Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16 (HPV16)-Immortalized Human...

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EXPERIMENTAL CELL RESEARCH 236, 304–310 (1997) ARTICLE NO. EX973729 Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16 (HPV16)-Immortalized Human Keratinocytes and Support HPV16-Mediated Immortalization without Affecting the Levels of HPV16 E6/E7 mRNA 1 Mohammad Ali Khan,* Alfredo J. Canhoto,² Paul R. Housley,‡ Kim E. Creek,* , § and Lucia Pirisi* ,2 *Department of Pathology, Department of Pharmacology, and §Children’s Cancer Research Laboratory, Department of Pediatrics, University of South Carolina School of Medicine, and ²Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208 and malignant lesions of the cervix [reviewed in 1]. Clinical and epidemiological evidence shows that HPV We investigated the effects of the glucocorticoids hy- drocortisone and dexamethasone on human papillo- infection alone is not sufficient to lead to malignancy mavirus type 16 (HPV16)-mediated human cell carci- and that other factors must contribute to producing nogenesis using normal human keratinocytes (HKc) a malignant phenotype. An increased risk of cervical and HKc immortalized by transfection with HPV16 neoplasia has been associated with long-term use of DNA (HKc/HPV16). Normal HKc did not require gluco- oral contraceptives [2] and pregnancy [3]. These obser- corticoids for proliferation. In contrast, growth of vations led to investigations of the effects of steroid early passage HKc/HPV16 strictly required these hor- hormones on HPV gene expression and HPV-mediated mones, although glucocorticoid dependence became transformation [4 – 10] and to the discovery and charac- less stringent during in vitro progression. Glucocorti- terization of progesterone and glucocorticoid-respon- coid dependence was acquired by HKc early after im- sive elements (GREs) in the upstream regulatory re- mortalization with HPV16 DNA, and glucocorticoids gion (URR) of various HPVs [11–16]. were required for efficient HKc immortalization. How- A powerful influence of glucocorticoids on HPV-medi- ever, treatment of HKc/HPV16 with hydrocortisone or ated transformation has also been described. Glucocor- dexamethasone did not increase the steady-state lev- ticoids have been shown to enhance neoplastic transfor- els of HPV16 E6/E7 mRNA or protein. Firefly luciferase mation of HPV16 immortalized human keratinocytes activity expressed under the control of the HPV16 up- (HKc) with the K-ras oncogene [5] and to increase the stream regulatory region and P97 promoter increased efficiency of formation of calcium/serum-resistant colo- by about fourfold following dexamethasone treatment nies in HKc electroporated with HPV16 or HPV18 DNA of HeLa, but only twofold in HKc/HPV16, and less than [6]. Glucocorticoids are important for HPV-mediated twofold in SiHa. However, all of these cell lines ex- transformation of rodent cells as well [7–10]. It has pressed sufficient endogenous glucocorticoid recep- been assumed that glucocorticoid dependence for effi- tors to allow for a dexamethasone response of the cient transformation by HPV was mediated by a direct mouse mammary tumor virus promoter. These results effect of these hormones on HPV gene expression, indicate that mechanisms other than a direct influence through the GREs in the HPV16 and 18 URR [11– by glucocorticoids on HPV16 early gene expression 16]. However, in cervical carcinoma-derived cell lines may contribute to the striking biological effects of containing integrated HPV18 DNA, cell growth and these steroids on HPV16-mediated human cell carcino- endogenous HPV18 E6/E7 transcription were not al- genesis. q 1997 Academic Press ways enhanced by dexamethasone. In some cell lines (C4-1 and C4-2) glucocorticoids increased growth and INTRODUCTION E6/E7 transcription [17]. In SW 756, however, dexa- Human papillomavirus type 16 (HPV16) and other methasone led to a significant reduction of cell growth oncogenic HPVs are etiologic agents of premalignant and E6/E7 expression [18], while in HeLa, despite the presence of sufficient levels of glucocorticoid receptors, 1 Supported by NIH Grants R29-CA48990 and RO1-CA62094 to dexamethasone had no effect on growth or on HPV18 L.P., the South Carolina Endowment for Children’s Cancer Research, gene expression [18]. Furthermore, in BRK cells and Grant RO1-DK47951 to P.R.H. The first two authors contributed HPV16 E6 and E7 transcription was not affected by equally to these studies. RU486, in spite of its significant inhibition of transfor- 2 To whom reprint requests should be addressed. Fax: (803) 733- 1515. E-mail: [email protected]. mation by HPV16 and Ha ras [8]. 304 0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

Transcript of Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16 (HPV16)-Immortalized Human...

