The EMBO Journal vol. 1 1 no.6 pp.2283 - 2291, 1992

Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor gene in nontumorigenic and tumorigenic HeLa hybrid cells Dusan Bartsch, Barbara Boye, Corinna Baust, Harald zur Hausen and Elisabeth Schwarz1 Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-6900 Heidelberg, FRG 'Corresponding author Communicated by H.zur Hausen

Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18-positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR-f and -'y. We show here that RA down-regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA-dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR-,B mRNA levels in non-tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR-,B gene or of the HeLa RAR-, promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR-,B control region was activated by RA more strongly in non-tumorigenic hybrid cells than in HeLa cells, thus indicating differences in tins-acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR-3 gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells. Key words: cervical carcinoma/HeLa hybrid cells/HPV18 E6 and E7/HPV 18 transcription/retinoic acid receptor

Introduction The human papillomavirus (HPV) group presently comprises 67 different virus types that infect epithelial cells and induce hyperproliferative lesions. Among the HPVs infecting the anogenital tract, several types, in particular HPV16 and HPV18, show a strong association with carcinomas of the cervix and induce cervical intraepithelial neoplasias that have a high tendency for malignant progression (for review see zur Hausen, 1991; Howley, 1991). The oncogenic potential of HPV 16 and HPV 18 has been attributed primarily to two early genes, E6 and E7. These genes are expressed in cervical carcinomas and carcinoma-derived cell lines (C Oxford University Press

harbouring integrated HPV16 or HPV18 DNA (Schwarz et al., 1985; Schneider-Gadicke and Schwarz, 1986; Smotkin and Wettstein, 1986; Baker et al., 1987; Choo et al., 1987), their expression is necessary for cell proliferation in vitro (von Knebel Doeberitz et al., 1988) and is linked to tumourigenic growth in vivo (Bosch et al., 1990). Furthermore, the E6 and E7 genes together induce immortalization of human epithelial cells (Barbosa and Schlegel, 1989; Hawley-Nelson et al., 1989; Munger et al., 1989; Hudson et al., 1990). Thus, E6 and E7 seem to play a role in the development of tumours as well as their maintenance. Transcription of E6 and E7 is regulated by the upstream regulatory region (URR) that contains the early E6/E7 promoter and enhancer elements with binding sites for various viral and cellular transcription factors (GarciaCarranca et al., 1988; Chong et al., 1991). Development of cervical carcinoma is neither an immediate nor an inevitable consequence of HPV infection. Furthermore, it is known that HPV gene expression is tightly linked to the state of differentiation of the host epithelial cells (Taichman and LaPorta, 1984). In mild cervical intraepithelial lesions, efficient synthesis of E6/E7 mRNAs is restricted to the highly differentiated non-dividing cells, whereas only low mRNA levels can be detected in the replication-competent basal cells. In contrast, higher levels of E6/E7 mRNAs are present in the abnormal basal-like cells of severe cervical intraepithelial neoplasias and carcinomas (Crum et al., 1988, 1989; Stoler et al., 1990; M.Durst, D.Glitz, A.Schneider and H.zur Hausen, submitted). It has been postulated that one essential event in the multistep process of HPV-linked cervical carcinogenesis is the activation of E6 and E7 gene expression due to loss or inactivation of negative cellular controls (zur Hausen, 1977, 1986). The cellular genes exerting this control have not yet been identified. However, it seems likely that some of them may be found within those networks regulating epithelial cell proliferation and differentiation. A key regulator of epithelial cell differentiation is vitamin A, with retinoic acid (RA) being one of the biologically active retinoids (for review see Roberts and Sporn, 1984). Retinoids also affect the proliferation and differentiation of preneoplastic and neoplastic cells and RA has been shown to be a potent inhibitor of the in vitro growth of various tumour cell lines including the cervical carcinoma cell line HeLa (Lotan, 1980; Lotan et al., 1980; Dion and Gifford, 1980). HeLa cells contain integrated HPV18 DNA and express the E6 and E7 genes (Boshart et al., 1984; Schneider-Gadicke and Schwarz, 1986). Cellular responses to RA are mediated by nuclear retinoic acid receptors (RARs) including subtypes RAR-ce, -(3 and -'y (Giguere et al., 1987; Petkovich et al., 1987; Benbrook et al., 1988; Brand et al., 1988; Krust et al., 1989, for the human RARs) and retinoid X receptors (RXRs) (Mangelsdorf et al., 1990). RAR and RXR are transcription factors and belong to the superfamily of nuclear hormone receptors (Evans, 1988). Upon ligand 2283

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activation, RARs activate transcription of target genes by binding to retinoic acid-responsive elements (RAREs). One target gene is RAR-(3 itself and a RARE has been identified in its promoter region (de The et al., 1989, 1990). We have addressed the question of whether RA-mediated growth inhibition of HeLa cells is associated with changes in the expression of HPV18 E6/E7 and cellular RAR genes. In addition to HeLa cells, we have analysed HeLa x fibroblast and HeLa x keratinocyte hybrids. HeLa cells fused with normal human cells retain a transformed phenotype in culture, but show a completely suppressed tumorigenic potential in vivo, i.e. upon inoculation into nude mice (Stanbridge et al., 1982), which is correlated with suppressed HPV18 E6 and E7 mRNA levels (Bosch et al., 1990). Since rare tumorigenic segregants have been isolated (Stanbridge et al., 1982), a model system of genetically closely related non-tumorigenic and tumorigenic cells is available which allows identification of intracellular changes linked to the proliferation potential in vivo. First, we analysed by Northern (RNA) blot hybridization the effect of RA on nmRNA levels of HPV18 and of the RAR-(3 and --y genes. The observed reduction of HPV18 mRNA levels prompted us to test, by nuclear run-on and transient transfection assays, whether and by what mechanism RA and the RARs affect transcription of the E6 and E7 genes. The Northern blot data furthermore indicated differences of RAR-,B gene expression between the non-tumorigenic and tumorigenic cells. We examined whether these differences are due to cis- or trans-effects.

