Mutagenesis, Vol. 18, No. 3, 243-247,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Increased cell proliferation is associated with genomic instability: elevated micronuclei frequencies in estradiol-treated human ovarian cancer cells
Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg and 2 Institute of Toxicology, Merck KGaA, Darmstadt, Germany
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Abstract |
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Estrogen-related cancers are often associated with the hormone’s tumor promoting activity. Recently, estradiol has also been demonstrated to induce gene mutations in the physiological concentration range. Mitotic disturbances are found at higher concentrations. In the present study we demonstrate data suggesting an additional mechanism for the induction of genetic damage, i.e. chromosomal breakage. Estrogen receptor-positive (BG-1) and -negative (UCI) human ovarian cancer cell lines were investigated for micronucleus formation after treatment with estradiol. BG-1 cells but not UCI cells showed an increase in micronucleus formation which correlated with the estradiol-induced cell proliferation. The specific estradiol receptor antagonist hydroxytamoxifen suppressed the formation of micronuclei in BG-1 cells. Increased micronucleus frequencies were also seen after normalization of the data to the number of cell divisions. Kinetochore analysis revealed a difference between micronuclei induced by picomolar concentrations of estradiol (kinetochore-negative) and micromolar concentrations (predominantly kinetochore-positive) leading to mitotic disturbances. In accordance with this finding, analysis of the cell cycle revealed decreased cell numbers in G2/M phase after treatment with picomolar concentrations, usually not found after mitotic disturbances. We hypothesize that hormone-specific forcing of responsive cells through the cell cycle leads to an override of checkpoints operating under homeostatic control of the cell cycle, resulting in genomic instability.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Neoplasias of hormone-responsive tissues currently account for >35% of all newly diagnosed cancers in men and >40% in women in the USA (Wingo et al., 1995
). The carcinogenic activity of estradiol has been demonstrated in several animal studies. Administration of estradiol to mice increased the incidence of mammary, vaginal, uterine and several other types of cancer (Highman et al., 1980, 1981; Nagasawa et al., 1980). Estradiol-related carcinogenicity is considered to be the result of a combination of hormonal and genotoxic mechanisms (reviewed in Roy and Liehr, 1999; Liehr, 2000).
Binding of estradiol to its receptors can result in the stimulation of cell proliferation. Cells containing damaged DNA might gain a growth advantage and thereby contribute to tumor formation (Feigelson and Henderson, 1996). However, administration of synthetic estrogens possessing equivalent hormonal activity to estradiol showed reduced carcinogenic activity in Syrian hamsters (Liehr et al., 1986). This indicates that inducing proliferation of damaged cells by estradiol might not be sufficient for tumor development. In addition, it seems that direct genotoxic effects of estradiol might play a role. In fact, several lines of evidence indicate a genotoxic potential of estradiol. Recently, Kong and co-workers reported the induction of gene mutations such as deletions and point mutations in Chinese hamster V79 cells at physiological concentrations (Kong et al., 2000). Furthermore, chromosomal damage such as ploidy changes and micronucleus formation has been reported (Wheeler et al., 1986; Eckert and Stopper, 1996; Schuler et al., 1998). Thus, the current concept of estradiol-induced carcinogenesis displays a dual role for estradiol. It can act as a hormone, stimulating cell proliferation, and in addition as an initiating carcinogen inducing direct genomic damage (Liehr et al., 1986; Liehr, 2000).
Recently, we have presented data suggesting an additional mechanism for the induction of genomic damage by estradiol (Fischer et al., 2001). In the human breast cancer cell line MCF-7 we have found an elevation in the formation of micronuclei after estradiol-stimulated cell proliferation at physiological concentrations. We hypothesized that forcing cells through the cell cycle might lead to an override of checkpoints operating under homeostatic control. High fidelity maintenance of genomic integrity is ensured by DNA repair and cell cycle checkpoints, surveillance pathways that respond to DNA damage by inhibiting critical cell cycle events (Weinert, 1998). Impaired fidelity of checkpoint control might give cells the opportunity to proceed through mitosis without adequate DNA repair, resulting in elevated genomic damage.
In order to further support our hypothesis, we extended our previous work. Here, we present data on the induction of micronuclei in estrogen receptor-positive (BG-1) and -negative (UCI) human ovarian cancer cell lines, indicating that increased cell proliferation might be associated with a decreased genomic stability.