Page 1: Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16 (HPV16)-Immortalized Human Keratinocytes and Support HPV16-Mediated Immortalization without Affecting the Levels of

EXPERIMENTAL CELL RESEARCH 236, 304–310 (1997)ARTICLE NO. EX973729

Glucocorticoids Stimulate Growth of Human Papillomavirus Type 16(HPV16)-Immortalized Human Keratinocytes and Support

HPV16-Mediated Immortalization without Affectingthe Levels of HPV16 E6/E7 mRNA1

Mohammad Ali Khan,* Alfredo J. Canhoto,† Paul R. Housley,‡ Kim E. Creek,*,§ and Lucia Pirisi*,2

*Department of Pathology, ‡Department of Pharmacology, and §Children’s Cancer Research Laboratory, Departmentof Pediatrics, University of South Carolina School of Medicine, and †Department of Chemistry and Biochemistry,

University of South Carolina, Columbia, South Carolina 29208

and malignant lesions of the cervix [reviewed in 1].Clinical and epidemiological evidence shows that HPVWe investigated the effects of the glucocorticoids hy-

drocortisone and dexamethasone on human papillo- infection alone is not sufficient to lead to malignancymavirus type 16 (HPV16)-mediated human cell carci- and that other factors must contribute to producingnogenesis using normal human keratinocytes (HKc) a malignant phenotype. An increased risk of cervicaland HKc immortalized by transfection with HPV16 neoplasia has been associated with long-term use ofDNA (HKc/HPV16). Normal HKc did not require gluco- oral contraceptives [2] and pregnancy [3]. These obser-corticoids for proliferation. In contrast, growth of vations led to investigations of the effects of steroidearly passage HKc/HPV16 strictly required these hor- hormones on HPV gene expression and HPV-mediatedmones, although glucocorticoid dependence became transformation [4–10] and to the discovery and charac-less stringent during in vitro progression. Glucocorti- terization of progesterone and glucocorticoid-respon-coid dependence was acquired by HKc early after im- sive elements (GREs) in the upstream regulatory re-mortalization with HPV16 DNA, and glucocorticoids gion (URR) of various HPVs [11–16].were required for efficient HKc immortalization. How- A powerful influence of glucocorticoids on HPV-medi-ever, treatment of HKc/HPV16 with hydrocortisone or ated transformation has also been described. Glucocor-dexamethasone did not increase the steady-state lev- ticoids have been shown to enhance neoplastic transfor-els of HPV16 E6/E7 mRNA or protein. Firefly luciferase mation of HPV16 immortalized human keratinocytesactivity expressed under the control of the HPV16 up- (HKc) with the K-ras oncogene [5] and to increase thestream regulatory region and P97 promoter increased efficiency of formation of calcium/serum-resistant colo-by about fourfold following dexamethasone treatment nies in HKc electroporated with HPV16 or HPV18 DNAof HeLa, but only twofold in HKc/HPV16, and less than

[6]. Glucocorticoids are important for HPV-mediatedtwofold in SiHa. However, all of these cell lines ex-transformation of rodent cells as well [7–10]. It haspressed sufficient endogenous glucocorticoid recep-been assumed that glucocorticoid dependence for effi-tors to allow for a dexamethasone response of thecient transformation by HPV was mediated by a directmouse mammary tumor virus promoter. These resultseffect of these hormones on HPV gene expression,indicate that mechanisms other than a direct influencethrough the GREs in the HPV16 and 18 URR [11–by glucocorticoids on HPV16 early gene expression16]. However, in cervical carcinoma-derived cell linesmay contribute to the striking biological effects ofcontaining integrated HPV18 DNA, cell growth andthese steroids on HPV16-mediated human cell carcino-endogenous HPV18 E6/E7 transcription were not al-genesis. q 1997 Academic Press

ways enhanced by dexamethasone. In some cell lines(C4-1 and C4-2) glucocorticoids increased growth and

INTRODUCTION E6/E7 transcription [17]. In SW 756, however, dexa-Human papillomavirus type 16 (HPV16) and other methasone led to a significant reduction of cell growth

oncogenic HPVs are etiologic agents of premalignant and E6/E7 expression [18], while in HeLa, despite thepresence of sufficient levels of glucocorticoid receptors,

1 Supported by NIH Grants R29-CA48990 and RO1-CA62094 to dexamethasone had no effect on growth or on HPV18L.P., the South Carolina Endowment for Children’s Cancer Research, gene expression [18]. Furthermore, in BRK cellsand Grant RO1-DK47951 to P.R.H. The first two authors contributed HPV16 E6 and E7 transcription was not affected byequally to these studies.

RU486, in spite of its significant inhibition of transfor-2 To whom reprint requests should be addressed. Fax: (803) 733-1515. E-mail: [email protected]. mation by HPV16 and Ha ras [8].

