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. 2002 Jun;160(6):1945–1951. doi: 10.1016/S0002-9440(10)61143-1

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

Renske D M Steenbergen *, Vanessa E OudeEngberink *, Debbie Kramer *, Henri F J Schrijnemakers *, Rene H M Verheijen †, Chris J L M Meijer *, Peter J F Snijders *
PMCID: PMC1850837  PMID: 12057898

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. 2-4 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 full-length 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 telomerase activity in immortal HPV-transformed cells. 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 identify 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. In all cell lines the acquisition of an immortal phenotype was well defined and shown to be associated with an arrest of telomere shortening and activation of telomerase. 4

Materials and Methods

Cell Lines

The cell lines FK16A, FK16B, FK18A, and FK18B were established by transfection of primary human foreskin keratinocytes (EK94-2) with the entire HPV 16 and HPV 18 genome, respectively, and cultured as described previously. 4 The cervical carcinoma cell lines SiHa and HeLa were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies Inc., Breda, The Netherlands) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mmol/L, Life Technologies). The breast carcinoma cell line MCF-7 was also obtained from the American Type Culture Collection and cultured in RPMI (Life Technologies Inc.) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mmol/L, Life Technologies, Inc.). Of all cell lines cells were harvested by trypsinization at different passages before and after immortalization when the cells were growing exponentially.

Differential Display Analysis

Four pre-immortal stages of cell lines FK18A and FK18B (FK18A p12, FK18A p13, FK18B p13, and FK18B p18; P = passage number) were compared with seven immortal stages of the different cell lines (FK16A p27, FK16A p30, FK16B p37, FK18A p25, FK18A p28, FK18B p27, and FK18B p61). Rat RNA was included as a control for aspecific bands.

Total RNA was isolated using RNAzolB (Tel-Test Inc., Friendswood, TX) and treated with RNase-free RQ1-DNase (Promega Corp., Leiden, The Netherlands) to remove residual DNA. Differential display analysis was performed on 200 ng of RQ1-DNase-treated RNA using the RNAimage-mRNA differential display system (GenHunter Corporation, Nashville, TN), according to the manufacturer’s protocol.

Automated Sequencing

Reamplified differential-display polymerase chain reaction (PCR) products were sequenced directly by cycle sequencing using the Thermosequenase dye terminator cycle sequencing kit (Amersham Life Science, Cleveland, OH). Primers provided with the differential display kit were used and sequences were analyzed using the ABI 373 XL sequencer and Sequence analysis 3.3 Software (Applied Biosystems, Perkin Elmer Corp., Foster City, CA).

Reverse Transcriptase (RT)-PCR

RT-PCR was performed using GATA-3-specific primers spanning nucleotides 1227 to 1499 of the GATA-3 sequence (GenBank accession number NM_002051); (forward primer: 5′-AAG GCATCCAGACCAGAAACCG-3′; reverse primer: 5′-AGCATCGAGCAGGGCTCTAACC-3′). RT-PCR for the housekeeping gene encoding the U1 small nuclear ribonucleoprotein-specific A protein (snRNP U1A) 12 served as a reference for the semiquantitative assessment of GATA-3 mRNA levels. RT-PCR was performed as described previously 12 on 50 ng of target RNA for 28 PCR cycles.

To avoid amplification of residual genomic DNA the RNAs were pretreated with RQ1-DNase (Promega) and reactions without reverse transcriptase added during cDNA synthesis were included. To quantify RNA expression, RT-PCR products were hybridized to a radiolabeled GATA-3-specific oligonucleotide probe (5′-AGACACATGTCCTCCCTGAGCCACATCTCG-3′) or an snRNP U1A-specific oligonucleotide probe, 12 respectively. Signals were quantified by Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA). mRNA expression levels were normalized to the levels measured in primary donor keratinocytes according to the following formula: intensity ratio (GATA-3/snRNP U1A) of analyzed cell culture/intensity ratio (GATA-3/snRNP U1A) of primary donor keratinocytes × 100%.

Western Blot Analysis

Primary keratinocytes (EK00-12, 2.106 cells), SiHa (2.106 cells), and MCF-7 (0.5.106 cells) cells were lysed in 40 μl of lysis buffer (0.2 mol/L Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 0.18% v/v glycerol, 0.02% v/v mercaptoethanol) for 5 minutes at room temperature and centrifuged at 14,000 rpm for 5 minutes. Supernatants were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Blots were probed with murine monoclonal anti-GATA-3 antibody (HG3-31, dilution 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG1 antibody (dilution 1:5000; Southern Biotechnology Associates, Birmingham, AL) was used for visualization.