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Materials and methods |
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Cell culture and micronucleus test
BG-1 and UCI cells were cultured in DMEM/F12 (BG-1) or RPMI 1640 medium (UCI) supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate, human insulin (0.2 ng/ml) and 5% fetal bovine serum (FBS) (all from Sigma Chemie GmbH, Deisenhofen, Germany). Cell cultures were grown in a humidified atmosphere with 5% CO2 in air at 37°C. For experiments, cells (0.5–0.7x105/ml) were seeded in culture flasks (T25). Twenty-four hours later the medium was exchanged. The fresh medium (Phenol red-free) contained 5% charcoal/dextran-treated FBS (Hyclone) and test compunds as indicated. At 96 h the medium was exchanged again and cells were harvested at 168 (BG-1) or 100 h (UCI). Cell numbers were determined by Coulter counter. In experiments with cytochalasin B cells were incubated for 48 h in the presence of 2.5 µg/ml cytochalasin B.
After harvesting, cells were placed on glass slides by cytospin centrifugation and fixed in methanol (-20°C, 1 h). For staining nuclei and micronuclei the slides were incubated with acridine orange (0.00625% w/v in Sorensen buffer, pH 6.8, 4 min). Slides were washed twice with buffer and mounted for microscopy. Numbers of nuclei and micronuclei were scored at a magnification of 500x. In experiments with cytochalasin B micronuclei were evaluated in binucleated cells only. Each data point represents the mean of three slides from one experiment with 1000 cells/binucleate cells analyzed for the number of micronuclei per slide. Objects were classified as micronuclei if they were clearly separated from the nucleus, were round or oval, had an area of less than one-quarter of the area of a nucleus and showed staining characteristics similar to those of the nuclei.
Gene expression assay
Transient transfection of cells with the plasmids pSVGal and ptkERE2Luc was performed by the Lipofectamine method according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). Briefly, cells were seeded in 96-well plates at a density of 16 000 cells/well in Phenol red-free medium containing 5% charcoal/dextran-treated FBS. The next day cells were transfected in serum-free, Phenol red-free medium using Lipofectamine and plasmids for 6.5 h. After transfection, the medium was aspirated from the wells and replaced with Phenol red-free medium containing 5% charcoal/dextran-treated FBS and the indicated test compounds. Twenty hours later the medium was removed and cells were incubated in lysis buffer for 30 min at 25°C. Collected supernantants were divided into two portions. One was analyzed for luciferase activity while the other was used for determination of the ß-galactosidase activity. Luciferase activity was determined in a Berthold LB96P luminometer using the manufacturer’s reagents and instructions. ß-Galactosidase activity was measured after addition of 40 µg galactosidase substrate and subsequent incubation at 37°C for 1 h. Optical density was measured at 420 nm. Luciferase activity, i.e. gene expression activity, was normalized for ß-galactosidase activity and protein concentration. Luciferase activity of samples treated with test compounds divided by the normalized luciferase activity of those treated with vehicle was used to determine the induction. The data shown are representative of three independent experiments, each with three replicates.
Kinetochore staining
Kinetochore staining was performed by incubating the fixed cell preparations [after rinsing with phosphate-buffered saline (PBS)/0.1% Tween 20] with CREST serum (60 min) in a humidified chamber at 37°C. After rinsing with PBS/0.5% Tween 20 cells were incubated (30 min) with FITC-conjugated goat anti-human antibody (diluted 1:100 in PBS), rinsed again (PBS/0.1% Tween 20) and counterstained with bisbenzimide 33258 (1 µg/ml, 5 min). Slides were mounted for microscopy with antifade (Oncor). At least 100 micronuclei were analyzed for the presence of kinetochore signals.
Flow cytometry
After harvesting the cells were fixed in ethanol (70%, 4°C, 60 min). After washing with PBS cells were stained with propidium iodide (0.25 mg/ml) containing RNase (25 U/ml) for 1 h at room temperature. Fluorescence was measured with a FACScan using CELLQuest software (BD, Heidelberg, Germany). If cells were not analysed immediately they were stored at 4°C.
Statistical analysis
The statistical analysis of data was determined by the Student’s t-test. The level of significance was set at P < 0.05.