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Influence of RA on cell proliferation RA was reported to have the capacity to inhibit HeLa cell proliferation in vitro. Using RA concentrations of 10-9-10-5 M, growth inhibition was shown to be dosedependent and not caused by cytotoxic effects of RA, even at 10-5 M RA. A 7-day treatment with 10-5 M RA resulted in about 90% growth inhibition as compared with untreated control cells (Dion and Gifford, 1980; Lotan et al., 1980). In our experiments, we have first examined whether 10-5 M RA also exerts a growth inhibitory effect on HeLa hybrid cells. Two pairs of hybrid cell lines were compared with HeLa cells: (i) the non-tumorigenic HeLa x fibroblast hybrid 444 and the tumorigenic 444 segregant CGL3 and (ii) the non-tumorigenic HeLa x keratinocyte hybrid P6 and the tumorigenic P6 segregant P6TR1.2. RA-induced growth inhibition was observed for the cells of all five cell lines reaching 70-90% after a 6 day treatment. Growth inhibition became apparent after 3 days of RA treatment and could be reversed within -2 days after removal of RA (data not shown). RA regulation of mRNA levels of HPV18 and cellular genes We next examined the influence of RA on expression of the HPV18 genes E6 and E7, and of the cellular genes encoding the RA receptors RAR-( and RAR-'y. For comparison, two other cellular genes, c-myc (which is involved in regulation of cell proliferation) and ,B-actin (which is a structural gene) were also analysed. Northern blot analysis was performed using RNAs extracted from cells cultured in the presence or absence of 10-5 M RA for 1-6 days (Figure 1). In all cells, an RA-induced down-regulation of HPV18 mRNA levels was observed (Figure 1, panels h). The mRNA

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Fig. 1. Effects of RA on mRNA levels of HPV18 and cellular genes in HeLa and HeLa hybrid cells. Cells (seeded at 1 x 105 cells/25 cm2 flask) were grown in the absence (-) or presence (+) of 10-5 M RA for 1, 2, 3, 4 or 6 days, respectively. Cytoplasmic RNA or poly(A)+ RNA was extracted and separated in 1% agarose gels (upper panels).

Filters were hybridized with the 32P-labelled DNA probes indicated beside the panels: HPV18 E6-E7 (h), RAR-,3 (b), RAR--y (g), c-myc exon 3 (m) and (3-actin (a). (A) Analysis of HeLa, 444 (nontumorigenic HeLaxfibroblast) and CGL3 (tumorigenic 444 segregant) cells. The filters with cytoplasmic RNA were first hybridized with the RAR-,B probe and rehybridized with RAR--y, HPV18 and (3-actin. (B) Analysis of 444 poly(A)+ RNA. (C and D) Analysis of P6 (nontumorigenic HeLaxkeratinocyte) and P6TRI.2 (tumorigenic P6-segregant). The filters were hybridized with the indicated probes in the order RAR-B, RAR--y, HPV18 (C) or c-myc, HPV18, ,3-actin (D). Filters were exposed to X-ray films from 1 to 10 days depending on the abundance of the transcripts to be detected. Exposure times of more than 1 week were necessary for detection of RAR-,B transcripts in cytoplasmic RNA, whereas 1-3 day exposures were usually sufficient for detection of the more abundant RAR--y transcripts. The triangles indicate the positions of the 28S and 18S RNA.

decrease was apparent after 24 h and became stronger with the duration of RA treatment. However, a certain amount of HPV18 mRNA remained detectable even after a 6-day treatment. Decreased mRNA levels were also observed for the c-myc gene in RA-treated cells at all time points examined (data shown for P6 HeLa x keratinocyte hybrid cells in

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Fig. 3. Effect of protein synthesis inhibition on mRNA levels in RAtreated 444 cells. Cells were seeded at densities of 2 x 105 cells/25 cm2 and 4x 105 cells/25 cm2 (indicated by 2 and 4, respectively) and were cultured for 4 days in the presence (+) or absence (-) of 10-5 M RA. Subsequently, cycloheximide (CH) was added for 1, 2, 6 and 9 h, respectively, to RA-treated cells. RNAs were separated in a 1 % agarose gel (upper panel) and blotted. The filter was hybridized successively with the radiolabelled DNA probes in the order c-myc exon 3 (m), HPV18 E6-E7-E1 (h), (3-actin (a).