Raft Cultures and Tissue Specimens

Organotypic raft cultures of the primary keratinocytes and of the HPV-transformed cell lines have been described previously. 13

Formalin-fixed, paraffin-embedded tissue specimens of normal cervix (n = 14), CIN I (n = 6), CIN II (n = 2), and CIN III (n = 9) lesions, and cervical carcinomas (n = 12), collected during the course of routine clinical practice, were obtained from women undergoing biopsy or surgery at the gynecology department of the Vrije Universiteit Medical Center in Amsterdam. Tissue specimens were previously HPV typed using the general primer GP5+/6+ PCR immunoassay (EIA) as described by Jacobs and colleagues. 14 HPV positivity was found in 25% of normal cervical epithelium specimens analyzed (HPV type 16), in 50% of CIN I lesions (HPV types 16, 39, and 42), and in all CIN II lesions (HPV types 16, 51, and 58), CIN III lesions (HPV types 16 and 31), and cervical squamous cell carcinoma (HPV types 16, 18, and 35). Also, formalin-fixed tissue specimens of two estrogen receptor-positive breast carcinomas were subjected to immunohistochemical analysis because these have a high likelihood of overexpressing GATA-3. 15

Immunohistochemistry

Immunohistochemical staining was performed on 4-μm sections of raft cultures, and cervical and mammary tissue specimens. Endogenous peroxidase was inactivated by incubation with 0.3% H2O2 in methanol for 30 minutes. For GATA-3 detection slides were pretreated with 1 mmol/L of ethylenediaminetetraacetic acid, 0.1 mol/L Tris, pH 8.0, in an autoclave, followed by successive rinses in 0.5% Triton X-100 and 0.1 mol/L of glycine. MIB-1 detection was performed as described previously. 13 Consecutive slides were incubated with a primary antibody against GATA-3 (HG3-31, dilution 1:200; Santa Cruz Biotechnology Inc.) and a primary antibody against the proliferation marker Ki-67/MIB-1 (MIB-1, dilution 1:100; DAKO, Glostrup, Denmark), at 4°C overnight. Antibodies were detected using the Ultravision Large Volume Detection System (Labvision Corp., Fremont, CA) and 3-amino-9-ethyl carbazole was used as a chromogen. Sections were counterstained with hematoxylin.

To ensure the quality of the sections, GATA-3 staining was performed within 3 days after cutting of the sections. In addition, the majority of samples were stained with a second antibody against GATA-3 (HG3-35, dilution 1:100; Santa Cruz Biotechnology). Both GATA-3 antibodies gave very similar staining patterns in all sections analyzed, although HG3-31 staining tended to be somewhat stronger in some sections. Positive staining of T lymphocytes in the cervical stroma, as was seen in all sections, served as an internal control. Moreover, the integrity of the paraffin material was tested by staining consecutive slides for the proliferation marker Ki-67/MIB-1. All sections included in this study showed epithelial cells staining positive for Ki-67/MIB-1. Two expert pathologists performed histological examination of the stained sections.

Results

Identification of Differentially Expressed Genes in Pre-Immortal Versus Immortal HPV-Transformed Keratinocytes

By differential display analysis a panel of RNAs isolated from FK18A and FK18B cells at the pre-immortal stage was compared with RNAs isolated from FK16A, FK16B, FK18A, and FK18B cells at the immortal stage. Eight different arbitrary primers were combined with three defined anchoring primers, resulting in 24 primer combinations. On average 65 bands were obtained for each primer combination, giving rise to an overall representation of ∼1500 mRNA species.

PCR fragments were only isolated and sequenced when differential expression was observed in a major subset of pre-immortal versus immortal cells involving more than a single cell line. This does not only allow the preferred selection of genes that are more generally associated with HPV-mediated immortalization, but also reduces the chance of isolating false-positive PCR products.

We isolated two PCR fragments, which displayed a reduced intensity in the immortal stages of all four cell lines (Figure 1A) ▶ . On sequencing one of these fragments was found to represent a yet unidentified gene, whereas the second one was found to be identical to sequences encoding the GATA-3 transcription factor.

Figure 1.

Figure 1.

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Differential expression of GATA-3 in monolayer cultures. A: Identification of differentially expressed GATA-3 sequence by differential display PCR. The RNAs of the pre-immortal and immortal cells used for PCR analysis are indicated above the lanes. Rat is a control lane for aspecific bands. The excised bands in the pre-immortal cells representing the GATA-3 sequence are located between the dots. B: Semiquantitative RT-PCR for GATA-3 on pre-immortal and immortal cells. Indicated are primary keratinocytes (EK94-2); pre-immortal and immortal stages of cell lines FK16A, FK18A, and FK18B; and the cervical cancer cell lines SiHa and HeLa. Top: GATA-3 RT-PCR products. Bottom: RT-PCR products of housekeeping gene snRNP U1A. Levels of GATA-3 transcripts relative to primary keratinocytes (set to 100%) are indicated below the lanes. C: Western blot analysis of GATA-3 protein (50 kd) expression in MCF-7 cells (lane 1), SiHa cells (lane 2), and primary keratinocytes (EK00–12, lane 3).

