[CANCER RESEARCH 52, 6848-6852, December 15, 1992] Stimulates Human Papillomavirus Type 16 Gene Expression and Cell Proliferation in a Cervical Cancer Cell Line 1

Jin-Yun Hwang, 2 Bi-Yu Lin, 3 Fen-Mei Tang, 3 and Winston C. Y. Yu 3 Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China

ABSTRACT genes such as PR and collagenase in cultured MCF-7 cells (31, 32). These side effects of tamoxifen are important to take The widely adopted use of tamoxifen as a chemotherapeutic agent is into account when considering the long-term clinical usage of primarily based on its inhibition of cancer cell growth. However, we tamoxifen for adjuvant therapy and as preventive medicine. The report that tamoxifen at low concentrations (10 -9 and 10 -1~ M) causes stimulation of cell proliferation in a cervical cancer cell line, SFR. The purpose of this study was to examine the effect of tamoxifen on facts that SFR cells do not contain receptors and are estrogen cell growth using a cervical cancer cell line (SFR) as a model nonresponsive imply the existence of an -specific binding and to elucidate the possible mode of action of tamoxifen protein and suggest that the effect of tamoxifen is possibly mediated through a specific gene, HPV-16, which is crucial in regulating through a pathway other than estrogen receptors. Tamoxifen at low cervical cancer cell growth. concentrations stimulated human papiilomavirus type 16 (HPV-16) HPV-I 6 is the most prevalent virus associated with the gen- gene transcription and E7 protein production. Levels of HPV-16 mRNA esis of cervical malignant lesions (33-35). Open reading frames and E7 protein reached a peak at approximately 2-4 h after tamoxifen E6 and E7 are present and integrated in the majority of cervical treatment, persisted for several hours, and subsequently decreased to cancer biopsy specimens (36). Laboratory studies have also their prestimulation levels by about 24 h after treatment. Our results demonstrated that E6 and E7 genes can transform or immor- indicate for the first time that tamoxifen stimulates cell proliferation of cervical cancer cells, and we suggest that the enhanced HPV-16 mRNA talize human keratinocytes and embryonic fibroblasts (37, 38). and E7 protein levels are probably responsible. Specific inhibition of E6 and E7 gene expression in cervical cancer cells by antisense RNA leads to significantly reduced cell INTRODUCTION growth (39). The effects of E6 and E7 on cell proliferation are further supported by studies using dexamethasone-treated cer- The inhibitory effect of tamoxifen on estrogen action has vical cancer cells, whose growth rate is positively correlated been studied quite extensively (1-4). Tamoxifen retards estro- with the expression of papillomavirus E6 and E7 genes (40). gen-stimulated tumor growth and has been used clinically not Taken together, these data suggest that gene expression of E6 only as a chemotherapeutic agent (2-4) but also for preventive and E7 is a limiting factor in both the regulation of cell growth treatment of in recent years (5). It has been pro- and maintenance of the malignant state. Various factors have posed that the mechanism of action of tamoxifen is mediated been reported to play important roles in regulating viral gene through ER 4, as suggested by the results of competitive binding expression in host cells. Factors identified so far include gluco- studies (1, 6). However, many breast tumors are ER negative, corticoid hormones and progesterone (41, 42), gene products of and 10-13% may exhibit a positive response to antiestrogen open reading frame E2 (43), and a keratinocyte-specific enhanc- therapy (7-9). Laboratory studies have revealed that the active ing factor (38). In this report, we provide evidence that tamox- metabolite of tamoxifen, OHT, strongly inhibits an