RAR species expressed in human skin (Krust et al., 1989; Elder et al., 1991). Northern blot hybridization experiments performed with radiolabelled RAR probes showed that the RAR--y gene is expressed at much higher mRNA levels than the RAR-,3 gene in both HeLa cells and HeLa hybrid cells (Figure 1, panels b and g). No changes in RAR--y mRNA levels were induced by RA. In contrast, RA treatment resulted in a strong increase of RAR-,B mRNA levels in the cells of the two non-tumorigenic hybrid cell lines 444 and P6 (Figure IA and C), whereas RAR-,B mRNA levels remained very low in RA-treated tumorigenic hybrid segregant CGL3 and P6TRI.2 cells as well as HeLa cells (Figure lA and C). These results indicate that the nontumorigenic HeLa hybrid cells differ from the tumorigenic hybrid segregants and parental HeLa cells in ligand regulation of RAR-4 gene expression. RA-induced up-regulation of RAR-,B mRNAs has been reported to occur with a latency of -1 h in human hepatoma

Fig. 4. Nuclear run-on analysis of HPV18 and c-myc transcription in HeLa cells. (A) Cells were cultured for 3 days in the absence or presence of 10-5 M RA. Isolation of nuclei and transcript elongation were performed as described in Materials and methods. The radiolabelled RNA probes from untreated cells (-RA) and RA-treated cells (+RA) were hybridized to filters containing plasmid DNAs that were cleaved with appropriate restriction enzymes to excise the genespecific inserts from the vectors. The upper part shows the DNAs after electrophoretic separation in a 1.2% agarose gel. The genespecific inserts are: HPV18 E6-E7-E1 (lane 1, first band from top), HPV18 E6-E7 separated from El (lane 2, first and third band from top, respectively), HPV18 E6*-E7 (lane 3, lower band), c-myc exon 1 (lane 4, lower band), c-myc exon 3 (lane 5, lower band), purified fragment of ,B-actin cDNA (lane 6). (B) Schematic representation of the HPV18, c-myc and ,B-actin fragments (not drawn to scale). The numbers refer to the lanes in part A.

cells (de The et al., 1989). We have examined the time course of RAR-4 induction in 444 cells. Cells were grown in medium containing either 10% FCS and 10-5 M RA or 10% charcoal-stripped FCS and l0-7 M RA. Under both conditions, the induction had a latency of < 2 h (shown for l0-7 M RA, Figure 2). Effects of protein and RNA synthesis inhibitors on HPV18 mRNA level In order to examine possible mechanisms of the RA-induced down-regulation of HPV18 E6 and E7 mRNAs, we first analysed whether inhibition of protein synthesis in RA-treated cells could reverse the negative effect of RA. However, Northern blot hybridization revealed no elevation of HPV18 mRNA levels after addition of cycloheximide to RA-treated cells (Figure 3). In contrast, a cycloheximide-induced increase of c-myc mRNA was observed which was probably caused by mRNA stabilization (Dani et al., 1984). To analyse whether RA treatment leads to destabilization of HPV18 mRNAs, cells were cultured in the presence or absence of RA for 3 days, actinomycin D was then added for inhibition of RNA synthesis and RNA was extracted after 0.5-9 h of actinomycin D treatment. Northern blot 2285

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Fig. 5. Effect of RARs on viral and cellular transcriptional control regions. Relative luciferase activity (RLU) was measured (as described in Materials and methods) following transfection of HeLa cells with luciferase reporter plasmids containing the following control regions: HPV18 URR (HPV18), HPV16 URR (HPV16), RAR-42 control region (RAR(3), human cytomegalovirus immediate early promoter/enhancer (HCMV), cellular HMG promoter (HMG) and SV40 early promoter/enhancer (SV40). 10 Ag of luciferase reporter plasmid DNA was cotransfected with 2 jig of RAR expression plasmid DNA (hRAR-a, hRAR-3 or hRAR--y) or with 2 yg pBluescript DNA. Luciferase activity was determined after treatment of the cells with 10-5 M RA (filled bars) or DMSO only (open bars) for 24 h (also indicated by - and + below the columns). Values are the means of at least eight independent transfections, the standard deviations are indicated.

hybridization indicated that HPV18 mRNAs have a similar half-life in RA-treated and untreated cells (data not shown). RA effect on endogenous HPV18 transcription We next examined by nuclear run-on analysis whether decreased transcription could be the cause of the reduced HPV18 mRNA levels in RA-treated cells. Transcription of the HPV18 E6 -E7 -El region was compared with that of c-myc and 3-actin. In the case of HPV18 and c-myc, but not fl-actin, the hybridization signals produced by the labelled run-on transcripts

from RA-treated cells were clearly weaker than those produced by the RNA probe from untreated cells (Figure 4). These data indicate that the RA-mediated downregulation of HPV18 (and c-myc) mRNAs as observed by Northern blot analysis is due to inhibition of transcription. Effects of cotransfected RARs on the regulatory region of HPV18 E6/E7 and of other viral and cellular genes

In order to study whether RARs play a role in inhibition of HPV18 transcription, we performed transient transfection experiments in HeLa cells using the luciferase reporter gene assay. The response of the HPV18 URR to cotransfected

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Fig. 6. Effect of RAR-3 receptor on HPV18 URR in transiently transfected HeLa cells. Relative luciferase activity (RLU) was measured following cotransfection of 10 jg p18URRL DNA and 4 Ag pAc-Gal DNA with increasing amounts of hRAR-,B DNA. Cells were incubated in the absence (open circles, -RA) or presence (filled circles, +RA) of 10-5 M RA. Values are the means of eight independent transfections, the standard deviations are indicated.

