The HPV16 E2 transcriptional regulator mode of action depends on the

Human papillomavirus (HPV) infection is a major risk factor for the development of cervical cancer. The HPV-induced immortalization of epithelial cell usually requires integration of the viral DNA into the host cell genome. The integration event causes disruption of the E2 gene and this is followed by overexpression of the E6 and E7 oncoproteins. The E2 protein is a transcription factor that regulates expression of the E6 and E7 oncoproteins by binding to four sites within the viral long control region. We used an in vitro cell culture model to explore the role of the E2 protein in the transcriptional control of the HPV16 long control region. Employing transient and stable transfection experiments we simulated the episomal and integrated states of the viral genome, respectively. We show that the E2 protein up-regulates E6/E7 transcription from episomal DNA but represses it in the case of integrated DNA. The activator function of the E2 protein seems to counteract the repressive chromatin structure formed over episomal DNA. Steroid hormones and retinol also modulate oncogene transcription differently depending on the physical structure of the viral DNA. Our data suggest regulatory mechanisms involving interactions between the E2 protein and nuclear hormone receptors.


Vol. 52 No. 4/2005, 823-832
on-line at: www.actabp.plHuman papillomaviruses (HPV) are epitheliotropic small dsDNA viruses with a circular genome of approximately 8 kbp.More than 80 different HPV types are known and new ones are currently being isolated.These viruses can cause benign proliferative lesions (e.g., skin warts and anogenital condylomata) and anogenital cancers (especially cervical cancer).HPV types have been classified as either high risk (e.g.  or low risk (e.g.HPV6 and -11) on the basis of the clinical lesions they are associated with and the relative predisposition of these lesions to progress to cancer (zur Hausen, 1996;2000;Longworth & Laimins, 2004).The viruses encode two oncoproteins, E6 and E7, whose expression extends the life span of the normal host cell (squamous epithelial cells).Overexpression of these oncoproteins leads to immortalization and transformation of the host cell.The main transforming properties of the E6 and E7 oncoproteins are their ability to inactivate the p53 and retinoblastoma (pRB) tumor suppressor pro-teins, respectively.HPV-induced carcinogenic progression is frequently related to integration of the viral genome into the human chromosome in cancer cells.Such an event o�en results in the loss of expression of the viral E2 gene but preserves E6/E7 expression or even increases it (Barbosa & Schlegel, 1989;Choo et al., 1987;Longworth & Laimins, 2004;Nakagawa et al., 2000;Sashiyama et al., 2001).
E2 is a regulatory protein able to either activate or repress transcription from the promoter directing expression of the E6 and E7 oncogenes (Cripe et al., 1987;Thierry & Yaniv, 1987;Nishimura et al., 2000;Bouvard et al., 1994).This 42-kDa protein is composed of an N-terminal transcriptional activation domain, a hinge region and a C-terminal DNA-binding and dimerization domain.The viral regulatory protein binds to multiple copies of the ACCN 6 GGT motif that occur in the long control region (LCR) of all HPVs (McBride & Myers, 1997;Antson et al., 2000;Hou et al., 2002).The LCR regulatory sequence contains also multiple binding sites 2005 M.T. Schmidt and others for cellular transcription factors that modulate its action positively (TFIID, SP-1, AP-1, NF1, Oct-1, TEF-1, steroid receptors) or negatively (YY1, CDP, NF-κB, C/EBPβ).The transcription factors interacting with their specific motifs form a keratinocyte-specific enhancer and silencer, respectively (O'Connor et al., 1995).
Correlation studies (de Villiers, 2003) and in vitro experiments have shown that steroid hormones and retinol influence cervical cancer cells (Khare et al., 1997;Narayanan et al., 1998;Webster et al., 2001).The effect is mainly a�ributed to the presence of hormone response element sequences in the viral LCR (Khare et al., 1996), but HPV-independent actions have also been observed (Khan et al., 1997).
