinomas revealed a complete absence of GATA-3 immunostaining. Hence, complete down-regulation of GATA-3 expression represents a rather late event during cervical carcinogenesis. Whether GATA-3 down-regulation is etiologically involved in HPV-mediated immortalization and cervical carcinogenesis remains to be examined. Infection with high-risk human papillomavirus (HPV) types is the most significant risk factor for the development of cervical cancer and HPV DNA can be detected in almost all cervical squamous cell carcinomas. 1 HPV functions are, however, not sufficient for the development of cervical cancer and additive oncogenic events involving host cell genes are required, consistent with a multistep process of carcinogenesis. To gain better insight in HPV-mediated carcinogenesis in vitro model systems have been proven very valuable. High-risk HPV types, in particular HPV 16 and HPV 18, can induce immortalization of primary human epithelial cells in vitro. [2][3][4] Studies involving somatic cell fusions and microcell-mediated chromosome transfers have indicated that particularly recessive gene alterations are crucial for immortalization of HPV-containing epithelial cells. 5,6 HPV-mediated immortalization is associated with an arrest of telomeric shortening and activation of the telomere lengthening enzyme telomerase. 4,7 Moreover, activation of telomerase, by up-regulation of its catalytic subunit human telomerase reverse transcriptase gene, seemed to be necessary and sufficient for immortalization of HPV-transfected keratinocytes. 6,8,9 In addition, expression of the viral oncogene HPV 16 E6 from a heterologous promoter has been shown to activate telomerase in primary keratinocytes. 10 However, expression of HPV 16 and HPV 18 E6 from their native promoter in the context of the fulllength virus genome did not result in telomerase activation, 4 indicating that additive events are required.Recent studies have shown that yet unknown genes residing at chromosomes 3, 4, and 6 can regulate telomer...

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