RARs in the presence and absence of 10-5 M RA was compared with that of various other transcriptional control regions: the HPV16 URR, the RAR-f control region, the immediate early promoter/enhancer of the human cytomegalovirus (HCMV), the SV40 early promoter/enhancer and the control region of the cellular housekeeping gene hydroxymethylglutaryl-coenzyme A reductase (HMG). The results are summarized in Figure 5. The activity of the HPV1 8 and HPV16 URRs was reduced by ligand-activated RARs. As compared with the activity in the absence of cotransfected RARs, the reduction was 3-fold in the case of RAR-f and up to 10-fold for RAR--y. In contrast, the RAR-f control region was induced -4-fold by RARs in the presence of ligand. The HMG control region showed no regulation by RARs. The HCMV promoter was induced by RAR-a and RAR-3, whereas the SV40 promoter was inhibited by RAR-j and RAR--y. These data indicate that the down-regulation of the HPV18 URR (and HPV16 URR) was a specific, and not merely a toxic, effect of cotransfected RARs. The RAR-mediated inhibition of transcription from the HPV 18 URR was dose-dependent, as tested for RAR-,B in the presence and absence of ligand (Figure 6). Ligandindependent inhibition of the HPV18 URR was observed in all cotransfection experiments, extending to 15-30% for RAR-,B (Figures 5 and 6), 50% for RAR-ca and 40% for RAR--y (Figure 5). -

Identification of the HPV18 URR segment involved in RAR-dependent transcrptional down-regulation The HPV18 URR has been divided into three segments separated by RsaI restriction sites: (i) the proximal promoter segment which contains the E6/E7 promoter and the binding sites for the viral E2 trans-repressor/activator protein, (ii) the central enhancer segment (often referred to as the constitutive or cell-type specific enhancer) which has been shown to require cellular, and not viral, factors for its activity and to be active specifically in epithelial cells (Cripe et al., 1987; Gloss et al., 1987; Garcia-Carranca et al., 1988; Gius et al., 1988) and (iii) the distal enhancer with unknown function. In order to characterize the HPV18 URR segment through which RAR-dependent repression is mediated,

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Fig. 7. Transcriptional activity of HPV18 URR deletion mutants in the presence and absence of RA and cotransfected RAR-(8. (A) Schematic representation of HPV18 URR and HPV18 URR deletion mutants. The three segments of the HPV18 URR, the distal enhancer (dashed box), central enhancer (open box) and proximal promoter/E2 responsive enhancer element (filled box) are indicated, as are the flanking open reading frames E6 and LI. HPV18 URR deletion mutants were either linked directly to the luciferase gene thus using the genuine HPV18 promoter (pI8, p436/18L) or cloned in enhancer configuration upstream of the truncated HSV tk* promoter (p230/tk*L). Nucleotide positions of the 5'- and 3-boundaries of the cloned HPV18 URR fragments are indicated above the boxes (numbered according to Cole and Danos, 1987). Binding sites for cellular transcription factors NFl, API, PVF, KRF and Oct-I in the central enhancer are indicated (according to Garcia-Carranca et al., 1988; Chong et al., 1990; Mack and Laimins, 1991; Hoppe-Seyler et al., 1991). (B) Luciferase activity measured after transfection of HeLa cells with the plasmid DNAs shown in part A. The numbers in the first column [basal luciferase activity (RLU)] represent the standardized luciferase activities measured in the absence of RA and cotransfected RAR-(.i. Relative luciferase activity represents the activity of the luciferase reporter construct (as a percentage of the basal luciferase activity, which is set as 100%) after treatment of the cells with 10- M RA (second column, +RA), after cotransfection with 2 ALg hRAR-, DNA (third column, +RAR,3), or cotransfection of 2 Ag hRAR-,B DNA and RA treatment (fourth column, RA+RAR,3). In the absence of RAR-,B, 2 jig pSV2neo DNA was cotransfected in order to compensate for possible squelching by RAR-3 expression plasmid.

different HPV 18 URR constructs were tested in transient luciferase assays in the presence and absence of RA and cotransfected RAR-,B (Figure 7). The assays showed that the HPV 18 central enhancer plus proximal promoter (construct p436/18L) was inhibited by ligand-activated RAR-,B -2to 3-fold, similar to the complete URR. When the HPV18 central enhancer was linked to the HSV thymidine kinase (tk) promoter (construct p230/tk*L), the repression mediated by ligand-activated RAR-,B was enhanced > 10-fold; repression by RAR-fl in the absence of ligand was enhanced 3-fold. These data indicate that regulatory elements involved in RAR-mediated repression are located within the 230 bp central enhancer fragment (positions 7510-7739).