3040014-4827/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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305GLUCOCORTICOIDS IN HPV16-MEDIATED TRANSFORMATION

Northern blot analysis. Normal HKc and HKc/HPV16 wereTaken together, these data suggest that, althoughplated in complete medium and grown until 50% confluent. Cellsfunctional GREs are present in the HPV16 and HPV18were then washed with Dulbecco’s phosphate-buffered saline (PBS),URR, the influence of glucocorticoids on the overall lev- incubated in the absence of glucocorticoids for 48 h, and then treated

els of transcription from the respective early promoters with medium containing glucocorticoids or with medium devoid ofsteroids for an additional 72 h. Total RNA was isolated by the guanid-may vary depending on the host cell type, the natureinium thiocyanate/cesium trifluoroacetate gradient centrifugationof the genomic sequences flanking integrated HPVmethod and subjected to Northern blot analysis with a specificDNA, or other factors. We therefore decided to studyHPV16 E6/E7 probe and a b2-microglobulin probe as previously de-

the role of glucocorticoids in HPV16-mediated transfor- scribed [25].mation in our model system for multistep human cell Quantification of E6 and E7 protein levels. Immunofluorescencecarcinogenesis in vitro [19–23]. These studies con- and adherent cell analysis (ACAS) (Meridian) were conducted as

previously described [25, 26] using an anti-HPV16 E7 monoclonalfirmed that glucocorticoids play a pivotal role in en-antibody (Ciba Corning) or an anti-HPV18/HPV16 E6 monoclonalhancing HPV16-mediated transformation of HKc, as inantibody (Oncogene Science). Western blot analysis for E7 was alsoother models [5–8]. However, we failed to demonstrate conducted using the anti-HPV16 E7 monoclonal antibody as de-

a modulation by glucocorticoids of HPV16 gene expres- scribed [26].sion in HKc/HPV16, indicating that a different mecha- Electroporation and luciferase assay. To produce the plasmidnism mediates the biological effects of glucocorticoids p16URR-Luc,3 a segment of the HPV16 genome encompassing most

of the URR and the p97 promoter (nucleotides 7232-119) was ampli-in this system.fied by PCR and cloned upstream of the firefly luciferase gene in theplasmid pGL2-Basic (Promega). Cells were cultured for 24 h in eitherMATERIALS AND METHODSMCDB153-LB devoid of glucocorticoids (HKc/HPV16) or MEM sup-plemented with 10% charcoal-stripped FBS (HeLa, SiHa) and elec-Materials. Hydrocortisone and dexamethasone were purchasedtroporated with 10 mg of plasmid DNA (pAH-Luc or p16URR-Luc).from Sigma Chemical Co. The 11a-hydrocortisone was obtained fromAfter electroporation the cells were split equally into six dishes, threeSteraloid Inc. RU486 was a gift from Roussel-UCLAF. [3H]-of which were treated with dexamethasone (1007 M) in steroid-freeTriamcinolone acetonide (30–50 Ci/mmol) and [3H]dexamethasoneMCDB153-LB or in MEM supplemented with charcoal-stripped FBS.(35–50 Ci/mmol) were purchased from New England Nuclear. TheThe remaining three dishes received ethanol (0.1%) in place of dexa-plasmid pAH-Luc, which contains the mouse mammary tumor virusmethasone, in the respective media. After 48 h of treatment the(MMTV) promoter upstream of the firefly luciferase gene [24], wascells were lysed and luciferase activity was determined using thea gift from Dr. Steve K. Nordeen. The cell lines SiHa and HeLa wereluciferase assay reagent (Promega).from American Type Culture Collection.

Glucocorticoid receptor binding assay. Glucocorticoid receptorCell culture and cell lines. Normal HKc were isolated from neona-binding assays were conducted as previously described [27]. Briefly,tal foreskin and cultured in serum-free MCDB153-LB medium ascells were cultured for 7 days either in serum-free MCDB153-LBpreviously described [19], except that trypsin (0.25% in Hanks’ buf-devoid of glucocorticoids (normal HKc and HKc/HPV16) or in MEMfered saline, at 47C, for 18–22 h) was used in place of collagenase(SiHa and HeLa) supplemented with 10% charcoal-stripped FBS.to separate the epidermis from the dermis. The HKc/HPV16 linesCells were lysed by sonication in ice-cold lysis buffer (10 mM Hepes,used in this study have been previously described and characterizedpH 7.35, 1 mM EDTA, 10 mM sodium molybdate, 0.2 mM phenyl-[19–23]. SiHa and HeLa were cultured in MEM supplemented withmethylsulfonyl fluoride, 1% aprotinin, 2 mM dithiothreitol) and the10% fetal bovine serum (FBS), penicillin, and streptomycin. Clonallysates cleared by centrifugation (130,000g for 15 min). Protein con-growth and mass culture growth assays were conducted as previouslycentration in the cell lysates was then determined by the DC proteindescribed [21–23]. Briefly, for clonal growth assays, cells were platedassay (Bio-Rad). Aliquots (0.1 ml) of each lysate were incubated onat low density (1000 cells/dish) in 60-mm dishes and then refed, 24ice for 3 h with 50 nM [3H]triamcinolone acetonide, or 50 nM [3H]-h after plating, with 8 ml of medium containing no glucocorticoidsdexamethasone, in the absence or in the presence of a 1000-foldor various concentrations of hydrocortisone or dexamethasone. Cellsmolar excess of unlabeled dexamethasone (to determine total andwere allowed to grow clonally for 10–12 days and then fixed in meth-nonspecific binding, respectively). Free 3H-labeled steroids were ad-anol and stained with Giemsa. The area occupied by colonies in eachsorbed when each sample was mixed with 0.15 ml of a charcoaldish was then measured by computerized image analysis. For masssuspension (1% activated charcoal, 0.1% dextran in 10 mM Hepes,culture growth assays, cells were plated (20,000 cells/dish) in 35-mmpH 7.35). Radioactivity in the supernatant was determined afterdishes and treated, 24 h after plating and every 48 h thereafter, withcentrifugation at 12,000g for 5 min.either glucocorticoid-free MCDB153-LB or MCDB153-LB supple-

mented with hydrocortisone or dexamethasone. Triplicate dishes/condition were trypsinized at various times after plating and the

