Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK1
Author for correspondence: Kevin Gaston. Fax +44 117 928 8274. e-mail Kevin.Gaston{at}Bristol.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The HPV E2 proteins are required for efficient virus replication and are thought to play a role in the regulation of HPV gene expression (reviewed by Thierry, 1996 ). More recently, the E2 proteins have also been shown to have dramatic effects on cell survival. The HPV-16 E2 protein can induce apoptotic cell death in a variety of HPV-transformed and non-HPV-transformed cell lines (Sanchez-Perez et al., 1997
; Webster et al., 2000
). Similarly, the E2 proteins from HPV-18 and HPV-33 have been shown to induce apoptosis in HeLa cells, an HPV-18-transformed cell line, and in normal human foreskin keratinocytes, respectively (Desaintes et al., 1997
; Frattini et al., 1997
). The bovine papillomavirus E2 protein has been shown to induce growth arrest in HeLa cells by down-regulating expression of the integrated HPV-18 oncogenes (Francis et al., 2000
). However, we have shown that the DNA-binding activity of the HPV-16 E2 protein is not required for induction of apoptosis in these cells (Webster et al., 2000
). Thus, the ability of the E2 proteins to induce cell death is not purely a consequence of their ability to bind DNA and alter viral gene expression. Taken together these observations suggest that following HPV-16 infection, the E2 protein could generate a pro-apoptotic signal within the cell.
The HPV-16 E7 protein brings about cell proliferation and can also induce apoptosis (reviewed by Crook & Vousden, 1996 ). E7 binds to the Rb tumour suppressor protein and the Rb-related proteins p107 and p130 (Dyson et al., 1989
). The binding of E7 to RbE2F complexes brings about the release of E2F and targets Rb for ubiquitin-dependent proteolysis (Boyer et al., 1996
; Jones et al., 1997
). Once released from Rb, E2F-1 activates the transcription of genes required for S-phase and can also induce apoptosis (Wu & Levine, 1994
; Qin et al., 1994
; Field et al., 1996
). Overexpression of the HPV-16 E6 protein can block both E2- and E7-induced apoptosis (Webster et al., 2000
). One function of the E6 protein is to bind the p53 tumour suppressor protein and target this protein for degradation (Scheffner et al., 1990
; Werness et al., 1990
; Lechner et al., 1992
; Hubbert et al., 1992
). Interestingly, the HPV-16 E2 protein can interact physically with p53 and both E2 and E7 are capable of inducing p53-dependent apoptosis (Massimi et al., 1999
; Webster et al., 2000
). Thus, during virus infection the pro-apoptotic signals generated by E7 and E2 are probably counter-balanced by the E6 protein. This is in agreement with studies that have shown decreased apoptosis and increased cell proliferation in HPV-infected cervical epithelium (Nair et al., 1999
). We have suggested previously that random integration events which disrupt the HPV-16 E2 gene upset this balance between pro-apoptotic and anti-apoptotic signals and result in cells that are more likely to proliferate and, therefore, more likely to produce cervical cancer (Sanchez-Perez et al., 1997
). Since the E2 protein regulates HPV gene expression, another possibility is that disruption of the E2 gene leads to the de-regulation of E6 and E7, which in turn leads to increased cell proliferation (Francis et al., 2000
, and references therein).