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RAR-,8 gene and promoter structure The different response of RAR-,B gene expression to RA in the non-tumorigenic and tumorigenic cells (see Figure 1) could result from mutations in the structural or regulatory part of the RAR-, gene. In order to examine this possibility, the genomic DNAs of HeLa cells and the four hybrid cell lines were compared by Southern blot hybridization analysis with radiolabelled RAR-f probes covering the exon sequences (cDNA) and the 5'-control region [5 kb HindIll-BamHI fragment contained within clone Dl, which represents the control region of the RAR-,32 isoform (Zelent et al., 1991)], respectively. Identical hybridization patterns were obtained (data not shown), indicating that no gross differences in RAR-,B structure exist in the different cells. Furthermore, the nucleotide sequence of the RAR-,32 control region was determined from HeLa and placenta DNA. After PCR amplification and BamHI cleavage, a 906 bp segment (extending from the BgIII up to the BamHI site; positions -746 to + 160, de ThM et al., 1990; Hoffmann et al., 1990), was cloned and DNA sequence analysis was performed with 13 HeLa and three placental clones. The HeLa sequence was identical to the placental sequence including the RAR-3 RARE element, TATA box and cap site (data not shown). These results render it very unlikely that the RA-

Fig. 8. Comparison of RA-mediated inducibility of RAR-(32 control region and RARE elements. Tumorigenic HeLa and non-tumorigenic 444 cells were transfected with 10 itg DNA of plasmid Dl (A) or RARE3-tk-luc (B) and 4 Ag pAc-Gal. Luciferase activity (RLU) was measured after treatment of cells with 10-5 M RA (filled bars) or DMSO only (open bars) for 24 h as described in Materials and methods. Values are the means of at least eight independent transfections, the standard deviations are indicated.

unresponsiveness of the RAR-(l gene in the tumorigenic cells is caused by mutations in the RARE or other regulatory elements of the RAR-,B2 promoter region. Activity of RAR- promoter and RARE in HeLa and non-tumorigenic hybrid cells Alternatively, differences in trans-acting factors could account for the different RA-responsiveness of the endogenous RAR-,B genes. Using the two luciferase reporter constructs Dl (containing the 5 kb HindJJ-BamHI RAR-(3 control region fragment) and RARE3 -tk-luc (containing three copies of a RARE oligonucleotide linked to the tk promoter; de The et al., 1990), we analysed the promoter activity in HeLa and non-tumorigenic 444 cells in the absence and presence of 10-5 M RA. RA-mediated increase of luciferase activity was significantly lower in HeLa cells (4-fold) than in 444 cells (20-fold) (Figure 8A). In contrast, RA-mediated induction of the chimeric RARE3 -tk promoter was similar in HeLa and 444 cells (Figure 8B). 2287

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Discussion This study has shown that RA-mediated inhibition of cell growth in vitro of HeLa and HeLa hybrid cells is associated with a reduction of HPV18 E6/E7 mRNA levels. The negative effect of RA on cell proliferation and HPV18 gene expression in vitro was seen in cells of all five cell lines irrespective of their tumorigenic (HeLa, HeLa x fibroblast hybrid segregant CGL3, HeLa x keratinocyte hybrid segregant P6TR1.2) or non-tumorigenic (HeLa x fibroblast hybrid 444, HeLaxkeratinocyte hybrid P6) phenotype in vivo. In contrast, the RA-mediated effects on RAR-fl gene expression in vitro were linked to the growth phenotype in vivo: RA-treatment of the non-tumorigenic cells (444, P6) resulted in induction of RAR-,B mRNA levels, whereas RAR(3 transcripts in RA-treated tumorigenic hybrid segregant (CGL3, P6TR1.2) and HeLa cells remained at very low levels. The RA-mediated reduction of HPV18 E6/E7 mRNA levels seems to be caused mainly by transcriptional repression. This conclusion is deduced, first, from nuclear run-on experiments that revealed a reduced transcription of the endogenous HPV18 E6 and E7 genes in RA-treated HeLa and HeLa hybrid cells. Second, transient transfection experiments in which the luciferase reporter gene under control of the HPV18 URR was cotransfected with RAR genes showed that the ligand-activated receptors could significantly reduce the activity of the HPV18 URR. In the absence of cotransfected RARs, RA treatment did not result in inhibition of expression of the HPV18 (and HPV16) URR-luciferase construct. This indicates that the endogenous levels of RARs in HeLa cells are not sufficient for inhibition of the exogenous HPV18 URR. A similar observation has been made for RA-induced activation of the RA-responsive element of the laminin B1 gene which was reported to require the presence of cotransfected RARs in transient transfection assays (Vasios et al., 1989). Analysis of HPV18 URR deletion mutants revealed that RAR-mediated repression probably occurs via regulatory elements located in the central enhancer of the HPV18 URR. The HPV18 and HPV16 central enhancers contain binding sites for several cellular transcription factors including NFl, Oct-I and API (Garcia-Carranca et al., 1988; Chong et al., 1991; Hoppe-Seyler et al., 1991; Mack and Laimins, 1991), and, in the case of HPV18, for a recently identified keratinocyte-specific transcriptional activator called KRF-1 (Mack and Laimins, 1991). It has been shown that API is an essential activator of the HPV16 and HPV18 enhancers (Chan et al., 1990; Chong et al., 1990; Cripe et al., 1990) and that KRF-1 interacts with API for activation of HPV18 E6/E7 gene transcription (Mack and Laimins, 1991). In contrast, Oct-I has been shown to repress the HPV18 enhancer in a manner that does not require DNA binding of Oct-I (Hoppe-Seyler et al., 1991). Interestingly, recent analyses of several cellular genes have shown that API-mediated transcriptional activation can be repressed by RARs (Nicholson et al., 1990; Schule et al., 1991) as can, vice versa, RAR-mediated activation be repressed by API (Schule et al., 1990) (for review see Schule and Evans, 1991; Miner and Yamamoto, 1991). Thus it seems an intriguing possibility that RAR-mediated repression of HPV18 E6/E7 transcription occurs through interference of RAR with API. Support for this possibility is given by our observation that a 42 bp fragment containing the APl binding 2288

