Functional Implications of Antiestrogen Induction of Quinone ...

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Molecular Endocrinology 17(7):1344–1355 Copyright © 2003 by The Endocrine Society doi: 10.1210/me.2002-0382

Functional Implications of Antiestrogen Induction of Quinone Reductase: Inhibition of Estrogen-Induced Deoxyribonucleic Acid Damage NICOLE R. BIANCO, GEORGE PERRY, MARK A. SMITH, DENNIS J. TEMPLETON, MONICA M. MONTANO

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Department of Pharmacology (N.R.B., M.M.M.) and Institute of Pathology (G.P., M.A.S.), Case Western Reserve University School of Medicine, Cleveland, Ohio 44106; and Department of Pathology (D.J.T.), University of Virginia Medical School, Charlottesville, Virginia 22908-0214 Recent studies have shown that the antiestrogens tamoxifen and raloxifene may protect against breast cancer, presumably because of a blockade of estrogen receptor (ER)-mediated transcription. Another possible explanation is that antiestrogenliganded ER transcriptionally induces genes that are protective against cancer. We previously reported that antiestrogen-liganded ER␤ transcriptionally activates the major detoxifying enzyme quinone reductase (QR) [NAD(P)H:quinone oxidoreductase]. It has been established that metabolites of estrogen, termed catecholestrogens, can form DNA adducts and cause oxidative DNA damage. We hypothesize that QR inhibits estrogeninduced DNA damage by detoxification of reactive catecholestrogens. We report here that physiological concentrations of 17␤-estradiol cause oxidative DNA damage, as measured by levels of 8hydroxydeoxyguanine, in ER-positive MCF7 breast cancer cells, MDA-MB-231 breast cancer cells

(ER␣ negative/ER␤ positive) and nontumorigenic MCF10A breast epithelial cells (very low ER), which is dependent on estrogen metabolism. Estrogeninduced 8-hydroxydeoxyguanine was inversely correlated to QR and ER␤ levels and was followed by downstream induction of the DNA repair enzyme XPA. Trans-hydroxytamoxifen, raloxifene, and the pure antiestrogen ICI-182,780 protected against estradiol-mediated damage in breast cancer cells containing ER␤. This is most likely due to the ability of these antiestrogens to activate expression of QR via ER␤. We conclude that upregulation of QR, either by overexpression or induction by tamoxifen, can protect breast cells against oxidative DNA damage caused by estrogen metabolites, representing a possible novel mechanism of tamoxifen prevention against breast cancer. (Molecular Endocrinology 17: 1344–1355, 2003)

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extrahepatic cytochrome P450 enzymes (mainly CYP1A1 and CYP1B1) to hydroxy-catecholestrogens and further oxidized to the semiquinone and quinone form (5–7). This metabolism is potentially harmful, given that the quinone-catecholestrogen can bind to DNA and form DNA adducts. Furthermore, redox cycling between the quinone and unstable semiquinone form causes hydroxyl radical formation that can lead to hydroxylated nucleotide bases [e.g. 8hydroxydeoxyguanine (8-OHdG)] and permanent mutation, if not repaired (2, 3, 8). Glutathione-S-transferase detoxifies these quinones by conjugation with glutathione, and catechol O-methyltransferase detoxifies the hydroxy-catecholestrogens by methylation (3). The estrogens and catecholestrogens can also be detoxified by conjugation to glucuronides and sulfates, although the significance of these processes in the breast is less clear (9). The ability of quinone reductase (QR) to detoxify quinone-catecholestrogens by reduction of the reactive quinone-catecholestrogen back to the hydroxy-catecholestrogen has been shown for two synthetic estrogens, diethylstilbestrol (10) and 4-hydroxyequilenin-o-quinone (11). The resulting hydroxy-catecholestrogen is then available a second