HPV-16 infection alone is probably insufficient to cause cervical cancer and several possible cofactors have been identified including the steroid hormones progesterone and oestrogen. Progesterone and progestins can act as cofactors in the transformation of baby rat kidney cells by HPV-16 and Ras (Pater et al., 1990 ). Furthermore, progesterone has been reported to increase HPV-16 and HPV-18 gene expression at the levels of transcription and mRNA stability (Chen et al., 1996
; Yuan et al., 1999
a; Mittal et al., 1993
). Most cases of cervical cancer arise in the most oestrogen-sensitive region of the cervix, an area known as the transformation zone (Autier et al., 1996
, and references therein). Furthermore, the incidence of HPV DNA in exfoliated cervical cells increases during pregnancy when oestrogen levels are elevated (Rando et al., 1989
; Schneider et al., 1987
) and prolonged use of oestrogen-containing oral contraceptives has been reported to double the risk of cervical cancer (Brisson et al., 1994
). Although the levels of E7 protein within HPV-transformed cell lines do not appear to change after oestrogen treatment (Selvey et al., 1994
), oestrogens have been reported to increase HPV gene expression and orally administered oestrogen can increase the proliferation of cervical cancer cells in vivo (Kim et al., 2000
; Mitrani-Rosenbaum et al., 1989
; Chen et al., 1996
; Bhattacharya et al., 1997
). In HPV-18 transgenic mice, HPV gene expression has been shown to vary during pregnancy and to be increased in the presence of oestrogen or progesterone (Michelin et al., 1997
). In addition, chronic oestrogen exposure has been shown to induce cervical carcinogenesis in transgenic mice expressing the HPV-16 E6 and E7 genes (Arbeit et al., 1996
). Interestingly, in these E6/E7 transgenic mice expression of E6 and E7 is under the control of the human keratin-14 promoter, suggesting that oestrogens do not induce cervical cancer simply by altering HPV gene expression (Arbeit et al., 1996
).
Although the mechanism whereby oestrogens act synergistically with E6 and/or E7 to induce cervical cancer is not known, one possibility is that oestrogens might act as survival factors, enabling the enhanced proliferation of cells expressing E6 and E7. This could in turn result in a larger pool of HPV-infected cells within which tumour cells could arise spontaneously. Another possibility is that high levels of DNA damage brought about by the oestrogen metabolite 16-hydroxyoestrone might result in the accumulation of mutations that eventually lead to carcinogenesis (Auborn et al., 1991
; Newfield et al., 1998
). Oestrogen is metabolized extensively within the body to produce a family of related compounds, including 16
-hydroxyoestrone. 16
-hydroxyoestrone is highly oestrogenic and has also been shown to be tumorigenic in mice (Swaneck & Fishman, 1988
; Telang et al., 1992
). The transformation zone of the cervix displays a constitutively high level of conversion of oestradiol to 16
-hydroxyoestrone and when these cells are immortalized by HPV-16 DNA, this activity increases around 8-fold (Auborn et al., 1991
).
Here we show that progesterone, oestrogen and 16-hydroxyoestrone increase the levels of apoptosis induced by the E2 and E7 proteins. We discuss the implications of these findings in terms of the biological roles of the E2 and E7 proteins in HPV-infected cells and in cervical carcinogenesis.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and transfections.
HeLa cells were maintained in Minimal Essential Medium (MEM; Sigma) supplemented with 10% foetal bovine serum (FBS; Sigma) and penicillin (100000 U/l) and streptomycin (100 mg/l) at 37 °C in 5% CO2. In some cases supplements were added to the media: progesterone (Sigma), RU486 (Sigma), 17--oestradiol (RBI), 3-hydroxytamoxifen (RBI), indole-3-carbinol (Sigma) and 16
-hydroxyoestrone (Sigma), at the concentrations stated in the figure legends.
Prior to transient transfection, the cells were seeded at 3x105 cells per well onto coverslips in six-well plates and incubated overnight to obtain a subconfluent culture. The liposome-based reagent Tfx-20 (Promega) was used at a 3:1 liposome:DNA ratio in 1 ml serum-free medium per transfection, according to the manufacturers instructions. After 18 h (E7 experiments) or 30 h (E2 experiments), the coverslips were washed in PBS and the cells fixed in 4% paraformaldehydePBS at 22 °C for 30 min. Following further washes in PBS, the cells were stained with bisbenzimide (Hoechst no. 33258; Sigma) for 30 min. Finally, the coverslips were washed in PBS and mounted onto microscope slides in 10 µl Mowiol (Calbiochem).
Fluorescence microscopy.
Fluorescence microscopy was carried out using a Leica DM IRBE inverted epifluorescent microscope with FITC and DAPI filter sets and a 20x air objective (Leica).
MTT assays.