The research reported here has three objectives.First, we wish to determine whether there is a difference in the chromatin structure over episomal and integrated reporter constructs resembling the different states of the HPV16 LCR.Second, we wish to analyze the effects of the E2 protein on the reporter constructs in these two physical states.Finally, we examine the influence of steroid hormones and retinol on HPV16 oncogene transcription in the presence and absence of the E2 protein.Our results indicate that the HPV16 genome in the episomal state undergoes severe repression by chromatin and that this repression is partially relieved by the E2 protein.After integration the chromatin over the HPV16 DNA goes through structural changes rendering it more open but totally altering the effect of the E2 protein.Furthermore, our work with steroid hormones and retinol indicates that the HPV16 genome is differently influenced by these factors depending on the physical structure of the viral DNA.
For stable transfection the LCR-luc-E6MAR reporter casse�e was cut out from pGL3-LCR/E6MAR reporter vector with SacI and SalI followed by agar-ose gel electrophoresis and purification from the gel using QIAquick Gel Extraction Kit (Qiagen).
Plasmids were isolated and purified with the Mobius 1000 Plasmid Kit (Novagen).
Cell culture.The HPV-negative human cervical carcinoma cell line C-33A was cultured in Dulbecco's Modified Eagle's medium (Sigma) supplemented with 10% foetal calf serum (Gibco) and gentamycin (Sigma) at 37 o C and 5% CO 2 .
Cell transfections.The day before transfection, 3 × 10 5 cells per well were seeded in a 12-well plate.The cells were transiently transfected using Tfx-20 Reagent (Promega) and 2 µg of pGL3-LCR/ E6MAR plasmid DNA at a 2:1 ratio in serum free medium for 1.5 h.In the case of co-transfections 2 µg of pGL3-LCR/E6MAR reporter vector and 1 µg pCMV-pt16E2 expression vector (or pCMV1 empty expression vector as a negative control) were used.
For the generation of stably transfected C-33A lines linear LCR-luc-E6MAR reporter casse�e with the neomycin resistance gene expression cas-se�e (Neo R ) at a 10:1 ratio was introduced to cells using the Effectene Transfection Reagent (Qiagen) according to the procedure recommended by the manufacturer.Transfectants were selected using the neomycin homologue G418 antibiotic (ICN) added gradually to the culture medium to the final concentration of 0.8 mg/ml and maintained for 3 weeks.The stably transfected C-33A cells represent a mixed population.A cell line stably transfected with LCRluc-E6MAR/Neo R was transiently transfected with 2 µg pCMV-pt16E2 or pCMV1 expression vector using Tfx-20 Reagent (Promega).
Cell treatment and luciferase assays.Cell cultures were supplemented, where necessary, with dexamethasone (100 nM), 17β-estradiol (100 nM), progesterone (100 nM) or retinol acetate (3 µM) in the form of cyclodextrin-encapsulated water-soluble complexes (Sigma), and trichostatin A (TSA, 300 ng/ml; ICN) 24 h a�er transfection.Forty-two hours a�er transfection the cells were washed with PBS and lysed in 100 µl Cell Culture Lysis Reagent (Promega).Cell lysate (20 µl) was mixed with 100 µl luciferin substrate (Promega) and the activity of luciferase was measured in a Biocounter M1500 (Lumac) with 2 s delay and 10 s integration period.Luciferase activity was standardized to total protein content of the cell lysates.Protein concentration was measured using the modified Lowry's method (Total Protein Kit; Sigma) a�er protein precipitation with trichloroacetic acid.