ER-nega- ifen is another factor that can affect HPV-16 viral gene expres- tive and a PR-negative breast cancer cell line (10, 11). It is sion and that an enhanced E7 protein level may be related to the apparent that the sensitivity of cells to the growth inhibitory long-suspected stimulatory effect of tamoxifen on cell growth. effect of tamoxifen does not always correlate with ER status (11-15). A saturable high-affinity antiestrogen-binding protein MATERIALS AND METHODS has been demonstrated in a wide variety of tissues and cell lines (16-20). Thus, antiestrogen-binding sites have been suggested Chemicals. [N-methyl-3H]-tamoxifen (82.6 Ci/mmol) was pur- as possible alternative mediators for tamoxifen function in chased from NEN (Boston, MA) and stored, protected from light, in some ERonegative cells (10, 18, 20, 21). The stimulatory effect ethanol at -20~ [35S]Methionine was purchased from Amersham of tamoxifen on cell growth is an even more complicated issue, Corp. (Buckinghamshire, England). Radioinert tamoxifen, 17~-estra- however. Numerous studies have revealed a partial estrogenic diol, progesterone, and triamcinolone acetonide were obtained from Sigma Chemical Co. (St. Louis, MO), prepared as l0 mM stock solu- effect exerted by tamoxifen and its active metabolite OHT in tions in ethanol, and stored at 4~ The GAPDH DNA-containing various model systems, including breast cancer cells both in plasmid was a gift from Dr. Selina Chen-Kiang (Mt. Sinai Medical culture (22-24) and in nude mice (25, 26), endometrial adeno- School, New York). Anti-HPV-16 E7 antibody was purchased from carcinoma cells in athymic mice (27), and uterine tissue in Triton Diagnostics (Alameda, CA). Epidermal growth factor was ovariectomized rats and mice (28-30). Moreover, tamoxifen purchased from Collaborative Research Inc. (Bedford, MS). Sigma and OHT can stimulate the expression of estrogen-regulated was also the source of insulin, transferrin, cholera toxin, kanamycin monosulfate, hydrocortisone, NP-40, sodium orthovanadate, protein Received 6/12/92; accepted 10/6/92. A-Sepharose, 2-mercaptoethanol, and aprotinin. Penicillin and strep- The costs of publication of this article were defrayed in part by the payment of tomycin were obtained from Gibco BRL (Gaithersburg, MD). The page charges. This article must therefore be hereby marked advertisementin accord- oligo(dT)-ceUulose, phenylmethylsulfonyl fluoride, and leupeptin were ance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by National Science Council Grant 81-0412-B-001-04 to obtained from Mannheim Boehringer GmbH (Mannheim, Germany). W. C. Y. Y. Sodium fluoride and tetrasodium pyrophosphate were bought from 2 National University Preparatory School for Overseas Chinese Students. Merck (Darmstadt, Germany). 3 To whom requests for reprints should be addressed. Cell Culture. SFR cells, a subline of the cervical carcinoma cell line 4 The abbreviations used are: ER, ; OHT, 4-hydroxytamoxifen; PR, progesterone receptor; HPV-16, human papiliomavirus type 16; NP-40, non- CaSki, were routinely cultured in serum-free media using T-75 flasks or idet P-40; SDS, sodium dodecyl sulfate; poly(A)+, polyadenylated; GAPDH, glyc- 150-cm 2 dishes and kept in a humidified incubator at 370C under a 5% eraldehyde 3-phosphate dehydrogenase. CO2-enriched atmosphere. Serum-free media consisted of F-12 basal 6848 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. TAMOXIFEN STIMULATION OF HPV-16 GENE EXPRESSION media supplemented with 5 ~tg/ml insulin, 5 ug/ml transferrin, 362.5 progesterone, 173-, and triamcinolone acetonide as indicated ng/ml hydrocortisone, 40 ng/ml cholera toxin, 25 ng/ml epidermal in the figure legends. All studies were performed in triplicate. growth factor, 100 units/ml penicillin, 100 ttg/ml streptomycin, and 1.25 #g kanamycin. SFR