RESULTScells counted on a hemocytometer.Immortalization assay. Normal HKc at passage 2 were plated

(25,000 cells/dish) and transfected, using Lipofectin reagent, with 10 Glucocorticoids Are Not Required for the Proliferationmg/dish of HPV16 DNA (pMHPV16d) [19] or high-molecular-weight of Normal HKc and Are Growth-Inhibitory forcalf thymus DNA, in triplicate 35-mm dishes, in MCDB153-LB con- Some Normal HKc Strainstaining 200 nM hydrocortisone (complete medium). The next dayeach plate was passed into two 100-mm dishes, again in complete We first determined the effects of glucocorticoids onmedium. The dishes were split into two groups, 24 h after plating:

the growth of 17 individual normal HKc strains usingOne group was fed with complete medium, while the other groupclonal growth assays. In 13 of 17 strains hydrocortisonewas fed with MCDB153-LB without hydrocortisone every 48 h. The

cells were allowed to grow until calf thymus DNA-transfected cellsdifferentiated and died. Colonies of transformed cells which devel-

3 A. J. Canhoto, G. R. Jenkins, L. Pirisi, and K. E. Creek, manu-oped in the presence and in the absence of glucocorticoids werecounted, after fixing with methanol and staining with Giemsa. script in preparation.

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TABLE 1

Clonal Growth of Normal HKc Strainsin the Absence of Glucocorticoids

Cells Relative colony areaa { SD

Normal HKc (n Å 13) 104 { 20Normal HKc K18 410 { 54Normal HKc K19 158 { 6Normal HKc K21 202 { 34Normal HKc K23 176 { 5

Note. Clonal growth assays were conducted on 17 different individ-ual normal HKc strains, in the absence of steroid or in the presenceof hydrocortisone (200 nM ), as described under Materials and Meth-ods. The total area of the dishes occupied by colonies was measuredby computerized image analysis.

a Relative colony area: (Colony area in the absence of steroid/Col-ony area in the presence of steroid) 1 100.

FIG. 1. Clonal growth of normal HKc and HKc/HPV16 in theabsence of glucocorticoids. Clonal growth assays were conducted us-ing normal HKc (NHKc) and the indicated HKc/HPV16d lines (HKc/did not significantly affect total colony area (Table 1).HPV16d-1 to -3) at low passage (õ40) as described under Materials

However, glucocorticoids inhibited growth of 4 normal and Methods, in medium devoid of steroids or containing hydrocorti-HKc strains, which grew significantly better in the ab- sone (200 nM). The average colony area was determined by computer-

ized image analysis of the dishes, after staining with Giemsa. Thesence than in the presence of hydrocortisone (Table 1).total colony area in the absence of glucocorticoids (0GC) is expressed11a-Hydrocortisone, an inactive steroid which does notas a percentage of the colony area in the presence of hydrocortisonebind to the glucocorticoid receptor, had no effect on (/GC) for each cell line. The data are averages { SD of at least three

clonal growth of normal HKc (data not shown). determinations.

Glucocorticoids Are Required for the Proliferation ofEarly Passage HKc/HPV16; However, Late Passage Acquisition of Glucocorticoid Dependence by HKcHKc/HPV16 and Growth Factor-Independent HKc/ after Immortalization with HPV16HPV16 Become Relatively Glucocorticoid

To demonstrate that the growth dependence of HKc/IndependentHPV16 on glucocorticoids was a consequence of immor-

We next investigated the effects of glucocorticoids onthe growth of independently derived HKc/HPV16 lines.Early passage (õ40) HKc/HPV16 did not proliferatewell in the absence of glucocorticoids in either clonalgrowth (Fig. 1) or mass culture growth assays (Fig. 2).11a-Hydrocortisone alone had no effect on the growthof HKc/HPV16, could not replace the active steroidsin supporting growth, and did not modify the growthstimulatory effect of glucocorticoids in these cells (datanot shown). The growth dependence of HKc/HPV16 onglucocorticoids was not absolute: HKc/HPV16d-1 atearly passage (passage 10) were more dependent onhydrocortisone than at late passage (passage 140) (Ta-ble 2). In addition, growth factor-independent HKc/HPV16 (HKc/GFI), which represent a more advancedstage of transformation in our in vitro model of HPV16-induced human cell carcinogenesis [20–23], were lessdependent on glucocorticoids than their parental HKc/HPV16 lines (Table 2). Furthermore, a glucocorticoid- FIG. 2. HKc/HPV16 require glucocorticoids for mass culture

growth. HKc/HPV16d-2 were plated in 35-mm dishes, in mediumindependent line, HKc/HPV16d-2 NS, was derivedcontaining hydrocortisone (200 nM). Cells were then refed 24 h afterfrom HKc/HPV16d-2 by selection in glucocorticoid-freeplating and cultured in the absence (open circles) or in the presencemedium. HKc/HPV16d-2 NS no longer required gluco- (solid circles) of hydrocortisone (200 nM). At the indicated times cells

corticoids for proliferation and in fact grew better in from triplicate dishes were trypsinized and cell numbers determinedby counting in a hemocytometer. Bars, SD.the absence of steroid (Table 2).