After removal of medium, cells were incubated in 5 µg/ml MTT [3-(4,5 dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide] for 2 h at 37 °C. The blue formazan crystals were then extracted and dissolved in DMSO, and the absorbance was measured at 560 nm with an ELISA plate reader (Mosmann, 1983 ).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Progesterone increases E2- and E7-induced apoptosis via the progesterone receptor
Progesterone binds to the progesterone receptor and induces a conformational change in the receptor that allows the hormonereceptor complex to bind to specific DNA sequences and regulate gene expression. The anti-progesterone compound RU486 (Mifepristone) binds with high affinity to the progesterone receptor and acts as a partial antagonist (reviewed by Cadepond et al., 1997 ). To determine whether the progesterone receptor is required for the increase in E2- and E7-induced cell death seen in the presence of progesterone, we cultured HeLa cells in 2 µM progesterone for 1 week prior to transfection with the plasmids pWEB-E2 and pWEB-E7. The transfected cells were then incubated in media containing 2 µM progesterone and increasing concentrations of RU486. As expected, the cell populations transfected with either the E2 or the E7 expression plasmid and incubated in 2 µM progesterone show high levels of apoptosis (Fig. 2a
, b
, respectively). In contrast, in the presence of progesterone and 50 nM RU486 the levels of E2- and E7-induced apoptosis are reduced almost to the background levels seen in the population transfected with the empty vector. RU486 can also bind to the glucocorticosteroid receptor and act as an anti-glucocorticosteroid. However, the glucocorticosteroid dexamethosone has little if any effect on the levels of E2- or E7-induced apoptosis, suggesting that in this case the effects of RU486 are not due to its binding to the glucocorticosteroid receptor (not shown). Taken together, these experiments suggest that the progesterone receptor is required for progesterone to elicit its effect on E2- and E7-induced apoptosis.
|
Oestrogen increases the levels of E2- and E7-induced apoptosis
To investigate any effects of oestrogen on E2- and E7-induced apoptosis we transiently transfected HeLa cells with pWEB-E2, pWEB-E7 or the empty pWEB vector and then incubated the cells in media containing different concentrations of 17--oestradiol, the primary active oestrogen. As expected, transfection with the E7-expressing plasmid results in a significant increase in the level of apoptosis when compared to cells transfected with the empty vector (Fig. 3a
). In the presence of oestrogen there is a significant increase in the level of E7-induced apoptosis. The percentage of E7-expressing cells undergoing apoptosis increases from around 20% in the absence of oestrogen to around 40% in the presence of 100 nM oestrogen. In contrast, the level of apoptosis in the cells transfected with the empty pWEB vector remains at around 57% in both the presence and absence of oestrogen. Oestrogen also brings about a significant increase in the level of E2-induced apoptosis (Fig. 3b
). However, in this case higher concentrations of oestrogen (between 200 and 400 nM) are required to bring about an increase from around 20% apoptotic cells in the absence of oestrogen to around 40% in the presence of oestrogen. These higher oestrogen concentrations do not increase the level of apoptosis in cells transfected with the empty vector. Thus, oestrogen increases the levels of E7- and E2-induced apoptosis but does not increase the background level of apoptosis in these cells.