cells were subcultured by trypsinization every 8 days, with media changed on the fourth and sixth day. The seeding RESULTS density of cells was 6 x 10 6 cells per T-75 flask. Cell growth studies Effect of Tamoxifen on Cell Growth. Various concentrations were conducted in 6-well plates with 7 x 104 cells seeded/well. Cells of tamoxifen were tested for their effects on SFR cell growth were allowed to grow and attach for 24 h before experiments started. Northern Blotting. Total RNA from SFR cells was isolated by the during a 5-day period (Fig. 1). Tamoxifen exhibited a biphasic acid guanidium thiocyanate-phenol-chloroform extraction method action according to dose; it stimulated cell growth at low con- (44). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatogra- centrations (10 -9 and 10 -ll M), whereas high concentrations of phy as previously described (45). Poly(A)+ RNA (2.5 ~tg) was analyzed tamoxifen (10 -6 and 10 -7 M) showed inhibitory effects. The by agarose gel (1%) electrophoresis and transferred to Hybond-C extra increased cell growth could only be observed toward the later cellulose membrane, which was then hybridized with a 32p-labeled stage of the growth curve after 4 to 5 days of treatment, sug- HPV-16 genomic probe. Hybridizations were performed at 65~ in gesting a cumulative effect of tamoxifen. We have observe a hybridization buffer (0.5 M sodium phosphate, pH 7.4-7% SDS-I% similar stimulatory effect of tamoxifen at a concentration of nonfat milk-2.5 mM EDTA-100 ttg/ml single-strand salmon sperm 10 -9 M in at least 3 separate experiments (data not shown). DNA). At the end of hybridization, blots were washed sequentially in Effect of Tamoxifen on HPV-16 Gene Expression. The effect buffer A (0.2% SDS-2x standard sodium citrate containing 0.3 M NaCl- of tamoxifen on HPV-16 gene expression in SFR cells in their 0.03 M sodium citrate, pH 7.0) twice at 42~ for 10 min, twice at 50"C third day of growth was first revealed by a Northern blot using for 10 min, and then in buffer B (0.04% SDS-0.4x standard sodium poly(A) + RNA as shown in the time-course study (Fig. 2). The citrate) at 50~ for 20 min and at 55~ for 20 min. Autoradiography was conducted at -70~ with an intensifying screen. After HPV-16 was level of HPV-16 mRNA was elevated and reached a peak ap- detected, the same membrane was again hybridized with GAPDH, proximately 2-4 h after the addition of tamoxifen. It subse- which was used as an internal control. Before rehybridization, mem- quently decreased to the prestimulation level at approximately branes were washed using removing buffer (2 mM Tris-HCI/EDTA, pH 24 h. A prominent increase in HPV-16 gene transcription oc- 8.2-0.1% SDS) at 65~ for 15 min. Images on autoradiograms were curred again at 48 h. This second peak, however, was not quantitated by densitometer. tamoxifen specific and has been related to general nutrient de- Immunoprecipitation. Cells under various treatments were labeled ficiency which occurs when serum-free media are used for >48 with [aSS]methionine (71.43 Ci/ml) for 1.5 h. These cells were then h. 5 A specific tamoxifen effect was also demonstrated by the lysed in cold buffer (0.15 M NaCI-I% NP-40-0.5% sodium deoxycho- dose-response curve as shown in Fig. 3. Tamoxifen at a con- late-0.1% SDS-50 mM Tris-HCl, pH 8.0-1 mM EDTA). Protease inhib- centration of 10 -9 M exhibited the highest stimulatory effect itors (0.05 mg/ml aprotinin, 50 UM leupeptin, and 1 mM phenylmeth- when compared with various concentrations from 10 -13 to ylsulfonyl fluoride) and phosphatase inhibitors (30 mM tetrasodium 10 -6 M. pyrophosphate, 50 mM NaF, 100 #M sodium orthovanate) were in- The stimulatory effect of tamoxifen on HPV-16 gene expres- cluded in the buffer. After the supernatants were mixed and centrifuged, they were collected