site of the HPV 1 8 central enhancer confers RAR-dependent repression on the heterologous tk promoter (unpublished data). Work is in progress in order to determine the mechanism of repression, the mode of RAR action and the

involvement of API and/or other transcription factors. The data presented in this study indicate that the RARs can act as suppressors of HPV genes at least under certain conditions. In this study, a pharmacological concentration of RA (10-5 M) was used for most experiments. This experimental condition was chosen because earlier work by others had shown that inhibition of HeLa cell proliferation by RA is dose-dependent with 10-5 M RA being most effective yet not cytotoxic (Lotan et al., 1980; Dion and Gifford, 1980). Retinoids are used as therapeutic agents for treatment of various epithelial diseases, including HPVlinked cervical intraepithelial neoplasias (Lippman et al., 1987). Our data provide a hint towards at least one mechanism by which retinoic acid may act at the molecular level in the therapy of HPV-induced lesions. It remains to be clarified whether RARs play a role in the negative regulation of HPV gene expression at physiological RA concentrations and in HPV-infected epithelia in vivo. Furthermore, it will be of interest to analyse the possible participation of other nuclear receptors in RA-mediated regulation of HPV E6/E7 gene expression. Recent reports of heterodimer formation between RAR and the retinoid X receptor RXR (Bugge et al., 1992; Kliewer et al., 1992; Leid et al., 1992; Yu et al., 1991; Zhang et al., 1992) may be particularly relevant in this respect. The other result of our study is that the non-tumorigenic and tumorigenic cells of the HeLa/HeLa hybrid cell system showed a clear difference in the response of RAR-,B gene expression to RA. In contrast to the non-tumorigenic hybrid cells, the two tumorigenic hybrid segregants of HeLa x fibroblast and HeLa x keratinocyte origin, respectively, and HeLa cells failed to respond to RA treatment with induction of RAR-f mRNA levels. The molecular changes which are the cause of the differences in RAR-j3 expression are not yet known. Our data, however, indicate that differences in trans-regulating factors are more likely than cis-effects. First, by Southern blot hybridization and nucleotide sequence analysis, we could not detect any obvious structural abnormalities of the protein-coding and regulatory parts of the RAR-(3 gene in the tumorigenic cells. Second, transient transfection experiments (using construct Dl with the region from -5 kb to + 155, de The et al., 1990) revealed that the RA responsiveness of the RAR-,B2 control region was 5-fold lower in HeLa cells than in nontumorigenic 444 cells (Figure 8). Interestingly, this difference was not observed with a construct containing a triple RARE element linked to the tk promoter. These results suggest that transcription factors not acting through the RARE element and thus probably different from the RARs are essential regulators of RAR-( promoter activity and that either quantitative or qualitative differences in these transacting factors account for the different RAR-,B expression in the tumorigenic and non-tumorigenic cells. The factors remain to be identified. Possible candidates are API or nuclear receptors able to interact with RARs. The correlation between RA-mediated inducibility of RAR-f in vitro and the non-tumorigenic phenotype in vivo implies that the RAR-j3 gene may be causally involved in determination of the non-tumorigenic phenotype of HeLa hybrid cells. The RAR-,B gene is discussed as a candidate -

Retinoic acid regulation of HPV18 E6/E7 and

tumour suppressor gene since it maps to a region on the short arm of chromosome 3 that is frequently deleted in carcinomas of the lung (Naylor et al., 1987; Houle et al., 1991), kidney (Zbar et al., 1987; Kovacs et al., 1988) and uterine cervix (Yokota et al., 1989). In addition, RAuninducibility of RAR-3 mRNAs with no apparent changes in gene structure has also been demonstrated in cell lines derived from oral squamous cell carcinomas (Hu et al., 1991) and lung tumours (Gebert et al., 1991). At present, however, the alternative possibility that the abnormally low expression of RAR-3 in the tumorigenic cells is only a symptom of alterations linked to tumorigenicity cannot be ruled out. With regard to cervical carcinomas, our data establish the HeLa hybrid cells as a model system particularly suited for deciphering the role of the RAR-j3 gene itself and/or transcription factors regulating RAR-,B gene expression in carcinogenesis. Since growth inhibition and down-regulation of HPV18 mRNA levels were observed after RA treatment in both the non-tumorigenic and tumorigenic cells in vitro, these two responses are obviously not caused by the increased RAR-fl mRNA levels. Under in vivo conditions after heterografting of cells into nude mice, down-regulation of HPV E6/E7 mRNAs has been demonstrated in the non-tumorigenic HeLa x fibroblast hybrid cells as well as in HPV 16immortalized non-malignant keratinocytes (Bosch et al., 1990; Durst et al., 1991). It remains to be analysed whether the RAR-,B gene and/or other nuclear receptors are involved in this down-regulation in vivo. It should be mentioned that chromosome 11 has clearly been implicated in the suppression of tumorigenicity of cervical carcinoma cell lines HeLa and SiHa and of HeLa x fibroblast hybrids (Saxon et al., 1986; Srivatsan et al., 1986; Koi et al., 1989). Thus, the potential role of RAR-,B in regulation of cell proliferation should also be viewed in the context of these findings.