|
Oestrogen increases E2- and E7-induced apoptosis via an oestrogen receptor
Oestrogen binds to oestrogen receptors resulting in a conformational change that allows the hormonereceptor complexes to regulate the expression of target genes. The oestrogen receptor antagonist 3-hydroxytamoxifen (3-OHT) binds to oestrogen receptors and blocks receptor activity. To determine whether oestrogen receptors are required for the increase in E2- and E7-induced cell death seen in the presence of oestrogen, we transfected E2 and E7 expression plasmids into HeLa cells and then incubated the cells in media containing oestrogen and increasing concentrations of 3-OHT. As expected, cells transfected with pWEB-E2 or pWEB-E7 and incubated in the presence of 400 and 100 nM oestrogen respectively, show high levels of apoptosis (Fig. 4a, b
). However, the percentage of apoptotic cells is reduced from around 35% in the presence of E2 and oestrogen to around 13% in the presence of E2, oestrogen and 400 nM 3-OHT (Fig. 4 a
). Similarly, the percentage of apoptotic cells is reduced from around 35% in the presence of E7 and oestrogen to around 15% in the presence of E7, oestrogen and 400 nM 3-OHT (Fig. 4b
). In each experiment the percentage of apoptotic cells in the pWEB-transfected population is not altered in the presence of oestrogen and 3-OHT. In the absence of oestrogen, 3-OHT has little or no effect on the levels of E2- or E7-induced apoptosis (Fig. 4c
). Similarly, MTT assays show that 3-OHT and the combination of 3-OHT and oestrogen have little or no effect on the proliferation of these cells (Fig. 4d
, e
, respectively). Taken together these data suggest that the effects of oestrogen on the levels of E2- and E7-induced apoptosis are mediated by an oestrogen receptor.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several previous studies have shown that HPV gene expression can be increased in response to either progesterone and oestrogen (Mitrani-Rosenbaum et al., 1989 ; Mittal et al., 1993
; Chen et al., 1996
; Yuan et al., 1999a
; Kim et al., 2000
). Thus one way in which these hormones might bring about an increase in the levels of E2- and E7-induced cell death in HeLa cells is via a direct effect upon expression of the integrated HPV-18 E6 and/or E7 genes. At the concentrations used in our experiments neither progesterone nor oestrogen has any effect on the levels of apoptosis seen in cells that are not expressing E2 or exogenous E7. These data suggest that the ability of these hormones to augment the levels of E2- and E7-induced cell death may not simply be a consequence of their effects on expression of the integrated E6 and E7 genes. However, whether progesterone and oestrogen increase E2- and E7-induced cell death via direct effects on HPV gene expression, or via indirect pathways, our experiments demonstrate that these hormones are important in determining the outcome of E2 and E7 expression.
The oestrogen receptor antagonist 3-hydroxytamoxifen blocks the increases in E2- and E7-induced cell death seen in the presence of oestrogen, but has no effect on the levels of E2- or E7-induced cell death seen in the absence of oestrogen, or on the background levels of cell death. Similarly, the anti-progesterone RU486 (Mifepristone) blocks the increases in E2- and E7-induced cell death seen in the presence of progesterone. Somewhat surprisingly, RU486 can also block the increases in E2- and E7-induced cell death seen in the presence of oestrogen. These data suggest that oestrogen acts upstream of progesterone (or at least the progesterone receptor) in a pathway that leads to increased cell death in the presence of overexpressed E2 or E7. One possibility is that oestrogen increases the levels of progesterone receptor within HeLa cells and that in the presence of progestins this leads to increased E2- and E7-induced cell death. Another possibility is that RU486 blocks E2- and E7-induced cell death indirectly. Both E2 and E7 induce p53-dependent apoptosis in at least some experimental systems (Webster et al., 2000 ) and RU486 has been shown to decrease the levels of p53 in breast cancer cells (Hurd et al., 1995
). However, RU486 has been shown to have little or no effect on p53 levels in C4-1 cervical carcinoma cells (Kamradt et al., 1999
).
Oestrogen metabolism produces a family of related compounds including the highly oestrogenic 16-hydroxyoestrone and the weakly anti-oestrogenic 2-hydroxyoesterone. Cells in the transformation zone of the cervix exhibit a constitutively high level of conversion of oestrogen to 16
-hydroxyoestrone (Auborn et al., 1991
). We have shown that 16
-hydroxyoestrone can also increase the levels of both E2- and E7-induced apoptosis. Indole-3-carbinol is a dietary compound that induces 2-hydroxylation of oestrogen at the expense of 16
-hydroxylation and that has been proposed as a potential preventative of cervical cancer (Michnovicz & Bradlow, 1990
; Yuan et al., 1999b
). We have shown that indole-3-carbinol blocks the stimulatory effect of oestrogen on the levels of E2- and E7-induced cell death but does not block the stimulatory effect of 16
-hydroxyoestrone. Therefore, the 16
-hydroxylation of oestrogen is probably required for the effect of this hormone on E2- and E7-induced cell death. However, indole-3-carbinol has been shown to induce cell cycle arrest in breast cancer cells through the inhibition of cyclin-dependent kinase-6 gene expression (Cover et al., 1998
). Thus it is also possible that it might block the increase in E2- and E7-induced apoptosis seen in the presence of oestrogen via a mechanism that does not involve changes in oestrogen metabolism.