DNA extraction and real-time PCR copy number determination.Cells were washed three times with TEN buffer (10 mM Tris/Cl, pH 7.4, 5 mM EDTA, 0.9% NaCl) to remove extracellular E2 function depends on the physical state of the viral genome plasmid DNA prior to cell lysis in the case of transiently transfected cells.Total DNA from stably and transiently transfected cells was isolated using the Genomic Mini Kit (A&A Biotechnology, Poland).The quantitative real-time PCR assay was based on primers that specifically amplify the luc luciferase gene (forward: 5'-TGTGGACGAAGTACCGAAAG-GT-3' and reverse: 5'-CCTTCTTGGCCTTTATGAG-GATCT-3') and a TaqMan probe (5'-6FAM-CCG-GAAAACTCGACGCAAGAAAAATCAG-TAMRA-3'; IBB PAS, Warszawa, Poland).Using the Smart-Cycler (Cepheid) the PCR was performed in a total volume of 25 µl in capillary tubes containing 300 nM of each primer, 100 nM of TaqMan probe, 25 ng of genomic DNA, and 1× Smart Reaction Buffer (Smart Kit, Eurogentec).The PCR program was initiated with a step of 50 o C for 2 min and 95 o C for 10 min, followed by 45 cycles of 95 o C for 15 s and 60 o C for 1 min.Real-time PCR quantitation was performed three times.The quantification of the luc gene (integrated transgene copies or plasmid molecules that entered cells) was performed to self prepared standards.The standard was made by serial dilutions of pGL3-Basic plasmid in 5 mM Tris buffer pH 8.0, containing genomic C-33A DNA.

Chromatin represses the HPV16 LCR to different extents in episomal and integrated states
Nucleosomal organization of eukaryotic DNA is required to store the bulky genome and to facilitate gene regulation.Most o�en the effect of chromatin is suppressive for transcription (van Holde & Zlatanova, 1996;Berger, 2002).To test how the chromatin structure influences transcriptional activity of the p97 promoter in episomal and integrated state we made use of the histone deacetylase inhibitor trichostatin A (TSA).Transiently transfected C-33A cells containing pGL3-LCR/E6MAR and stably transfected C-33A cells containing LCR-luc-E6MAR simulating the episomal and integrated states of the viral genome, respectively, were treated with TSA.A significant increase in luciferase activity was observed in both cases, about 8.5-fold for the episomal and about 3.3-fold for the integrated reporter (P < 0.01, Fig. 1).The transiently transfected cells contain over 1000-fold more copies per cell of the reporter construct plasmid than the stably transfected line (about 3.8 reporter construct copy/cell genome) as measured by real-time PCR (not shown).
These data indicate that the chromatin structure in the LCR-E6MAR region in the episomal and integrated states represses the p97 promoter-driven transcription to very different extents.These findings suggest that the HPV16 episomal genome undergoes much higher repression by chromatin structure than the integrated genome.

The HPV16 E2 protein regulates p97 transcription differently depending on the chromatin structure over the HPV16 LCR
To study the effects of the E2 protein on the activity of the LCR in the episomal and integrated states the stably transfected LCR-luc-E6MAR C-33A cells were transiently transfected with the E2 expression construct pCMV-pt16E2, and C-33A cells were transiently co-transfected with pGL3-LCR/E6MAR and pCMV-pt16E2.Additional TSA treatment allowed an assessment of the influence of E2 on chromatin.
Expression of the HPV16 E2 protein increases (about 1.19-fold, P < 0.01) the transcriptional activity of the episomal reporter and decreases (about 1.19fold, P < 0.01) the activity of the integrated reporter (Fig. 2).As in the previous experiment TSA treatment increases the transcription about 8.5-fold and about 3.3-fold for the episomal and integrated reporter, respectively (P < 0.01).Expression of E2 protein in conjunction with TSA treatment brings about a further enhancement of the reporter activity in the case of the episomal reporter (about 11-fold, P < 0.01).However, E2 and TSA together have li�le additional effect over TSA alone in the case of the integrated reporter (about 3.5-fold, P < 0.01).
These results demonstrate that the effect of the E2 protein depends on the chromatin structure formed on the HPV16 LCR.In the episomal state E2 protein acts as a transcriptional activator but a�er integration E2 represses the p97 promoter.Trichostatin A treatment relieves the repressive action of chromatin and allows a further increase of the p97 transcriptional activity by E2 in the episomal state.In contrast, the activity of the integrated reporter construct does not respond significantly to E2 in the presence of TSA.