Materials and methods Cell lines and cell culture conditions Beside HeLa cells, two pairs of hybrid cell lines derived from fusions of HeLa cells with either human fibroblasts or human keratinocytes were used: (i) the non-tumorigenic HeLaxfibroblast hybrid cell line 444 and the tumorigenic segregant line CGL3 (Stanbridge, 1976) and (ii) the nontumorigenic HeLaxkeratinocyte hybrid cell line ESH100 P6 and the tumorigenic segregant P6TRI.2 (Peehl and Stanbridge, 1982). The hybrid cells were kindly provided by Dr Eric Stanbridge (Irvine, USA). HeLa cells and the hybrid cells were grown in tissue culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, glutamine and 100 U penicillin/1000 U streptomycin. All-trans-RA (Sigma, Munchen, FRG) was dissolved in dimethylsulphoxide (DMSO) at a concentration of 10-2 M. One day after seeding of cells, the normal medium was replaced by medium supplemented with i0-5 M RA and 0.1% (v/v) DMSO, or for control cultures with 0.1% (v/v) DMSO alone. Medium was changed every third day. Alternatively, cells were grown for 12 h in medium containing 10% charcoal-stripped serum before they were treated with 10-7 M RA. Cycloheximide (Boehringer, Mannheim, FRG) and actinomycin D (Sigma, Munchen, FRG) were used at concentrations of 50 and 10 Ag/ml, respectively. The percentage of growth inhibition by RA was calculated after determination of cell numbers in RA-treated and control cultures. RNA isolation and Northern blot analysis Cytoplasmic RNA was isolated by lysis of cells in a solution containing 0.5% NP-40, 0.15 M NaCl, 10 mM Tris-HCI (pH 7.5) and 1 mM EDTA (Meinkoth and Wahl, 1984). Alternatively, total RNA was isolated with acid guanidinium isothiocyanate as described (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography. For Northern blot analysis, RNA samples were denatured and fractionated in 1 % agarose gels under non-denaturing conditions

RAR-f

(Khandijian and Meric, 1986). RNAs were transferred to GeneScreen nylon membranes (Dupont NEN, Dreieich, FRG) using the Posiblot pressure blotter (Stratagene, Heidelberg, FRG) and attached by UV exposure plus baking. DNA fragments were labelled with 32P by random priming (Feinberg and Vogelstein, 1983). Hybridization was performed in S xSSC (1 xSSC is 0.15 M NaCl, 0.015 M sodium citrate) either at 42°C in the presence of 50% formamide or at 68°C. Filters were washed in 2 x SSC, 0.1 % SDS at 680C and were exposed to Kodak XAR films at -70°C using intensifier screens. DNA isolation and Southern blot analysis DNA was isolated from cells by proteinase K treatment and phenol extraction (Sambrook et al., 1989). For Southern blot analysis, DNAs were cleaved with appropriate restriction endonucleases, separated in 0.8% agarose gels and transferred to nylon membranes. Hybridization and washing conditions were as described for RNA.

Recombinant plasmids and DNA probes Plasmid p2.2 harbouring the HPV18 cDNA H4 derived from HeLa cells (Schneider-Gadicke and Schwarz, 1986) and plasmid pBE2.4 containing a 2.4 kb BamHI-EcoRI insert of genomic HPV18 DNA were used for preparation of fragments specific for HPV18 open reading frames E6* + E7 and E6 + E7 + El, respectively. Plasmid p18 contains the complete HPV18 URR (cloned from HeLa HPV18) linked to the luciferase reporter gene; plasmid p436/18L contains the HPV18 URR central enhancer plus proximal promoter (Hoppe-Seyler et al., 1991). In construct p230/tk*L, the HPV 18 URR central enhancer is cloned upstream of a truncated HSV tk promoter fragment covering positions -79 to -1 (tk*). The HPV18URR-luciferase constructs were kindly provided by Karin Butz and Felix Hoppe Seyler. Plasmid pl6L was constructed by cloning the HPV16 URR (nucleotide positions 7007-102) into the PstI and BamHI sites of pBL. A 790 bp RAR-,B specific DNA fragment was prepared by cleavage of RAR-,B-ER-CAS plasmid DNA with XhoI and BamHI (Brand et al., 1988). Plasmids hRAR-a, hRAR-,B and hRAR--y contain the cDNAs of the human RAR-c 1, RAR-132 and RAR--y receptor, respectively, cloned into the eukaryotic expression vector pSG5 (Petkovich et al., 1987; Brand et al., 1988; Krust et al., 1989). Samples of all RAR plasmid DNAs were kindly provided by Dr Pierre Chambon (Strasbourg, France). Plasmid DI contains a 5 kb insert with the RA-responsive RAR-032 promoter linked to the luciferase reporter gene. Construct RARE3 -tk-luc includes three copies of a RARE-containing 27mer oligonucleotide inserted upstream of the tk promoter (de The et al., 1990). DNA of plasmids Dl and RARE3-tk-luc was kindly provided by Dr Anne Dejean (Paris, France). Plasmids pCMVLuc, pHMG-Luc, pAc-Gal and pBL were kindly provided by Martin Rentrop and Andrea Klotzbucher (DKFZ, Heidelberg) and are described in HoppeSeyler et al. (1991). Plasmid pSV40-Luc was constructed by subcloning a PvuII-HindIll fragment of the SV40 early promoter/enhancer from pSV2neo (Southern and Berg, 1982) into pBL digested with SnaI and Hindlf. Plasmid pRC 1.3 containing a ClaI-EcoRI fragment of c-myc exon 3 and plasmid 421 -1 containing c-myc exon 1 were kindly provided by Axel Pollack and Georg Bornkamm (GSF, Munchen), plasmid pHF,3 A-I with a full-length cDNA of human ,B-actin was kindly provided by Peter