In HPV-induced cervical cancer, HPV DNA is often integrated into the host genome and this frequently results in the loss of the E2 protein. Since the E2 protein can modulate HPV gene expression, virus integration results in deregulated expression of E6 and E7. Furthermore, since HPV E2 protein is a potent inducer of apoptosis, the loss of E2 might be expected to result in increased cell proliferation (Sanchez-Perez et al., 1997 ; Webster et al., 2000
). We have shown here that oestrogen and progesterone increase the levels of E2-induced cell death. One possibility is that in the presence of E2 these hormones might be protective against cervical cancer via their upregulation of cell death. In contrast, in the absence of E2 these hormones might be a risk factor in cervical carcinogenesis either via their effects on HPV or cellular gene expression, or via other, as yet ill-defined, pathways. Agents such as indole-3-carbinol and 3-hydroxytamoxifen that block the effects of oestrogens, and RU486 that blocks the effects of progestins, could also have different effects on cancer risk before and after HPV integration. Our results highlight the need for further work in this area.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Auborn, K. J., Woodworth, C., DiPaolo, J. A. & Bradlow, H. L.(1991). The interaction between HPV infection and estrogen metabolism in cervical carcinogenesis. International Journal of Cancer 49, 867-869.
Autier, P., Coibion, M., Huet, F. & Grivegnee, A. R.(1996). Transformation zone location and intraepithelial neoplasia of the cervix uteri. British Journal of Cancer 74, 488-490.[Medline]
Baker, C. C., Phelps, W. C., Lindgren, V., Braun, M. J., Gonda, M. A. & Howley, P. M.(1987). Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. Journal of Virology 61, 962-971.[Medline]
Bhattacharya, D., Redkar, A., Mittra, I., Sutaria, U. & MacRae, K. D.(1997). Oestrogen increases S-phase fraction and oestrogen and progesterone receptors in human cervical cancer in vivo. British Journal of Cancer 75, 554-558.[Medline]
Bosch, F. X., Manos, M. M., Munoz, N., Sherman, M., Jansen, A. M., Peto, J., Schiffman, M. H., Moreno, V., Kurman, R. & Shah, K. V.(1995). Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. Journal of the National Cancer Institute 87, 796-802.[Abstract]
Boyer, S. N., Wazer, D. E. & Band, V.(1996). E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitinproteasome pathway. Cancer Research 56, 4620-4624.[Abstract]
Brisson, J., Morin, C., Fortier, M., Roy, M., Bouchard, C., Leclerc, J., Christen, A., Guimont, C., Penault, F. & Meisels, A.(1994). Risk factors for cervical intraepithelial neoplasia: differences between low- and high-grade lesions. American Journal of Epidemiology 140, 700-710.[Abstract]
Cadepond, F., Ulmann, A. & Baulieu, E.-E.(1997). RU486 (Mifepristone): mechanisms of action and clinical uses. Annual Review of Medicine 48, 129-156.[Medline]
Chen, Y.-H., Huang, L.-H. & Chen, T.-M.(1996). Differential effects of progestins and estrogens on long control regions of human papillomavirus types 16 and 18. Biochemical and Biophysical Research Communications 224, 651-659.[Medline]
Corden, S. A., Sant-Cassia, L. J., Easton, A. J. & Morris, A. G.(1999). The integration of HPV-18 DNA in cervical carcinoma. Molecular Pathology 52, 275-278.[Abstract]
Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F. & Firestone, G. L.(1998). Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. Journal of Biological Chemistry 273, 3838-3847.
Crook, T. & Vousden, K. H. (1996). In Papillomavirus Reviews: Current Research on Papillomaviruses, pp. 5560. Edited by C. Lacey. Leeds: Leeds University Press.
Desaintes, C., Demeret, C., Goyat, S., Yaniv, M. & Thierry, F.(1997). Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis. EMBO Journal 16, 504-514.