The HPV16 E2 protein is a potent activator of the p97 promoter in the episomal state HPV16 is a steroid hormone-responsive virus.There are three steroid response motifs within the HPV16 LCR sequence and these are able to bind with different affinities both steroid and retinol receptors (Khare et al., 1997;Yokoyama et al., 2001).To examine the effects of steroid hormones and retinol combined with the HPV16 E2 protein on the HPV16 p97 promoter activity in the episomal state we used the above-described experimental model.
In the absence of the E2 protein retinol increases about 1.13-fold (P < 0.05) and progesterone decreases about 1.12-fold (P < 0.10) p97 promoter activity, whereas dexamethasone and estrogen have no significant effect (Fig. 3).In the presence of E2 and steroid hormones or retinol p97 activity is in-creased significantly (Fig. 3).The combined action of estrogen, progesterone or retinol with the E2 protein statistically significantly increases the transcriptional activity of the viral promoter.The data analysis shown in Table 1 shows that dexamethasone appears to maintain p97 transcriptional activity at an unchanged level despite the presence of E2.This suggests that dexamethasone blocks the activity of E2.Estradiol has no effect on the p97 promoter either in the presence or absence of the E2 protein.
In contrast, E2 counteracts the repressive effect of progesterone, changing the transcription level from about 0.89 (P < 0.10) to about 1.17 (P < 0.05) that of control, thus causing an overall about 1.30-fold increase (P < 0.05) in the reporter activity.Retinol (about 1.13-fold increase, P < 0.05) and E2 (about 1.19fold increase, P < 0.05) seem to operate in an additive manner giving together about 1.33-fold (P < 0.05) upregulation of the p97 promoter activity.
These data show that the E2 protein is a transcriptional activator of the episomal reporter construct.E2 modulates positively the HPV16 LCR p97 promoter activity in the presence of progesterone and retinol, but does not function in the presence of dexamethasone.The episomal reporter construct is not responsive to estradiol treatment in these experimental conditions but neither does estradiol treatment block the effect of E2.

The p97 promoter in the integrated HPV16 LCR state is repressed by E2
To investigate the effects of E2 in the case of an integrated reporter construct, we used stably transfected LCR-luc-E6MAR C-33A cells transiently transfected with the empty pCMV1 vector or the E2 expressing pCMV-pt16E2 construct and measured the transcriptional activity in the presence or absence of steroid hormones and retinol (Fig. 4).
Dexamethasone treatment of the cells with the integrated reporter construct brings about downregulation of the promoter activity by 11% (P < 0.10) and this is unaffected by the presence of the E2 protein (about 0.87-fold of the nominal activity, P < 0.05; Fig. 4).Although estradiol treatment alone does not influence the activity of the integrated reporter construct, estradiol treatment partially relieves the E2-mediated repression (about 1.07-fold increase as compared to E2 action alone, P < 0.10).Similarly, progesterone has li�le if any influence on the p97 promoter activity but also seems to partially relieve the E2-mediated repression.Finally, retinol treatment has li�le or no effect on the reporter activity in the absence of E2 but almost abolishes the E2-mediated repression.
In summary, the HPV16 E2 protein-mediated repression is affected by the steroid hormones in the case of the integrated reporter construct, although

E2 function depends on the physical state of the viral genome
the protein shows no significant action in dexamethasone-treated cells when compared to the treatment alone.Retinol has li�le effect on the integrated reporter and it blocks repression by E2.The outcome of estradiol and progesterone treatment appears to be dependent on the chromatin structure over the reporter construct (physical state).Both treatments have li�le influence on the activity of the episomal reporter construct, but partially relieve the E2-mediated repression in the integrated state.These results together show that the effect of steroid hormone receptors and the HPV16 E2 protein depends on the chromatin structure over the HPV16 regulatory sequences.