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Results and discussion |
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In the present study we investigated the induction of cell proliferation and micronucleus formation in human ovarian cancer cells after estrogen treatment. Our model consisted of estrogen receptor-positive BG-1 cells and as a control estrogen receptor-negative UCI cells. To verify the expression and biological activity of the estrogen receptor in the BG-1 cells, transient transfection with a plasmid carrying the estrogen-responsive element conjugated with the reporter enzyme luciferase was performed (Klotz et al., 1996
). Treatment of transfected BG-1 cells with estradiol (1 nM) resulted in an increased activity, i.e. gene expression (Figure 1). The known estrogen receptor antagonist hydroxytamoxifen (50 nM) significantly reduced the activity. In control experiments with transfected UCI cells no induction of gene expression was observed (data not shown). Treatment of BG-1 cells with estradiol (10 pM) resulted in a significant increase in cell proliferation as well as micronucleus formation (Figure 2A and B). The observed effect was found to be reversible after addition of the estrogen receptor antagonist hydroxytamoxifen (0.5 nM), indicating specificity. The effect induced by estradiol showed dose dependency (data not shown). In contrast, UCI cells showed neither elevated cell proliferation nor increased micronucleus frequencies (Figure 2C and D). Even after treatment with 10 times higher estradiol concentrations (100 pM) no increase in micronucleus frequency was observed (19 MN/1000 cells in controls versus 17 MN/1000 cells after treatment). Together, these data indicate that an increased genomic instability might be caused by an increased rate of cell proliferation after hormonal stimulation. This idea is supported by our recent study, where we demonstrated a positive correlation between the rate of cell proliferation and micronucleus formation in the human breast cancer cell line MCF-7 after hormonal stimulation (Fischer et al., 2001). Furthermore, Davis and co-workers reported a similar finding, where estrogen-stimulated cell proliferation enhanced benzo[a]pyrene-induced micronucleus formation in MCF-7 cells (Davis et al., 2002).
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In order to further scrutinize the formation of micronuclei in BG-1 cells we investigated induced micronuclei for the presence of kinetochores (K). Spindle disturbing compounds induce K+ micronuclei, whereas clastogenic substances induce predominantly K- micronuclei (Antoccia et al., 1991
). Therefore, we analyzed micronuclei presumably derived from increased cell proliferation (low concentration, 100 pM) and those induced by ‘genotoxic’ concentrations (high concentration, 30 µM) of estradiol or by the known clastogenic compound mitomycin C (MMC). Since disturbances of the mitotic spindle apparatus is considered a mechanism in micronucleus induction by estradiol in receptor-negative cells, we also scrutinized micronuclei induced by the non-clastogenic and spindle inhibiting hormone diethylstilbestrol (DES). As depicted in Table I, MMC induced 13.8% K+ micronuclei, whereas DES induced 60.2% K+ micronuclei. High concentrations of estradiol resulted in 63.6% K+ micronuclei, in accordance with the spindle inhibiting mechanism. In contrast, when cells were stimulated with a low concentration of estradiol (100 pM) only 16.7%/12.9% of micronuclei were found to be K+. Since the number of induced micronuclei was higher when cells were incubated with 30 µM (Table I), the difference in the percentage of K+ micronuclei cannot be due to a higher contribution of spontaneous micronuclei (12.9% K+). Taken together, these findings support the idea that low concentrations of estradiol (100 pM) induce micronuclei via a mechanism(s) independent of the disturbance of the spindle apparatus.
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As suggested previously, an increase in the rate of cell proliferation may lead to impaired fidelity of control and/or repair of genomic integrity during the cell cycle, due to time limitations. Since the induction of increased cell proliferation by estradiol results in increased numbers of mitoses when compared with controls, we normalized the analysis of micronuclei to the number of mitoses by applying cytochalasin B (Fenech, 1993
). This compound inhibits cell division but not mitosis, yielding binucleate cells. If the analysis of micronucleus frequencies is limited to these binucleate cells, all analyzed cells have divided exactly once during the experiment. Frequency of micronuclei was found to be higher by a factor of 1.6 in estradiol-stimulated cells than in controls (Figure 3). Therefore, regarding the genomic stability, i.e. number of micronuclei, these data demonstrate a difference in the quality of cell division between cells treated with estradiol and controls. To further elucidate this relationship we performed additional experiments without the use of cytochalasin B (Figure 4). When treated and untreated cells were harvested at the same time point (see arrow a in Figure 4) the estradiol-stimulated cells showed higher numbers of micronuclei (38 versus 15 MN) as well as a higher cell number compared with controls. When cells were harvested at different time points (see arrow b in Figure 4), micronuclei frequencies were still different (42 versus 15 MN), whereas the cell numbers were identical under these experimental conditions. Again, this clearly demonstrates higher genomic damage in cells which have been ‘pushed’ through the cell cycle via hormonal stimulation.