and incubated with a 1:100 dilution of HPV-16 E7 sion was further supported by the result of our time-course antibody (0.1 mg/ml) at 4~ for 3 h. Protein A-Sepharose was added, study of E7 protein levels (Fig. 4). Metabolically labeled E7 and the material was incubated at 4~ for 2 h. Beads with attached protein was immunoprecipitated by anti-HPV-16 E7-specific immunocomplexes were then washed five times in washing solution monoclonal antibody and analyzed by SDS-polyacrylamide gel (100 mM NaCI-25 mM Tris-HCl, pH 8.0-0.5% sodium deoxycholate- electrophoresis. Like the pattern of mRNA changes, E7 protein 0.2% NP-40). Proteins were electrophoresed after boiling in sample production reached a maximum approximately 3 h after tamox- buffer (1.25 mM Tris-HCl, pH 6.75-4% SDS-20% glycerol-10% 2-mer- ifen was added and then declined toward the 24-h time point. captoethanol-0.2% bromphenol blue) for 10 min using a 10% polyacryl- The nutrient depletion-related E7 peak at 48 h persisted for amide gel. For autoradiography, the gels were fixed in 50% methanol another 24 h, at which time SFR cell proliferation had stopped plus 10% glacial acetic acid, dried, and exposed to Kodak X-ray film at (data not shown). -70~ Radioisotope-labeled protein bands were quantitated by densi- Since the studies of mRNA and E7 levels were all conducted tometric scanning of X-ray film using a densitometer. Tamoxifen-binding Assay. Cytosol extracted with a high concentra- using cells on the third day of growth, the increases in mRNA tion of salt was collected from SFR cells by first homogenizing them in and E7 levels most probably correspond to the enhanced cell buffer (25 mM sodium phosphate, pH 7.4-1 mM EDTA-I mM freshly proliferation (on day 4 and 5) that we observed and depict in added dithiothreitol-10% glycerol-10 mM sodium molybdate) con- Fig. 1. When the effect of 10 -9 M tamoxifen continuously taining 0.3 M KC1 using a Polytron homogenizer until >90% of the present in the culture medium was studied, daily E7 levels in cells were disrupted. The cytosol was collected after centrifugation at tamoxifen-treated SFR cells were the same as in control cells 105,000 x g for 1 h. All procedures were performed at 0-4~ The (data not shown). saturation point assay was performed by incubating 200 #1 of cytosol Tamoxifen-binding Protein. A tamoxifen-specific binding with various concentrations of [3H]tamoxifen (2.5-10 riM). Nonspecific protein was detected in the cytosol of SFR cells. It was saturable binding was determined by parallel incubation of the sample with a at ligand concentrations of 10 nM and displayed high affinity for 500-fold excess of radioinert tamoxifen. Binding was performed at tamoxifen, as demonstrated in Fig. 5. Tamoxifen binding in 30~ for 30 min. Bound radioactivity was separated from free activity SFR cells could not be displaced by estradiol or by triamcino- by incubating with 500 ~tl of dextran-coated charcoal suspension (0.5% of Norit A charcoal-0.05% dextran in buffer containing 25 mM sodium lone acetonide, and it required high concentrations of proges- phosphate, pH 7.4-1 mM EDTA-1 mM freshly added dithiothreitol-10% terone to cause the same degree of inhibition as that caused by glycerol-10 mM sodium molybdate) for 10 min, followed by centrifuga- tamoxifen. tion at 1800 x g for 10 min. The radioactivity in the supernatant was counted in a beta counter after mixing with scintillation solution. The competition assay was performed by incubating 200 ~tl of cytosol with s B-Y. Lin, F-M. Tang, and W. C. Y. Yu, Cell growth and HPV-16 E7 produc- [SH]tamoxifen and increasing concentrations of radioinert tamoxifen, tion in cervicalcarcinoma cells, submitted for publication. 6849 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. TAMOXIFEN STIMULATION OF HPV-16 GENE EXPRESSION