DISCUSSION
The HPV E2 protein has been shown to both activate and repress transcription in transient re-  Table 1.The effect of steroid hormones or retinol on the HPV16 LCR p97 promoter activity in the presence or absence of E2 protein.
Fold change in the HPV16 p97 activity in the episomal and integrated states of the reporter construct as influenced by the HPV16 E2 protein, steroid hormones and retinol.Values in bold designate statistical significance at P < 0.05, and values with asterisks -at P < 0.1.Control, represents C-33A cells co-transfected with empty pCMV1 expression vector, E2, cells co-transfected with the E2 protein expression vector pCMV-16ptE2, Dex, Est, Prg, and Ret -cells treated with dexamethasone, estradiol, progesterone and retinol, respectively.SH/R, depicts corresponding steroid hormone or retinol treated cells co-transfected with pCMV1 vector.porter assays (Phelps & Howley, 1987;Bernard et al., 1989;Lees et al., 1990;Parton et al., 1990;Romanczuk et al., 1990;Bouvard et al., 1994;Tan et al., 1994;Demeret et al., 1994;1997;Kovelman et al., 1996;Alloul & Sherman, 1999;Lee et al., 2000;DeFilippis et al., 2003;Thierry et al., 2004).The various findings of several research groups point to the dual action of the E2 protein, but can also be a�ributed to variations in the experimental models used, since transcriptional regulation is strongly context-dependent (reviewed in Fry & Farnham, 1999).However, when expressed in cells that hold integrated HPV genomes, E2 represses the transcription of E6 and E7 (Dowhanick et al., 1995;Desaintes et al., 1997;Francis et al., 2000;Goodwin & DiMaio, 2000;Nishimura et al., 2000).The repressor action of E2 on integrated HPV genomes is indirectly confirmed by the observation that in most cervical cancer cells the E2 ORF is disrupted (Schwarz et al., 1985;Romanczuk & Howley, 1992;Chen et al., 1994;Park et al., 1997;Yoshinouchi et al., 1999).Our experiments have shown that the E2 protein represses the integrated but activates the episomal HPV16 LCR-p97 promoter.These findings are to some extent in agreement with the results of the Beard group (Bechtold et al., 2003), which used the pair of isogenic cell lines W12 and S12 (containing episomal and integrated HPV16 DNA, respectively).They found that the E2 protein represses transcription from the integrated HPV16 genome but has no influence on the episomal viral DNA transcription.
The inconsistency can be ascribed to the possible difference in DNA conformation between the reporter plasmids and the viral minichromosome.Another possibility is that additional factors introduced in the course of recombinant adenovirus infection used to express the E2 protein have effects on HPV transcription (Leppard, 1997;Yew & Perricaudet, 1997;Steinwaerder et al., 2001).Organization of DNA into chromatin and the interaction of DNA with the nuclear matrix influence various aspects of the regulation of transcription and viral DNA copy number control and partition (van Driel et al., 1995;Tan et al., 1998;Stunkel & Bernard, 1999;Hancock, 2000;Stunkel et al., 2000).The episomal HPV DNA inside the viral capsid is packed into nucleosomes forming a viral mini-chromosome (Favre et al., 1977).Small circular DNA introduced into the cell acquires nucleosomes (Jeong & Stein, 1994) and interacts with the nuclear matrix (Mearini et al., 2004), thus plasmids transfected into cells can simulate the episomal form of the viral DNA.A common hallmark of cell transformation by HPV is viral integration into the host cell genome.This occurs in a random fashion but preferentially into fragile and transcriptionally active sites (Choo et al., 1996;Klimov et al., 2002;Thorland et al., 2000;2003;Wentzensen et al., 2004).Consequently, stable transfection can be used to model the