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307GLUCOCORTICOIDS IN HPV16-MEDIATED TRANSFORMATION

TABLE 2

Clonal Growth of HKc/HPV16 Lines at Various Stagesof Progression in the Absence of Glucocorticoids

Cells Relative colony areaa { SD

HKc/HPV16d-1, passage 10 24 { 10HKc/HPV16d-1, passage 140 73 { 14HKc/HPV16d-2 8 { 2HKc/GFId-2 39 { 11HKc/HPV16d-2 NS 140 { 12HKc/HPV16d-4 13 { 3HKc/GFId-4 67 { 14

Note. Clonal growth assays were conducted on HKc/HPV16 at theindicated passage numbers, HKc/GFI, and HKc/HPV16d-2 NS, inthe absence of steroid or in the presence of hydrocortisone (200 nM ),as described under Materials and Methods. The total area of the

FIG. 4. Glucocorticoids do not increase E6/E7 mRNA levels indishes occupied by colonies was measured by computerized imageHKc/HPV16. Northern blot analysis of RNA from HKc/HPV16d-3, d-analysis.4, and d-5 (passages 16, 21, and 23, respectively) incubated in thea Relative colony area: (Colony area in the absence of steroid/Col-absence of glucocorticoids (0GC) for a total of 5 days, or for 72 h inony area in the presence of steroid) 1 100.the presence of 200 nM hydrocortisone (/GC) after a 48-h preincuba-tion in the absence of steroids. Probes specific for HPV16 E6/E7 andb2-microglobulin (b2M) were prepared as described under Materialsand Methods.talization by HPV16, we studied the effects of hydrocor-

tisone on clonal growth of HKc both before and aftertransformation by HPV16 DNA. Before transformationwith HPV16 DNA, the normal HKc strain W21 grew about the same rate in the absence and in the presencewell in the absence of glucocorticoids (Fig. 3). After of hydrocortisone (data not shown).immortalization, however, these cells grew poorly inthe absence of hydrocortisone (Fig. 3). This experiment Hydrocortisone Stimulates HPV16-Mediatedwas repeated using normal HKc strain K18, which was Immortalization of Normal HKcgrowth inhibited by about 75% by glucocorticoids be-

The effects of hydrocortisone on HPV16-mediatedfore immortalization (Table 1): after immortalizationimmortalization of normal HKc were determined us-with HPV16 DNA the growth inhibitory effect of gluco-ing the immortalization assay described under Mate-corticoids on these cells was lost, and cells grew atrials and Methods. The number of immortalized colo-nies/dish averaged 22.3 { 6.2 in the presence of hy-drocortisone (200 nM), but only 1.1 { 1.5 in theabsence of glucocorticoids, showing that these hor-mones are required for efficient immortalization ofHKc by HPV16 DNA.

HPV16 E6/E7 mRNA and Protein Levels Are NotAffected by Glucocorticoids

Three independently derived HKc/HPV16 lines(HKc/HPV16d-3, -4, and -5) were cultured in the ab-sence of glucocorticoids for 48 h and then treated for72 h with or without hydrocortisone (200 nM). RNAextraction and Northern blot analysis with an HPV16E6/E7-specific probe were then performed. In none ofthe cell lines was an increase in the steady-state levelsof E6/E7 mRNA observed in the presence of hydrocorti-sone (Fig. 4). Treatment for 72 h with or without hydro-

FIG. 3. Glucocorticoid requirement of HKc before and after im- cortisone (200 nM), in the presence or in the absencemortalization by HPV16. Clonal growth assays were conducted as of 100 nM RU486, also did not affect E6/E7 mRNAdescribed under Materials and Methods, using normal HKc strain levels in HKc/HPV16d-2 (data not shown). In addition,W21 before (NHKcW21) and after immortalization with HPV16 DNA

HPV16 E6 and E7 protein levels, measured by immu-(HKcW21/HPV16). Total colony area in at least triplicate dishes/condition was measured by computerized image analysis. Bars, SD. nofluorescence and ACAS, were not affected by gluco-

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308 KHAN ET AL.

