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

Abstract

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...

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

“…In our previous studies we already showed that both morphologically and genetically, the HPV-transfected cell lines closely resembled cervical (pre)malignant lesions (Steenbergen et al, 1996(Steenbergen et al, , 1998a(Steenbergen et al, , 2002van Duin et al, 2003). The current study indicates that also with respect to DNA methylation, this model system nicely mimics cervical carcinogenesis in vivo.…”

“…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.…”

“…With increasing passage, these cell lines revealed increasing severity of dysplastic features in organotypic cultures that are reminiscent of the various stages of premalignant cervical lesions (Steenbergen et al, 1998b). Moreover, consecutive passages showed accumulation of chromosomal alterations overlapping with those found in cervical (pre)malignant lesions (Steenbergen et al, 1998a(Steenbergen et al, , b, 2005Wilting et al, 2006) and shared altered expression of certain genes with these (pre)cancer stages (Steenbergen et al, 1996(Steenbergen et al, , 2002van Duin et al, 2003). It is currently, however, unknown to what extent this system also provides a valuable model for studying epigenetic alterations that are common in cervical carcinomas.…”

The LIM-only protein, LMO4, and the LIM domain-binding protein, LDB1, expression in squamous cell carcinomas of the oral cavity

“…The C. elegans homologue of GATA, Elt, is a prerequisite for ectodermal cell differentiation (Gilleard and McGhee, 2001). GATA3 is expressed in the normal cervical squamous epithelial cells, but downregulated in progressive carcinoma cells (Steenbergen et al, 2002), suggesting a role of GATA in epithelial cell differentiation. The cis-acting E-box element is found in the Ecadherin promoter region and binding of the zinc-finger protein, SNAIL or SIP1, represses gene expression.…”

GATA-3 Expression in Breast Cancer Has a Strong Association with Estrogen Receptor but Lacks Independent Prognostic Value

“…In breast epithelial cells, it has been proposed that GATA-3 acts to maintain a differentiated state. Aberrant expression of GATA-3 has been reported in breast cancer as well as in pancreatic and cervical cancers (12)(13)(14).…”