Reduced GATA-3 mRNA and Protein Expression in HPV-Immortalized Cell Lines

To confirm differential expression of GATA-3 mRNA semiquantitative RT-PCR analysis was performed on RNAs isolated from the primary donor keratinocytes, different passages of pre-immortal FK16A, FK18A, and FK18B cells, and immortal cells of the four cell lines. No RNA was available from FK16B cells at the mortal stage. In addition, RNAs isolated from two cervical carcinoma cell lines, SiHa and HeLa, containing HPV 16 and HPV 18, respectively, were included in the analysis.

Compared to the primary donor keratinocytes in which the percentage of GATA-3 expression was set to 100%, cells of all pre-immortal passages expressed similar levels (range, 80 to 98%) of GATA-3 mRNA. In contrast, GATA-3 expression was strongly reduced in all passages of immortal cells (range, <1 to 33%) (Figure 1B) ▶ . Moreover, GATA-3 expression was markedly reduced in the cervical carcinoma cell lines SiHa and HeLa compared to the primary keratinocytes (<1%) (Figure 1B) ▶ .

To analyze whether the reduced mRNA expression resulted in a reduction in GATA-3 protein expression Western blot analysis was performed on total cell lysates of primary keratinocytes (EK00-12) and SiHa cells. Protein extract of the breast carcinoma cell line MCF-7, which is known to abundantly express GATA-3 and in which GATA-3 protein can specifically be detected with the monoclonal GATA-3 antibody HG3-31, 15 was included as a positive control. In primary keratinocytes GATA-3 protein was detected, although at a much lower level as in MCF 7 cells. In contrast, GATA-3 protein expression was undetectable in SiHa cells (Figure 1C) ▶ . Analysis of GATA-3 protein expression in three-dimensional organotypic raft cultures of the primary donor keratinocytes, immortal FK18B cells, and SiHa cells by immunohistochemistry showed an almost complete absence of GATA-3 expression in immortal FK18B and SiHa cells. In contrast, primary keratinocytes showed clear GATA-3 staining (Figure 2) ▶ . Taken together, these data show that both GATA-3 mRNA and protein expression is markedly reduced in HPV-immortalized cells and cervical cancer cells compared to primary keratinocytes and pre-immortal HPV-transformed cells.

Figure 2.

Figure 2.

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Differential expression of GATA-3 in three-dimensional organotypic cultures. Immunohistochemical detection of GATA-3 (a, c, e) and Ki-67/MIB-1 (b, d, f) in organotypic raft cultures of primary human keratinocytes (passage 3) (a, b), immortal FK18B cells (passage 83) (c, d), and SiHa cells (e, f).

GATA-3 Expression in Normal Cervical Epithelium, CIN Lesions, and Cervical Carcinomas

We subsequently tested whether differential expression of GATA-3 also occurs in cervical lesions. To this end GATA-3 expression was analyzed by immunohistochemistry in 14 normal cervical squamous epithelial specimens, 17 CIN lesions (6 CIN I, 2 CIN II, and 9 CIN III) and 12 cervical squamous cell carcinomas.

In normal cervical epithelia nuclear GATA-3 expression could be clearly detected in all tissue samples analyzed. Staining was most pronounced in the lower half of the squamous epithelium and was decreased in apical, more differentiated, epithelial layers. No staining was seen in columnar epithelial cells of the endocervix. GATA-3 immunostaining was also observed in all CIN I lesions (n = 6) both in basal and suprabasal cell layers, although two of these lesions revealed somewhat weaker staining compared to normal epithelium. Both CIN II lesions analyzed showed a weak GATA-3 staining in basal and suprabasal layers. Interestingly, one of the nine CIN III lesions was completely negative for GATA-3, whereas normal epithelium in the same section and lymphocytes underneath the CIN lesion were strongly positive. Another CIN III lesion showed focal staining for GATA-3 in some areas and four CIN III lesions showed sporadically weak GATA-3-positive cells. The three remaining CIN III lesions showed clear GATA-3 staining; one in the basal layer, one in the lower half of the epithelium, and one throughout the epithelium. Of 12 carcinomas analyzed, 8 revealed complete absence of GATA-3 staining in the tumor cells, whereas in surrounding stroma clear GATA-3-positive lymphocytes could be observed. Two other carcinomas showed focal GATA-3 expression with certain tumor areas staining very weakly positive whereas other areas were completely negative. In all these cases clear positive lymphocytes were detected in the surrounding stroma. The two remaining cervical carcinomas showed clear positive GATA-3 staining in the tumor areas. Representative examples of GATA-3 immunostaining on tissue specimens are shown in Figure 3 ▶ . Both estrogen receptor-positive breast carcinomas analyzed showed GATA-3 immunopositivity with staining intensities that were markedly higher than that observed in normal cervical epithelium (data not shown). This is in line with the different expression levels seen in the Western blot for primary keratinocytes compared to the estrogen receptor-positive MCF-7 cell line.