FIG. 5. Glucocorticoid modulation of luciferase activity expressed under the control of the MMTV promoter or the HPV16 URR/P97promoter in HeLa, SiHa, and HKc/HPV16. Cells were incubated in the absence of glucocorticoid for 24 h and then electroporated witheither the pAH-Luc plasmid, in which the MMTV promoter drives the expression of the luciferase gene, or the p16URR-Luc plasmid, whichcontains the HPV16 URR and P97 promoter upstream of the luciferase reporter gene. After electroporation, cells were incubated in theabsence (0GC) or in the presence of 100 nM dexamethasone (/GC), for 48 h. Cells were harvested and lysed, and luciferase assays wereconducted as described under Materials and Methods. The data are averages { SD of triplicate determinations.

corticoid removal or treatment with dexamethasone (1 of this receptor could explain the differences observednM–1 mM) for 72 h, and E7 levels measured by immu- between SiHa, HeLa, and HKc in the regulation ofnoprecipitation and Western immunoblot analysis HPV16 gene expression by glucocorticoids and whetherwere not modified by glucocorticoid removal or treat- glucocorticoid receptor levels changed with progressionment with glucocorticoids and/or RU486, for 72 h (data in vitro. No change in glucocorticoid binding was appar-not shown). ent in HKc/HPV16, compared to normal HKc. Specific

binding was decreased moderately in HKc/GFI, com-Firefly Luciferase Expressed under the Control of thepared to HKc/HPV16, while both SiHa and HeLa exhib-HPV16 URR and the P97 Promoter Is Activatedited significantly lower binding than HKc/HPV16 andDifferently by Glucocorticoids in HKc/HPV16,normal HKc (Table 3).HeLa, and SiHa

To investigate further the effects of glucocorticoids onDISCUSSIONHPV16 early gene expression we cloned the HPV16 URR,

including the promoter P97, upstream of the firefly lucif-erase gene, to produce the plasmid p16URR-Luc.3 This The results of growth assays conducted on normalconstruct was electroporated into low-passage (glucocor- HKc treated with or without glucocorticoids point toticoid-sensitive) HKc/HPV16, SiHa, or HeLa, and lucifer- some degree of inter-individual variability in the re-ase activity was determined 48 h after transfection in sponse of normal epidermal keratinocytes to glucocorti-cells incubated without glucocorticoids or with dexameth-asone (100 nM). As a positive control we used the plasmidpAH-Luc, in which the MMTV promoter drives luciferase

TABLE 3expression. As shown in Fig. 5, dexamethasone stimu-Glucocorticoid Receptor Binding in Normal HKc,lated several-fold the expression of luciferase activity

HKc/HPV16, HKc/GFI, SiHa, and HeLafrom the MMTV promoter (pAH-Luc) in all cell lines. Theextent of activation of the MMTV promoter varied in the

Specific bindingdifferent lines, with increases of 8-, 12-, and 59-fold in Cells (fmol/mg protein)HKc/HPV16, SiHa, and HeLa, respectively (Fig. 5). Incontrast, dexamethasone increased the activity of Normal HKc 327 { 13

HKc/HPV16d-1 334 { 66p16URR-Luc only about 5-fold in HeLa, 2-fold in HKc/HKc/GFId-1 204 { 21HPV16, and 1.5-fold in SiHa (Fig. 5).SiHa 121 { 6

Normal HKc and HKc/HPV16 Contain Comparable HeLa 80 { 10Levels of Glucocorticoid Receptors, while SiHa and

Note. Glucocorticoid receptor binding assays were conducted onHeLa Have Lower Glucocorticoid Receptor Levelsextracts prepared from normal HKc, HKc/HPV16, HKc/GFI, SiHa,Than HKcand HeLa after a 7-day incubation in their respective media devoid

We conducted glucocorticoid receptor binding assays of steroids, as described under Materials and Methods. The data areaverages { range of duplicate determinations.to determine whether variations in the cellular levels

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309GLUCOCORTICOIDS IN HPV16-MEDIATED TRANSFORMATION

coids. While most normal HKc strains did not require the levels of cellular factors that control the activity ofthe glucocorticoid receptor between normal or precan-glucocorticoids to proliferate, in some strains these hor-

mones caused growth inhibition. We observed, as oth- cerous cells and cancer cells [28]. An alternative possi-bility is that these findings reflect differences in theers have previously reported [6], that efficient HPV16-

induced immortalization of HKc is dependent on the availability of intracellular steroid, due to differencesin the metabolism of steroids in the different cell types.presence of glucocorticoids. In addition, our results di-

rectly link immortalization with HPV16 to a profound Along these lines, it will be of interest to explore theresponse to glucocorticoids of pAH-Luc and p16URR-change in the proliferative response of HKc to glucocor-

ticoids. Normal HKc which were indifferent to glucocor- Luc in cells at all stages of our model for HPV16-medi-ated multistep carcinogenesis in vitro [19–23]. Basalticoids became strictly dependent on these hormones

for proliferation upon transformation with HPV16 luciferase activity in cells electroporated with pAH-Lucand incubated in the absence of dexamethasone wasDNA, while in a normal HKc strain that was inhibited

by glucocorticoids, growth inhibition was lost after barely detectable, while luciferase activity fromp16URR-Luc was substantial in the absence of gluco-HPV16-mediated immortalization.