LTHOUGH IT IS widely accepted that the risk of developing breast cancer is directly related to one’s lifetime exposure to estrogen, the precise role of estrogen in the initiation and progression of breast cancer has yet to be determined. It has been hypothesized that initiation may result from induction of DNA damage by estrogen metabolites and preexisting lesions, although progression may be facilitated by estrogen receptor (ER)-mediated up-regulation of mitogenic genes. There is already significant evidence that estrogen metabolites are tumorigenic in animal models (1–3). More recently, the direct action of estrogen has been shown by its ability to transform normal breast epithelial cells in culture (4). In certain cell types, including both normal and breast cancer cells, estrogens may be oxidized by Abbreviations: DMSO, Dimethylsulfoxide; DNase, deoxyribonuclease; E2, estradiol; EpRE, electrophile response element; ER, estrogen receptor; NAC, N-acetylcysteine; 8-OHdG, 8-hydroxydeoxyguanine; 2- or 4-OH-E2, 2- or 4-hydroxyestradiol; QR, quinone reductase; QRAS, QR antisense; RAL, raloxifene; RNase, ribonuclease; TOT, transhydroxytamoxifen; XPA, xeroderma pigmentosum complementation group A.

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time for conjugation and excretion. Alternatively, if conjugation is impaired, then the metabolite may reenter the redox cycle. Figure 8 includes a schematic of estrogen metabolism for reference. In 1998, the antiestrogen tamoxifen became the first drug to be approved for the reduction of risk for breast cancer, showing about a 50% reduction in both noninvasive and invasive breast cancer (12). A more recent Italian study has seen a dramatic decrease in breast cancer specifically in high-risk women, based on risk factors such as nulliparity and early age at menarche, with a previous hysterectomy (13). Raloxifene (RAL), a similar selective ER modulator, may also protect against breast cancer (14). Antiestrogens have traditionally been thought to protect against breast cancer by blocking ER-mediated transcription of mitogenic genes. However, it is also possible that antiestrogens protect cells against tumor-promoting events by inducing detoxification enzymes such as QR. We have previously shown that antiestrogens induce QR via transcriptional regulation by the ER (mainly ER␤) at the antioxidant response element/electrophile response element (EpRE) located in the promoter region of QR and other phase II detoxification genes (15, 16). Thus, our primary goal was to determine whether antiestrogens could block estrogen-induced oxidative DNA damage via activation of QR. We report here that physiological concentrations of estradiol (E2) cause an increase in oxidative DNA damage that is dependent upon estrogen metabolism in MCF-7 breast cancer cells (ER␣/␤ positive) (17), MCF10A normal breast epithelial cells (very low ER) (18), and MDA-MB-231 cells (ER␣ negative/ER␤ positive) (19). This damage is inversely related to QR and ER␤ levels and can also be blocked by antiestrogens if ER␤ is present. We have also found a significant correlation between levels of E2-induced oxidative DNA damage and the downstream induction of the DNA repair enzyme xeroderma pigmentosum complementation group A (XPA).

RESULTS Estrogen-Induced Oxidative DNA Damage in Breast Epithelial Cells Is Dependent on Estrogen Metabolism, But Not the ER Although previous studies have shown estrogeninduced oxidative DNA damage in cultured breast cells, typically high concentrations of estrogen have been used (20, 21). Also, these studies have used in vitro methods such as HPLC-electrochemical detection to determine oxidative damage to DNA, which has produced controversial results (22). Thus, to determine the effect of physiological concentrations of estrogen, we treated breast cells with physiological doses of E2 (10⫺10 to 10⫺8 M) and then measured the oxidative DNA marker 8-OHdG by quantitative immunocytochemistry. This is a previously established