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In a next set of experiments we investigated whether speeding up cell cycle progression by estradiol is reflected in the distribution of cells in single phases of the cell cycle. Therefore, cells were stimulated with estradiol (10 pM) for the indicated times followed by cell cycle analysis (Figure 5
). In fact, FACS analysis revealed that hormonal stimulation yielded elevated percentages of cells in S phase, decreased cell numbers in G2/M phase and only minor differences in G1/G0 phase. Since estradiol-treated cells proceed faster through G2/M phase, these data are in line with the hypothesis that cell cycle checkpoints might be overridden after hormonal stimulation. In a recent publication, Zhou and co-workers demonstrated that caffeine abolishes the mammalian G2/M DNA damage checkpoint, sensitizing cells for genomic damage (Zhou et al., 2000). In accordance with these oberservations, it has been shown that dysregulated cell cycle checkpoint control also sensitizes cells to DNA damage induction (Suganuma et al., 1999). Thus, it seems likely that our observations can be explained by a mechanism involving cell cycle control. Therefore, the control of genomic integrity might be impaired before cell division, resulting in increased numbers of micronuclei.
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DuMond and Roy (2001)
observed a reduction in DNA repair after treatment of hormone-responsive Leydig cells with DES. In addition, they observed a reduction in cell cycle duration. According to the authors, the reduced DNA repair may have been a result of direct inhibition of repair or merely reflect the reduced time that cells had available to perform the necessary repair of DNA alterations. Impaired DNA repair leads to increased micronuclei frequencies (Fenech and Neville, 1992). Therefore, the observations by DuMond and Roy are in line with our studies. However, estradiol does not impair DNA repair directly (Epstein and Smith, 1988; Coibion et al., 1989), further suggesting that shortening the duration of cell division is responsible for the increased genetic instability after hormonal stimulation of cell proliferation.
According to a recent publication estradiol can exert a direct mutagenic activity in concentrations as low as those used in our experiments (Kong et al., 2000). However, only gene mutations and no chromosomal mutations were investigated by Kong and co-workers. Gene mutations would not be detected in the micronucleus assay and are thus not responsible for the effects we observed. The occurrence of chromosomal damage after treatment with high concentrations of estradiol has been described in the past and was usually ascribed to mitotic disturbances. At concentrations of 10 µM and higher chromosomal ploidy changes were observed (Wheeler et al., 1986; Schuler et al., 1998). Micronucleus induction at 20 µM and higher was found and was explained by mitotic disturbances in accordance with ploidy changes reported in that concentration range (Eckert and Stopper, 1996). In the present investigation, this type of genotoxicity could be detected and clearly separated from the micronuclei induced by increased cell proliferation.
In summary, our findings contribute to the understanding of mechanisms involved in the process of carcinogenesis induced by hormones such as estradiol. It will be of interest to see whether the observed correlation between the rate of cell proliferation and genomic instability can also be observed in an experimental approach using other stimulators of cell division, implicating that forcing cells through the cell cycle per se might be a risk factor.
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Acknowledgments |
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S.F.Arnold and J.A.McLachlan are gratefully acknowledged for providing the plasmids used in the gene expression assays and M.Kessler for technical assistance.
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Notes |
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2 To whom correspondence should be addressed. Tel: +49 931 201 48894; Fax: +49 931 201 48446; Email: fischer{at}toxi.uni-wuerzburg.de
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References |
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Antoccia,A., Degerassi,F., Battistoni,A., Ciliutti,P. and Tanzarella,C. (1991) In vitro micronucleus test with kinetochore staining: evaluation of test performance. Mutagenesis, 6, 319–324.
Coibion,M., Kiss,R., Jossa,V., de Launoit,Y., Van Geloven,O., Malengreau,A., Mattheiem,W., Pasteels,J.L. and Paridaens,R. (1989) In vitro influence of estrdiol or progesterone on the thymidine labeling indices of human benign breast tumors. Anticancer Res., 9, 475–482.[ISI][Medline]
Davis,C., Bhana,S., Shorrocks,A.J. and Martin,F.L. (2002) Oestrogens induce G(1) arrest in benzo[a]pyrene-treated MCF-7 breast cells whilst enhancing genotoxicity and clonogenic survival. Mutagenesis, 17, 431–438.