All HKc/HPV16 lines at early passages were depen- corticoids in all cell lines. However, the extent of dexa-methasone activation of p16URR-Luc was much lessdent on the presence of glucocorticoids for proliferation.

In general, these results agree with several other re- than for pAH-Luc. These observations point to the factthat regulation of transcription from the HPV16 pro-ports on the need for glucocorticoids for proliferation

of cells transformed by HPV16 [8, 10]. Here, we also moter is complex, and in the absence of glucocorticoidsother combinations of transcription factors interactshow that this growth dependence decreased with in-

creasing passage number and with progression in vitro, with the enhancer core to maintain a considerable levelof activity. For example, AP1 is a main regulator ofindicating that a loss of requirement for glucocorticoids

may occur as a relatively late change in HPV16-trans- transcription from the P97 promoter, and intact AP1sites in the enhancer core are essential for P97 activityformed cells. Cell lines derived from cervical carcino-

mas have been reported to respond differently to gluco- [15]. Our most recent results show that while AP1 ac-tivity increases slightly in normal HKc with glucocorti-corticoids, as glucocorticoid-dependent growth is ob-

served in some, but not all, cervical carcinoma cells coid treatment, this modulation does not occur in HKc/HPV16 (unpublished results). We are currently explor-harboring HPV [17, 18]. Taken together, these observa-

tions raise the possibility that glucocorticoid require- ing further the possible connections between changesin AP1 activity and glucocorticoid responses in normalment may be present in early HPV16-induced lesions,

but not at more advanced stages of transformation. HKc and HKc/HPV16.While the glucocorticoid response of the p16URR-LucSurprisingly, glucocorticoids did not increase the

steady-state levels of HPV16 E6 and E7 mRNA and construct in HeLa cells was much lower than that ofpAH-Luc, it was within the order of magnitude pre-protein in HKc/HPV16. Also, no decrease of E6 or E7

protein levels upon removal of glucocorticoids from the viously reported for a similar construct, which also con-tained the P97 promoter [11]. It is noteworthy that themedium was observed. In addition, the activity of the

HPV16 P97 promoter, under the control of the HPV16 HPV16 enhancer core alone, in front of the thymidinekinase promoter, expressed a much greater glucocorti-URR, was stimulated by dexamethasone only 2-fold in

HKc/HPV16, less than 2-fold in SiHa, and about 5-fold coid inducibility in HeLa [11], again pointing to thefact that the glucocorticoid receptor is only one of thein HeLa. Interestingly, the extent of activation of the

MMTV promoter (pAH-Luc) by dexamethasone was many transcription factors that interplay on the URRand regulate its activity.greatest in HeLa cells (59-fold), less pronounced in

SiHa (12-fold), and lowest in HKc/HPV16 (8-fold). Overall, these results argue against the interpreta-tion that the stimulatory effects of glucocorticoids onThese differences could not be explained based on the

relative availability of glucocorticoid receptors, since growth of HKc/HPV16 and on HPV16 DNA-mediatedtransformation of HKc are due to a direct influence ofreceptor binding assays showed that SiHa and HeLa

have less glucocorticoid receptors than HKc. It has re- glucocorticoids on the expression of endogenous HPVoncogenes (E6 and E7), and point to the possibility thatcently been shown that transcriptional responses to

glucocorticoids are of greater magnitude in malignant glucocorticoids modulate the levels/activity of cellularfactors that E6 and/or E7 interact with or that arecells than in normal cells or in cells at earlier stages of

dermal fibrosarcoma development in BPV1-transgenic otherwise necessary for the continuous growth of E6/E7 transformed cells. Both E6 and E7 interact withmice, and in human cancer cells compared with normal

human cells [28]. These differences in glucocorticoid key regulatory proteins in the cell cycle, as well asfactors responsible for maintaining genetic stability.responses could not be explained by differences in glu-

cocorticoid receptor levels [28]. These observations led For example, E6 binds to and promotes the degradationof p53 [29] and interacts with other cellular proteinsto the hypothesis that there are intrinsic differences in

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310 KHAN ET AL.

11. Gloss, B., Bernard, H. U., Seedorf, K., and Klock, G. (1987)including E6BP [30], while E7 interacts with the un-EMBO J. 6, 3735–3743.derphosphorylated form of RB and the other proteins

12. Chan, W. K., Klock, G., and Bernard, H. U. (1989) J. Virol. 63,of the RB family, p107 and p130 [reviewed in 31]. The3261–3269.

interaction between E7 and RB leads to release of the13. Cripe, T. P., Haugen, T. H., Turk, J. P., Tabatabai, F., Schmid,

transcription factor E2F, and thus bypasses a key cell P. G., III, Durst, M., Gissmann, L., Roman, A., and Turek, L. P.cycle checkpoint between G1 and S [31]. The differen- (1987) EMBO J. 6, 3745–3753.tial effects of glucocorticoids on the growth of normal 14. Mittal, R., Pater, A., and Pater, M. M. (1993) J. Virol. 67, 5656–