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method for quantifying relative increases in cellular 8-OHdG levels (23, 24), and compared with methods that involve prior isolation and manipulation of DNA, this method does not create artificial oxidative modification during the procedure. This method also allowed us to quantify relative 8-OHdG immunoreactivity per cell rather than total 8-OHdG of a cell population. We see a dose-dependent increase in 8-OHdG levels over control after 24 h of estrogen treatment in ER positive MCF7 breast cancer cells (Fig. 1A) with significance occurring at 10⫺9 M E2 (P ⫽ 0.01). Treatment of MCF7 cells with the antioxidant N-acetylcysteine (NAC) prevents E2-induced accumulation of 8-OHdG, confirming an oxygen radical mediated process (Fig. 1A). To determine whether E2-induced damage could be a result of ER-induced proliferation, we tested two other cells lines, MCF10A nontumorigenic breast epithelial cells (very low ER) (18) and MDA-MB-231 breast cancer epithelial cells (ER␣ negative, ER␤ positive) (19). Neither cell line proliferates in response to E2 (25, 26). However, E2 still induces 8-OHdG formation in these cells (Fig. 1B). The E2-induced damage is also time dependent, with maximum levels of 8-OHdG occurring by 24 h post treatment in MCF7 and MCF10 cells (Fig. 1C, top). There is generally a maximum 3- to 4-fold enhancement of 8-OHdG in MCF7 cells after 24 h and slightly less in MCF10A cells. Interestingly, MCF10A cells appear to accumulate oxidative damage much more slowly than MCF7 cells. The 8-OHdG in both cell lines drops back to basal levels by approximately 36 h post treatment. To determine whether the decrease in damage at 36 h was from degradation of the estrogen, we supplied MCF7 cells with fresh media and estrogen after 36 h. The damage was not restored after 24 h of fresh estrogen treatment. When cells were treated for 24 h and then removed from estrogen, the maximum levels of 8-OHdG slowly decreased back to basal levels after approximately 24 h in both cell lines (Fig. 1C; bottom). The 1F7 8-OHdG antibody recognizes both RNAderived 8-hydroxyguanine and DNA-derived 8-OHdG (27). To verify that we were examining DNA oxidation, we pretreated the cells with deoxyribonuclease (DNase) or ribonuclease (RNase). We found the majority of the E2-induced immunoreactivity in the nucleus, suggesting that DNA is the major target. Confirming this hypothesis, DNase treatment significantly depletes the nuclear staining, whereas RNase only slightly reduces the immunoreactivity (Fig. 1D). Although the increase in 8-OHdG is independent of ER-mediated proliferation, it is dependent upon estrogen metabolism to catecholestrogens. Treating the cells with the estrogen metabolism inhibitor ␣-naphthoflavone, which inhibits CYP1A and 1B, blocked the E2 effect (Fig. 2A). ␣-Naphthoflavone or its solvent dimethylsulfoxide (DMSO) had no effects on basal levels of 8-OHdG. The two hydroxy-catecholestrogen metabolites formed in breast cells by CYP1A1 and CYP1B1 are 2- and 4-hydroxyestradiol (2- and 4-OH-

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Fig. 1. Estrogen Induces Oxidative DNA Damage in Breast Epithelial Cells A, MCF7 breast cancer cells were grown on coverslips and treated with vehicle (control) or increasing doses of E2 for 24 h. The cells were then immunostained for 8-OHdG using the 1F7 monoclonal antibody (1:100; Trevigen). Shown are the relative levels of 8-OHdG after quantification. Values are the means ⫾ SE of three adjacent fields from three or more separate experiments. *, Level of significance P ⫽ 0.01 vs. control; **, level of significance P ⬍ 1 ⫻ 10⫺6 vs. control as determined by t test. As a control for oxidative damage, cells were treated with NAC (300 ␮M) ⫾ E2. B, Same experiments using MCF10A or MDA-MB-231 cells treated with E2 (10⫺8 M). **, P ⱕ 0.001; *, P ⱕ 0.005 vs. control as determined by t test. C, Top, Time course of E2-induced oxidative damage in MCF7 (䉬) and MCF10A (f) cells. Far right panel shows 36-h E2 treatment followed by 24-h treatment with fresh E2. Bottom, Withdrawal time course 24 h after E2 treatment. Cells were treated with 10⫺8 M E2 for 24 h, washed twice with Hanks’ Balanced Saline Solution, and replaced with hormone-free media. Cells were then fixed at indicated time points and immunostained. D, Images of 1F7-stained MCF7 cells before and after 24-h E2 (10⫺8 M) treatment. As controls, cells were also treated with DNase or RNase before adding the primary antibody.

E2), respectively (3). In an effort to determine the catecholestrogen responsible for this damage, we treated cells with increasing doses of either 2-OH-E2 or 4-OHE2. Only the 4-OH-E2 metabolite was capable of inducing 8-OHdG in our system (Fig. 2B).