DuMond,J.W. and Roy,D. (2001) The inhibition of DNA repair capacity by stilbene estrogen in Leydig cells: its implications in the induction of instability in the testicular genome. Mutat. Res., 483, 27–33.[ISI][Medline]
Eckert,I. and Stopper,H. (1996) Genotoxic effects induced by ß-oestradiol in vitro. Toxicol. In Vitro, 10, 637–642.[CrossRef]
Epstein,R.J. and Smith,P.J. (1988) Estrogen-induced potentiation of DNA damage and cytotoxicity in human breast cancer cells treated with topoisomerase II-interactive antitumor drugs. Cancer Res., 48, 297–303.
Feigelson,H.S. and Henderson,B.E. (1996) Estrogens and breast cancer. Carcinogenesis, 17, 2279–2284.
Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[ISI][Medline]
Fenech,M. and Neville,S. (1992) Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes. Environ. Mol. Mutagen., 19, 27–36.[ISI][Medline]
Fischer,W.H., Keiwan,A., Schmitt,E. and Stopper,H. (2001) Increased formation of micronuclei after hormonal stimulation of cell proliferation in human breast cancer cells. Mutagenesis, 16, 209–212.
Highman,B., Greenman,D.L., Norvell,M.J., Farmer,J. and Shellenberger,T.E. (1980) Neoplastic and preneoplastic lesions induced in female C3H mice by diets containing diethylstilbestrol or 17 beta-estradiol. J. Environ. Pathol. Toxicol., 4, 81–95.[ISI][Medline]
Highman,B., Roth,S.I. and Greenman,D.L. (1981) Osseous changes and osteosarcomas in mice continuously fed diets containing diethylstilbestrol or 17 beta-estradiol. J. Natl Cancer Inst., 67, 653–662.[ISI][Medline]
Klotz,D.M., Beckman,B.S., Hill,S.M., McLachlan,J.A., Walters,M.R. and Arnold,S.F. (1996) Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays. Environ. Health Perspect., 104, 1084–1089.[ISI][Medline]
Kong,L.Y., Szaniszlo,P., Albrecht,T. and Liehr,J.G. (2000) Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells. Int. J. Oncol., 17, 1141–1149.[ISI][Medline]
Liehr,J.G. (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev., 21, 40–54.
Liehr,J.G., Stancel,G.M., Chorich,L.P., Bousfield,G.R. and Ulubelen,A.A. (1986) Hormonal carcinogenesis: separation of estrogenicity from carcinogenicity. Chem.-Biol. Interact., 59, 173–184.[CrossRef][ISI][Medline]
Nagasawa,H., Mori,T. and Nakajima,Y. (1980) Long-term effects of progesterone or diethylstilbestrol with or without estrogen after maturity on mammary tumorigenesis in mice. Eur. J. Cancer, 16, 1583–1589.
Roy,D. and Liehr,J.G. (1999) Estrogen, DNA damage and mutations. Mutat. Res., 424, 107–115.[ISI][Medline]
Schuler,M., Hasegawar,L., Parks,R., Metzler,M. and Eastmond,D.A. (1998) Dose-response studies of the induction of hyperdiploidy and polyploidy by diethylstilbestrol and 17ß-estradiol in cultured human lymphocytes using multicolor fluorescence in situ hybridization. Environ. Mol. Mutagen., 31, 263–273.[CrossRef][ISI][Medline]
Suganuma,M., Kawabe,T., Hori,H., Funabiki,T. and Okamoto,T. (1999) Sensitization of cancer cells to DNA damage-induced cell death by specific cell cycle G2 checkpoint abrogation. Cancer Res., 59, 5887–5891.
Weinert,T. (1998) DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell, 94, 555–558.[CrossRef][ISI][Medline]
Wheeler,W.J., Cherry,L.M., Downs,T. and Hsu,T.C. (1986) Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in chinese hamster cells in vitro. Mutat. Res., 171, 31–41.[CrossRef][ISI][Medline]
Wingo,P.A., Tong,T. and Bolden,S. (1995) Cancer statistics, 1995. CA Cancer J. Clin., 45, 8–30.
Zhou,B.-B.S., Chaturvedi,P., Spring,K., Scott,S.P., Johanson,R.A., Mishra,R., Mattern,M.R., Winkler,J.D.and Khanna,K.K. (2000) Caffeine abolishes the mammalian G2/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem., 275, 10342–10348.
Received on April 15, 2002; revised on January 20, 2003; accepted on January 30, 2003.
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