100 ER. However, SFR cells do not contain ER, and their cell ~ IO-IM growth is not regulated by estrogen (data not shown). The fact that a distinct antiestrogen-specific binding species is present in GO :2, the cytosol of many animal and human tissues strongly suggests 10-13M

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Days Tamoxifen (M:) Fig. 1. Effects of various concentrations of tamoxifen on SFR cell growth. Fig. 3. Dosage effects oftamoxifen on HPV-16 gene transcription. Cells on the Cells were cultured as described in "Materials and Methods." Concentrations of third day of growth were treated with tamoxifen for 3 h. Poly(A) § RNA isolation, tamoxifen used were 0, 10 -6, 10 -7, 10 -9, 10 -11, and 10 -~3 M. Cell numbers were Northern blot procedure, quantitation of the results of autoradiography, and all counted every day using a hemocytometer. All points represent means of triplicate other procedures were as described in "Materials and Methods." Tamoxifen determinations. exhibited the highest stimulatory effect at a concentration of 10 -9 M.

Tam. ~ - + ~~ ~. Tam. = + = Time 0 2 4 8 12 24 48 4 24 48 (hr) Time 0 3 6 12 24 48 72 M.W (hr) (Kga) T HPV-16

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Fig. 2. Effect of tamoxifen on HPV-16 gene transcription in SFR cells at various times after tamoxifen treatment. A Northern blot of poly(A) + RNA extracted from SFR cells was conducted as described in "Materials and Methods." - 32.5 Tamoxifen at a concentration of 10 -9 M was added to cells on day 3. Poly(A) § RNA was isolated from cells at various times thereafter. The relative transcription rate was expressed based on the GAPDH transcription and used as an internal standard. - 27.5

DISCUSSION

These studies demonstrate that antiestrogen tamoxifen inhib- its growth of SFR cervical cancer cells at concentrations of 10 -6 - 18.5 or 10 -7 M. A similar inhibitory effect has been extensively reported for many cell types, including breast cancer cells (46). It is generally believed that tamoxifen tends to bind with ER Fig. 4. A time-course study of the effect of tamoxifen on E7 protein. SFR cells (1, 47, 48) and thus prevents further estrogen binding, which on day 3 of growth were metabolically labeled with [35S]methionine and E7 eventually eliminates estrogen-stimulated cell growth. This log- protein and then were assayed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis as described in "Materials and Methods." Tam., tamoxifen; ical explanation is possibly correct for those cells containing M.W., molecular mass marker. 6850 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. TAMOXIFEN STIMULATION OF HPV-16 GENE EXPRESSION

the promoter for progesterone-responsive genes (42). It is, therefore, reasonable to suggest that HPV-16 gene expression can also be enhanced by tamoxifen through the PR-binding oL_ 4,* c 100 - element located in the long control region. The glucocorticoid o o response element is another enhancer-binding site common in o the long control region of papillomavirus. We do not know 8o- whether the tamoxifen-binding species also acts through the 1o glucocorticoid response element since no competitive glucocor- t- :~ 60- ticoid displacement of tamoxifen binding was observed. How- 0 .a ever, the ligand used was triamcinolone acetonide, which has r 0) been shown to be much more specific than hydrocortisone for 40- X the glucocorticoid receptor. The dose of tamoxifen is critical to o E observe both the effect on cell growth and HPV-16 mRNA I- 20- levels. Interestingly, tamoxifen at 10 -9 M stimulated the highest -r- cell growth and at the same time also exhibited the most HPV-16 mRNA. High concentrations of tamoxifen (10 -6 and ! ! o.oos o.os 0.5 ; s'o sop l0 -7 M), however, had no effect on HPV-16 gene expression, and they inhibited cell proliferation. In summary, we give evidence for the first time that cervical Competitor concentration (ira) cancer cell growth can be regulated by tamoxifen. Furthermore, Fig. 5. Competitive binding studies of tamoxifen (Tam.), 17~-estradiol (E2), tamoxifen could induce HPV-16 gene transcription and E7 progesterone (Prog.), and triamcinolone acetonide (T.A.) for tamoxifen-specific binding protein in SFR cells. Abscissa, various levels of competing hormones protein production in these cells. The stimulation of cell pro- used. All other procedures were as described in "Materials and Methods." liferation is possibly mediated through the observed HPV-16 gene expression. that an alternative pathway of antiestrogen function exists, via a separate binding protein (11, 12, 14). The ER-negative and ACKNOWLEDGMENTS estrogen nonresponsive SFR cells in this experiment do possess We are grateful to T. P. Hsu, L. W. Chen, and H. T. Lai for their a tamoxifen-specific intracellular binding molecule which technical assistance. We also thank Bon Bon P. T. Hsiao for typing the shows no competition by estrogen and is only partially com- manuscript and Cathy Fletcher for English editing. petitively inhibited by progesterone at high concentrations. Whether or not this antiestrogen-binding species is the media- REFERENCES tor of the action of tamoxifen on cell proliferation awaits fur- 1. Bardon, S., Vignon, F., Dercq, D., and Rochefort, H. The antiproliferative ther experimentation. The suggestion that ER concentration in effect of tamoxifen in breast cancer cells: mediation by the estrogen receptor. SFR cells may be very low and thus would be beyond the sen- Mol. Cell. Endocrinol., 35: 89-96, 1984. 2. Legha, S. S., Buzdar, A. U., Hortobagyi,G. 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6852 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. Tamoxifen Stimulates Human Papillomavirus Type 16 Gene Expression and Cell Proliferation in a Cervical Cancer Cell Line

Jin-Yun Hwang, Bi-Yu Lin, Fen-Mei Tang, et al.

Cancer Res 1992;52:6848-6852.

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