5659.HKc and HKc/HPV16 may be mediated by a modula-15. Chong, T., Cahn, W. K., and Bernard, H. U. (1990) Nucleic Acidstion by glucocorticoids of cell cycle regulatory proteins

Res. 18, 465–470.and/or interactions with p53. While there are examples16. Bernard, H. U., and Apt, D. (1994) Arch. Dermatol. 130, 210–of p53 inhibiting glucocorticoid receptor transactiva-

215.tion of cellular promoters [32], and glucocorticoids in-17. von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E., and Giss-duce G1 arrest in some cell types [33], little is currently mann, L. (1988) Cancer Res. 48, 3780–3786.

known about glucocorticoid effects on cell cycle regula- 18. von Knebel Doeberitz, M., Bauknecht, T., Bartsch, D., and zurtion in human keratinocytes. These issues require fur- Hausen, H. (1991) Proc. Natl. Acad. Sci. USA 88, 1411–1415.ther exploration before a molecular mechanism can be 19. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J., and DiPaolo,

J. A. (1987) J. Virol. 61, 1061–1066.identified to fully explain our results.20. Pirisi, L., Creek, K. E., Doniger, J., and DiPaolo, J. A. (1988)

Carcinogenesis 9, 1573–1579.The pAH-Luc plasmid was a gift of Dr. Steve K. Nordeen, Univer-21. Pirisi, L., Batova, A., Jenkins, G. R., Hodam, J. R., and Creek,sity of Colorado Health Sciences Center. We thank Ms. Cheryl DiCa-

K. E. (1992) Cancer Res. 52, 187–193.pua for technical assistance with the glucocorticoid receptor bindingassays. 22. Zyzak, L. L., MacDonald, L. M., Batova, A., Forand, R., Creek,

K. E., and Pirisi, L. (1994) Cell Growth Differ. 5, 537–547.23. Creek, K. E., Geslani, G., Batova, A., and Pirisi, L. (1995) InREFERENCES

‘‘Diet and Cancer: Molecular Mechanisms of Interactions’’ (TheAmerican Institute for Cancer Research, Eds.), Chap. 11, p.

1. Howley, P. M. (1996) In ‘‘Fundamental Virology’’ (Fields, B. N., 117, Plenum, New York.Knipe, D. M., and Howley, P. M., Eds.), 3rd ed. Chap. 29 p. 947,

24. Nordeen, S. K. (1988) BioTechniques 6, 454–457.Lippincott-Raven, Philadelphia.25. Khan, M. A., Jenkins, G. R., Tolleson, W. H., Creek, K. E., and2. Brinton, L. A., Huggins, G. R., Lehman, H. F., Mallin, K., Sav- Pirisi, L. (1993) Cancer Res. 53, 905–909.itz, D. A., Trapido, E., Rosenthal, J., and Hoover, R. (1986) Int.26. Khan, M. A., Tolleson, W. H., Gangemi, J. D., and Pirisi, L.J. Cancer 38, 339–344.

(1993) J. Virol. 67, 3396–3403.3. Schneider, A., Hotz, M., and Gissmann, L. (1987) Int. J. Cancer

27. Housley, P. R., Dahmer, M. K., and Pratt, W. B. (1982) J. Biol.40, 198–201.Chem. 257, 8615–8618.

4. Monsonego, J., Magdelenat, H., Catalan, F., Coscas, Y., Zerat,28. Vivanco, M. d. M., Johnson, R., Galante, P. E., Hanaham, D.,L., and Sastre, X. (1991) Int. J. Cancer 48, 533–539.

and Yamamoto, K. R. (1995) EMBO J. 14, 2217–2228.5. Durst, M., Gallahan, D., Jay, G., and Rhim, J. S. (1989) Virology 29. Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1991)173, 767–771. EMBO J. 10, 4129–4135.6. Schlegel, R., Phelps, W. C., Zhang, Y. L., and Barbosa, M. (1988) 30. Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995)

EMBO J. 7, 3181–3187. Science 269, 529–531.7. Lees, E., Osborn, K., Banks, L., and Crawford, L. (1990) J. Gen. 31. Munger, K., and Phelps, W. C. (1993) Biochim. Biophys. Acta

Virol. 71, 183–193. 1155, 111–123.8. Pater, M. M., Hughes, G. A., Hyslop, D. E., Nakshatri, H., and 32. Maiyar, A. C., Phu, P. T., Huang, A. J., and Firestone, G. L.

Pater, A. (1988) Nature 335, 832–835. (1997) Mol. Endocrinol. 11, 312–329.9. Crook, T., Storey, A., Almond, N., Osborn, K., and Crawford, 33. Ramos, R. A., Nishio, Y., Maiyar, A., Simon, K. E., Ridder, C. C.,

L. (1988) Proc. Natl. Acad. Sci. USA 85, 8820–8824. Ge, Y., and Firestone, G. L. (1997) Mol. Cell. Biol. 16, 5288–5301.10. Pater, M. M., and Pater, A. (1991) Virology 183, 799–802.

Received January 27, 1997Revised version received June 6, 1997

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