integrated form of the viral genome (Matzner et al., 2003).We used these approaches to study the role of chromatin structure in the control of HPV transcription.The reporter construct used for these experiments contains the entire HPV16 LCR (including 5'MAR) and the downstream E6MAR (the 3'MAR) flanking the enhancer and the p97 promoter (Tan et al., 1998) and therefore resembles the structure of the HPV16 regulatory region.Our findings demonstrate that the chromatin structure on the episomal DNA is over 5fold more repressing than on the integrated DNA.This is consistent with the data from Stunkel and coworkers who showed that MAR repressed HPV16 transcription in undifferentiated epithelial cells (Stunkel et al., 2000).The E2 protein stimulates the HPV16 LCR-p97 promoter in the episomal state and appears to counteract the repressive effects of chromatin.This E2 activity can be a�ributed to its ability to interact with the CBP transcriptional co-activator, a protein that exhibits histone acetyltransferase activity (Lee et al., 2000).E2 proteins of all HPV types probably have the ability to bind nuclear matrix as it has been shown for the HPV11 E2 protein (Zou et al., 2000).We have also shown that the E2 protein has an opposite action on the integrated HPV16 LCR-p97 promoter.The repression of transcription in the integrated state may be involved in the maintenance of the differentiation state of the cells.Tumor cells are not productive in virus replication as they do not differentiate (Kaur & McDougall, 1989;Zhao et al., 1997;Pei et al., 1998;Flores et al., 2000;McMurray et al., 2001;Hadaschik et al., 2003;Longworth & Laimins, 2004).An integration event leading to derepression of viral oncogenes may favor tumorigenic cells in the course of cervical cancer etiology (Jeon et al., 1995;Badaracco et al., 2002).However, a�er the initial integration event the remaining episomes can still express the E2 protein and repression of the integrated HPV genome may leave the cellular phenotype unaltered.Thus the total loss of E2 expression may lead to malignant lesions that contain only integrated HPV genomes (Daniel et al., 1997;Vernon et al., 1997;Tonon et al., 2001;Pe� et al., 2004).
Our findings suggest that the E2 activator function is based on stimulation of the basal transcriptional machinery.The HPV16 LCR-p97 promoter on episomal DNA is activated by E2 both in the presence and absence of trichostatin A. The E2 repressive action on the HPV16 LCR p97 promoter on integrated DNA is probably mediated by preservation of chromatin acetylation.The E2 protein appears to be a transcriptional factor that regulates in a positive manner oncogene transcription in the case of episomal HPV16 genome.Our finding is supported by analyses of cervical lesions containing episomal HPV DNA (Crum et al., 1989;Durst et al., 1992;Higgins et al., 1992).The activity may counteract the chromatin repressive form that develops on E2 function depends on the physical state of the viral genome exogenous DNA introduced into the nucleus that represents a cellular defense mechanism against viral infection.
Recently, it was demonstrated by Khare et al. (1996;1997) that glucocorticoids up-regulated HPV oncoprotein expression.However, research by Khan et al. (1997) showed that growth stimulation of HPVimmortalized cells by glucocorticoids was independent of the E6/E7 mRNA level.We have shown that dexamethasone treatment does not increase reporter activity in either the episomal or integrated states.However, E2 activates the episomal reporter and represses the integrated one in untreated cells, and treatment with dexamethasone abolishes the effects of E2 in both cases.Our results thus seem to confirm the findings of Khan and coworkers but not those of Khare and collaborators.