Figure 3.

Figure 3.

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Differential expression of GATA-3 in cervical (pre)malignant lesions. Immunohistochemical detection of GATA-3 in normal cervical squamous epithelium (a, b), CIN I lesions (c, d), CIN III lesions (e, f), cervical carcinomas (g, i, k, l), and Ki-67/MIB1 in cervical carcinomas (h, j). a and b: GATA-3 is expressed in nuclei of basal and parabasal cells of normal cervical epithelium. c and d: CIN I: GATA-3 expression is found in basal, parabasal, and more differentiated cells of cervical epithelium. e and f: CIN III: GATA-3 is weakly expressed in basal and upper layers of cervical epithelium. g: Cervical squamous cell carcinoma: GATA-3 expression is focal and weak in tumor areas. h: Consecutive section of g, showing strong staining for Ki-67/MIB-1 in tumor areas. i: Cervical squamous cell carcinoma: GATA-3 staining is absent in tumor areas. j: Consecutive section of i, showing strong staining for Ki-67/MIB-1 in tumor areas. k and l: Cervical squamous cell carcinomas: GATA-3 staining is absent in tumor areas, whereas lymphocytes stain positive.

Discussion

In this study we showed that HPV-mediated immortalization in vitro is associated with a down-regulation of the transcription factor GATA-3. The reduction in GATA-3 mRNA expression during immortalization seemed to be a common feature for all four different HPV-transformed cell lines and the cervical cancer cell lines analyzed. Further Western blot analysis and immunohistochemical analysis of organotypic raft cultures of the cell lines revealed that reduction in GATA-3 mRNA expression is translated into a clearly reduced protein expression in HPV-immortalized cells and cervical carcinoma cells compared to primary keratinocytes. In addition, immunohistochemical analysis of cervical tissue specimens revealed complete loss of GATA-3 staining in 11% of CIN III lesions and 67% of cervical carcinomas. These results indicate that loss of GATA-3 expression is not only associated with immortalization in vitro, but is also with cervical carcinogenesis in vivo. In addition, these data suggest that complete loss of GATA-3 expression occurs rather late in the CIN to cervical carcinoma sequence.

To our knowledge this is the first study showing expression of GATA-3 in primary human keratinocytes and normal squamous epithelium. GATA-3 was originally identified as a transcription factor that binds to and activates the T-cell receptor gene enhancer. 16 GATA-3 belongs to the GATA family of transcription factors, which bind to the consensus sequence (A/T)GATA(A/G) and to the related motifs CGATGG and (T/A)GAT(T/A)(A/G). 17 GATA-3 gene knockout studies revealed a critical role for GATA-3 in embryogenesis. 18 In adults, GATA-3 has been shown to act as a transcriptional activator in T-cell differentiation, 16 whereas it has been described to act as a transcriptional repressor during adipocyte differentiation. 19 Moreover, GATA-3 haplo-insufficiency was found to be involved in the etiology of human malformations, ie, the hypoparathyroidism, sensorineural deafness, renal anomaly syndrome (HDR). 20

The GATA-3 gene is located at 10p15, a region, which is more than incidentally altered in HPV-immortalized cells and in cervical carcinomas. 4,21-26 In addition, putative senescence and telomerase repressor loci have recently been identified at 10p14-p15 and 10p15, respectively. 27,28 Therefore, further functional studies are warranted to find out whether GATA-3 deregulation is involved in HPV-mediated immortalization and cervical carcinogenesis.

Although this is the first demonstration of a potential role for GATA-3 in the development of cervical cancer, recent reports have described altered expression of GATA transcription factors in esophageal carcinoma cells, 29,30 breast carcinomas, 15 and gastric carcinoma cell lines. 31

In conclusion, down-regulation of GATA-3 was correlated with HPV-mediated immortalization in vitro and advanced (pre)malignant cervical disease. Therefore, it may provide a marker for progressive CIN disease.

Acknowledgments

We thank Elly Fieret and Monique Egging for excellent technical assistance with the immunohistochemical analysis.

Footnotes

Address reprint requests to Dr. P. J. F. Snijders, Dept. of Pathology, Unit of Molecular Pathology, Vrije Universiteit Medical Center (VUMC), De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: pjf.snijders@vumc.nl.

Supported 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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