Dürst, M., Kleinheinz, A., Hotz, M. & Gissmann, L.(1985). The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours. Journal of General Virology 66, 1515-1522.[Abstract]
Dyson, N., Howley, P. M., Münger, K. & Harlow, E.(1989). The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937.[Medline]
Field, S. J., Tsai, F. Y., Kuo, F., Zubiaga, A. M., Kaelin, W. G.Jr, Livingston, D. M., Orkin, S. H. & Greenberg, M. E.(1996). E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85, 549-561.[Medline]
Francis, D. A., Schmid, S. I. & Howley, P. M.(2000). Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells. Journal of Virology 74, 2679-2686.
Frattini, M. G., Hurst, S. D., Lim, H. B., Swaminathan, S. & Laimins, L. A.(1997). Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumour suppressor protein. EMBO Journal 16, 318-331.
Groshong, S. D., Owen, G. I., Grimison, B., Schauer, I. E., Todd, M. C., Langan, T. A., Sclafani, R. A., Lange, C. A. & Horwitz, K. B.(1997). Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27Kip1. Molecular Endocrinology 11, 1593-1607.
Hubbert, N. L., Sedman, S. A. & Schiller, J. T.(1992). Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. Journal of Virology 66, 6237-6241.[Abstract]
Hurd, C., Khattree, N., Alban, P., Nag, K., Jhanwar, S. C., Dinda, S. & Moudgil, V. K.(1995). Hormonal regulation of the p53 tumor suppressor protein in T47D human breast carcinoma cell line. Journal of Biological Chemistry 270, 28507-28510.
Jones, D. L., Thompson, D. A. & Münger, K.(1997). Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis. Virology 239, 97-107.[Medline]
Kamradt, M. C., Mohideen, N. & Vaughan, A. T. M.(1999). RU486 increases radiosensitivity and restores apoptosis through modulation of HPV E6/E7 in dexamethasone-treated cervical carcinoma cells. Gynecologic Oncology 77, 177-182.
Kim, C. J., Um, S. J., Kim, T. Y., Kim, E. J., Park, T. C., Kim, S. J., Namkoong, S. E. & Park, J. S. (2000). Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells. International Journal of Gynecology & Obstetrics 10, 157164.
Lechner, M. S., Mack, D. H., Finicle, A. B., Crook, T., Vousden, K. H. & Laimins, L. A.(1992). Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription. EMBO Journal 11, 3045-3052.[Abstract]
Massimi, P., Pim, D., Bertoli, C., Bouvard, V. & Banks, L.(1999). Interaction between the HPV-16 E2 transcriptional activator and p53. Oncogene 18, 7748-7754.[Medline]
Michelin, D., Gissmann, L., Street, D., Potkul, R. K., Fisher, S., Kaufmann, A. M., Qiao, L. & Schreckenberger, C.(1997). Regulation of human papillomavirus type 18 in vivo: effects of estrogen and progesterone in transgenic mice. Gynecologic Oncology 66, 202-208.[Medline]
Michnovicz, J. J. & Bradlow, H. L.(1990). Induction of estradiol metabolism by dietary indole-3-carbinol in humans. Journal of the National Cancer Institute 82, 947-949.[Abstract]
Mitrani-Rosenbaum, S., Tsvieli, R. & Tur-Kaspa, R.(1989). Oestrogen stimulates differential transcription of human papillomavirus type 16 in SiHa cervical carcinoma cells. Journal of General Virology 70, 2227-2232.[Abstract]
Mittal, R., Tsutsumi, K., Pater, A. & Pater, M. M.(1993). Human papillomavirus type 16 expression in cervical keratinocytes: role of progesterone and glucocorticoid hormones. Obstetrics and Gynecology 81, 5-12.[Abstract]
Mosmann, T.(1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63.[Medline]
Nair, P., Nair, K. M., Jayaprakash, P. G. & Pillai, M. R.(1999). Decreased programmed cell death in the uterine cervix associated with high risk human papillomavirus infection. Pathology Oncology Research 5, 95-103.[Medline]
Newfield, L., Bradlow, H. L., Sepkovic, D. W. & Auborn, K.(1998). Estrogen metabolism and the malignant potential of human papillomavirus immortalized keratinocytes. Proceedings of the Society for Experimental Biology and Medicine 217, 322-326.[Abstract]
Pater, A., Bayatpour, M. & Pater, M. M.(1990). Oncogenic transformation by human papillomavirus type 16 deoxyribonucleic acid in the presence of progesterone or progestins from oral contraceptives. American Journal of Obstetrics and Gynecology 162, 1099-1103.[Medline]
Qin, X.-Q., Livingston, D. M., Kaelin, W. G.Jr & Adams, P. D.(1994). Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proceedings of the National Academy of Sciences, USA 91, 10918-10922.