Gunning (Stanford). PCR amplification and DNA sequence analysis A RAR-,32 fragment was amplified by PCR from HeLa and placental DNA, respectively, by using as primers the oligonucleotides

5'-GGGATCCGAAATCTCATTTTCTG-3' [corresponding to positions -746 to -724 of the RAR-(2 DNA sequence (Hoffmann et al., 1990) with a change of the genuine Bglll site into a BamHI site] and 5'-ACTGACAGAACATCCATACAGT-3' [an A-region antisense oligonucleotide, positions 350-329 (de The et al., 1987)]. Amplification was performed using TaqI polymerase in a DNA thermal cycler (Perkin-Elmer Cetus). The amplification product was digested with BamHI and cloned into pBluescript KS + . Sequencing was performed by the dideoxy method (Sanger et al., 1977) with universal primers (Pharmacia, Freiburg, FRG) and RAR-,B2 specific oligonucleotide primers.

Nuclear run-on analysis Cells were grown for three days in the presence or absence of l0o- M RA. After washing with PBS, cells were scraped with a rubber policeman in 50 ml ice-cold NP [10 mM Tris-HCI pH 7.4, 10 mM NaCI, 3 mM MgCl2, 0.5% (v/v) NP40], incubated for 10 min on ice and centrifuged at 500 g. The nuclear pellet was dissociated in 15 mi NP by 10 strokes of a tight pestle in a Dounce homogenizer and centrifuged again. Nuclei were resuspended at a concentration of 108/ml in RSB (10 mM Tris-HCI pH 8.0, 150 mM KCI, 5 mM MgCl2, 0.25 mM each of GTP, ATP and UTP). The nuclear run-on assay was performed accordinf to Eick and Bornkamm (1986) with slight modifications. Briefly, 2 x 10 nuclei in 200

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LI RSB were mixed with 150 U RNAsin (Promega, Heidelberg, FRG) and 200 ltCi Ia-32P]CTP (800 Ci/mmol, Amersham, Braunschweig, FRG) and incubated at 28°C for 30 min with gentle agitation. Subsequently, DNase I (100 U, Boehringer, Mannheim, FRG) was added and the mixture was incubated for an additional 5 min. The reaction was terminated by adding 200 Al 2xTSE (100 mM Tris-HCI pH 8.0, 50 mM EDTA, 2% SDS) and 200 jsg proteinase K. After digestion at 60°C for 1 h, nuclear run-on transcripts were purified according to Chomczynski and Sacchi (1987). 32Plabelled RNAs were dissolved in 5 xSSC/l % SDS and hybridized at 68'C to nylon membranes containing 10 sg each of recombinant plasmid DNAs digested with appropriate restriction enzyme and blotted from 0.8% agarose gels after electrophoretic separation. Following hybridization for 48 h, the filters were processed as described (Eick and Bornkamm, 1986). Transient transfection and luciferase expression analysis Transfection of cells was performed by calcium phosphate precipitation according to Chen and Okayama (1987). Briefly, 10 jig reporter luciferase plasmid, 4 jAg pAc-Gal plasmid and, when needed, either 2 yg RAR expression vector DNA or 2 jig pSV2neo (as a negative control) were mixed and adjusted to 20 itg by addition of pBluescript DNA. 106 cells in 10 cm Petri dishes were incubated for 18 h with calcium phosphate/DNA mixture at 35°C and 3% CO2. For RA treatment, cells were washed with PBS, trypsinized and divided into two new Petri dishes in DMEM with 10% serum supplemented with either 10-5 M RA or DMSO solvent only. Cells were harvested after 24 h and luciferase activity was measured according to Brasier et al. (1989). Photon counts were standardized on ,B-galactosidase activity of cotransfected pAc-Gal which was measured as described (Sambrook et al., 1989). Relative luciferase activity (RLU) was calculated as the ratio of standardized luciferase activity of reporter plasmid to standardized luciferase activity of either promoterless pBL plasmid or pBLtk* promoter plasmid (for plasmid p230/tk*L). At least eight independent transfections were performed for every reporter plasmid. Luciferase and ,B-galactosidase activity measurements were done in duplicate. Standard deviations for every experiment were determined and generally did not exceed 10%.

Acknowledgements We wish to thank Pierre Chambon and Anne Dejean for generously giving RAR and RAR-(3 promoter plasmids, respectively, Felix Hoppe-Seyler and Karin Butz for HPV18 URR plasmids and for discussion, Andrea Klotzbucher and Martin Rentrop for luciferase expression plasmids, H.G.Stunnenberg and M.Durst for communication of data prior to publication, and Wolfgang Weinig for oligonucleotide synthesis. This work was supported by a grant (Schw250-2/3) from the Deutsche Forschungsgemeinschaft to E.S. and D.B.

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