Similarly, there are conflicting reports in the literature regarding the ability of retinoids to modulate the p97 promoter.According to the results of Narayanan et al. (1998), all-trans-retinoic acid is able to repress the E6/E7 promoter in CaSki cells.However, in experiments reported by Agarwal et al. (1994), all-trans-retinoic acid increased the viral E6/E7 mRNA levels in the same cell line.It has also been demonstrated that retinoids do not necessarily inhibit the proliferation of HPV-immortalized cervical cells via effects on HPV E6/E7 transcription but rather they can act through suppression of the EGF signaling pathway (Eckert et al., 1995;Sah et al., 2002).The la�er, indirect mode of action is also in agreement with the finding that the response to retinoids (Narayanan et al., 1998) or progesterone (Yuan et al., 1999) are of the delayed type, requiring 3 to 7 days of treatment.Another finding that highlights an important issue in understanding the nature of the differences reported among various studies is that even closely related HPV16-immortalized cervical epithelial cell lines respond to all-transretinoic acid in different manners depending on the retinoids level (Choo et al., 1995).The HPV-driven immoralization of epithelial cells is a sophisticated process requiring several factors, which can operate via distinct pathways, adding further degrees of complexity to the well-known tumorigenesis model.Our observations suggest that E2 activity is dependent on chromatin structure and may be modulated by steroid and retinoid receptors.

Figure 1 .
Figure 1.The effects of TSA on HPV16 p97 promoter activity in the episomal and integrated states.The HPV16 LCR-E6MAR region was cloned into reporter vector pGL3-Basic.The reporter constructs were transiently or stably transfected into C-33A cells, simulating the episomal and integrated states of the viral genome.p97 transcriptional activity in different physical states and in the presence or absence of TSA were determined using luciferase assays.The data represent the averages of four independent experiments, with error bars indicating ±S.D.

Figure 2 .
Figure 2. The effects of the chromatin structure and the HPV16 E2 protein on the p97 promoter activity in the episomal and integrated states.The reporter constructs were transiently or stably transfected into C-33A cells, simulating the episomal and integrated states of the viral genome.p97 transcriptional activity in the different physical states and in the presence and absence of the HPV16 E2 protein (cotransfection with expression vector) and/or TSA were determined using luciferase assays.All luciferase activities were calculated in relation to control samples for transient (episomal) and stable (integrated) transfection experiments, respectively.The data represent averages of four independent experiments, with error bars indicating ± S.D. Control, indicates C-33A cells transiently transfected with pGL3-LCR/E6MAR and stably transfected with LCR-luc-E6MAR cotransfected with empty pCMV1 vector; E2, C-33A cells transiently transfected with pGL3-LCR/E6MAR and stably transfected with LCR-luc-E6MAR cotransfected with the pCMV-pt16E2 expression vector; TSA, and TSA+E2, as above but trichostatin A-treated.

Figure 3 .
Figure 3.The effects of steroid hormones or retinol and the HPV16 E2 protein on p97 promoter activity in the episomal state.The reporter construct HPV16 LCR-E6MAR was transiently transfected into C-33A cells, simulating the episomal state of the viral genome.p97 transcriptional activity in the presence of the steroid hormones or retinol and the HPV16 E2 protein (cotransfection with an E2 expression vector) were determined using luciferase assays.The data represent averages of four independent experiments, with error bars indicating ±S.D. Control, indicates hormoneuntreated C-33A cells transiently transfected with pGL3-LCR/E6MAR cotransfected with empty pCMV1 or the E2 expressing pCMV-pt16E2 vector, and Dex, Est, Prg, and Ret, designate transfected cells additionally treated with dexamethasone, estradiol, progesterone and retinol, respectively.

Figure 4 .
Figure 4.The effects of steroid hormones or retinol and the HPV16 E2 protein on p97 promoter activity in the integrated state.The HPV16 LCR-luc-E6MAR reporter construct was stably transfected into C-33A cells, simulating the integrated state of the viral genome.p97 transcriptional activity in the presence of steroid hormones or retinol and the HPV16 E2 protein (cotransfection with an E2 expression vector) were determined using luciferase assays.The data represent averages of four independent experiments, with error bars indicating ± S.D. Control, indicates hormone-untreated C-33A cells transiently transfected with pGL3-LCR/E6MAR cotransfected with empty pCMV1 or the E2 expressing pCMV-pt16E2 vector, and Dex, Est, Prg, and Ret -designate transfected cells additionally treated with dexamethasone, estradiol, progesterone and retinol, respectively.