Rando, R. F., Lindheim, S., Hasty, L., Sedlacek, T. V., Woodland, M. & Eder, C.(1989). Increased frequency of detection of human papillomavirus deoxyribonucleic acid in exfoliated cervical cells during pregnancy. American Journal of Obstetrics and Gynecology 161, 50-55.[Medline]
Sanchez-Perez, A.-M., Soriano, S., Clarke, A. R. & Gaston, K.(1997). Disruption of the human papillomavirus type 16 E2 gene protects cervical carcinoma cells from E2F-induced apoptosis. Journal of General Virology 78, 3009-3018.[Abstract]
Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M.(1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129-1136.[Medline]
Schneider, A., Hotz, M. & Gissmann, L.(1987). Increased prevalence of human papillomaviruses in the lower genital tract of pregnant women. International Journal of Cancer 40, 198-201.
Schneider-Maunoury, S., Croissant, O. & Orth, G.(1987). Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors. Journal of Virology 61, 3295-3298.[Medline]
Selvey, L. A., Dunn, L. A., Tindle, R. W., Park, D. S. & Frazer, I. H.(1994). Human papillomavirus (HPV) type 18 E7 protein is a short-lived steroid-inducible phosphoprotein in HPV-transformed cell lines. Journal of General Virology 75, 1647-1653.[Abstract]
Swaneck, G. E. & Fishman, J.(1988). Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization. Proceedings of the National Academy of Sciences, USA 85, 7831-7835.[Abstract]
Telang, N. T., Suto, A., Wong, G. Y., Osborne, M. P. & Bradlow, H. L.(1992). Induction by estrogen metabolite 16 alpha-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells. Journal of the National Cancer Institute 84, 634-638.[Abstract]
Thierry, F. (1996). HPV proteins in the control of HPV transcription. In Papillomavirus Reviews: Current Research on Papillomaviruses, pp. 2129. Edited by C. Lacey. Leeds: Leeds University Press.
van Ranst, M., Tachezy, R. & Burk, R. D.(1996). Human papillomaviruses: a neverending story? In Papillomavirus Reviews: Current Research on Papillomaviruses , pp. 1-19. Edited by C. Lacey. Leeds:Leeds University Press.
Walboomers, J. M. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J., Shah, K. V., Snijders, P. J. F., Peto, J., Chris, J. L. M., Meijer, C. J. L. M. & Muñoz, N.(1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. Journal of Pathology 189, 12-19.[Medline]
Webster, K., Parish, J., Pandya, M., Stern, P. L., Clarke, A. R. & Gaston, K.(2000). The HPV 16 E2 protein induces apoptosis in the absence of other HPV proteins and via a p53-dependent pathway. Journal of Biological Chemistry 275, 87-94.
Werness, B. A., Levine, A. J. & Howley, P. M.(1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79.[Medline]
Wu, X. & Levine, A. J.(1994). p53 and E2F-1 cooperate to mediate apoptosis. Proceedings of the National Academy of Sciences, USA 91, 3602-3606.[Abstract]
Yuan, F., Auborn, K. & James, C.(1999a). Altered growth and viral gene expression in human papillomavirus type 16-containing cancer cell lines treated with progesterone. Cancer Investigation 17, 19-29.[Medline]
Yuan, F., Chen, D., Liu, K., Sepkovic, D. W., Bradlow, H. L. & Auborn, K.(1999b). Anti-estrogenic activities of indole-3-carbinol in cervical cells: implication for prevention of cervical cancer. Anticancer Research 19, 1-8.[Medline]
Received 23 June 2000;
accepted 26 September 2000.