Antiestrogens and QR Protect against EstrogenInduced Oxidative DNA Damage Our previous studies have shown that antiestrogenliganded ER can induce the detoxifying enzyme QR in

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Fig. 2. Estrogen-Induced Oxidative DNA Damage Is Dependent on Estrogen Metabolism A, MCF7 cells were grown on coverslips and treated for 24 h with 10⫺8 M E2 ⫾ ␣-naphthoflavone (␣-naph) (5 ␮M in DMSO) and stained for 8-OHdG. As controls, cells were treated with the vehicles ethanol (control) or DMSO. *, Level of significance P ⬍ 0.00001 vs. control as determined by t test. B, MCF7 cells were treated with vehicle (C) or increasing concentrations (10⫺11 to 10⫺7 M) of 2-OH-E2 or 4-OH-E2 and stained for 8-OHdG. Shown are the relative levels of 8-OHdG after quantification. Values are the means ⫾ SE of three adjacent fields from three or more separate experiments.

ER-containing cells (15, 16). To determine the functional significance of this activation, we monitored the effect of antiestrogens and QR on estrogen-induced oxidative damage, because QR may be able to detoxify the reactive catecholestrogen quinones (10). First, we sought to determine whether antiestrogens have a protective effect on estrogen-induced DNA damage. For this and subsequent experiments, we used the optimal concentration of E2 (10⫺8 M) required to induce oxidative DNA damage so that we may be able to accurately test the inhibition of this process. MCF7, MDA-MB-231, and MCF10A cells were treated with E2 alone or in combination with the antiestrogens transhydroxytamoxifen (TOT), RAL, and ICI-182,780. Antiestrogens show no protective effect on the levels of E2-induced 8-OHdG in MCF10A cells, which have very low levels of ER (Fig. 3B). In ER-positive MCF7 breast cancer cells, the antiestrogens protect against E2mediated damage by approximately 50% (Fig. 3A). However, only the protection by TOT and RAL was

Fig. 3. Antiestrogens Protect against Estrogen-Induced Oxidative DNA Damage in ER-Containing Cells MCF7 (A), MCF10A (B), or MDA-MB-231 (C) cells grown on coverslips were treated with vehicle (control) or E2 (10⫺8 M) ⫾ the antiestrogens (AE) TOT (10⫺7 M), ICI-182,780 (ICI; 10⫺7 M), or RAL (10⫺7 M) for 24 h and immunostained for 8-OHdG. The data are separated according to the AE used for that experiment. Shown are the relative levels of 8-OHdG after quantification. Values are the means ⫾ SE of three adjacent fields from three or more separate experiments per treatment group. *, Level of significance P ⬍ 0.05; **, P ⬍ 0.001 vs. E2 alone as determined by t test.

significant (P ⬍ 0.05). In MDA-MB-231 breast cancer epithelial cells (ER␣ negative, ER␤ positive), the antiestrogens still protect against oxidative damage, al-

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though only TOT treatment is considered significantly protective. To determine whether QR can protect against E2induced 8-OHdG, we transiently overexpressed or underexpressed QR in MCF7 cells using the selfcontained tetracycline-regulated pBPSTR1 vector (28) containing sense or antisense QR cDNA, respectively. In cells overexpressing QR (average increase, 40%), E2-induced 8-OHdG is blocked (Fig. 4A). Alternatively, cells with reduced QR (average decrease, 50%) contained significantly higher levels of 8-OHdG with all treatments except for TOT (P ⱕ 0.05) (Fig. 4B). This effect may be attributable to the ability of TOT to up-regulate QR or other detoxification genes also regulated by the EpRE, thus negating the antisense effect. Supporting this hypothesis, TOT does not compensate for the increase in 8-OHdG levels in MCF10A cells (very low ER) (18) underexpressing QR (average decrease, 40%) (Fig. 4C). Figure 5 shows the immunofluorescence confirming overexpression and underexpression of QR in infected cells. We also reconfirmed TOT-induced QR expression using the same antibody (Fig. 5). For this and subsequent experiments, we only used TOT, because this was the antiestrogen used in the initial studies regarding QR regulation. ER␤ Is Necessary for Antiestrogen Protection against Estrogen-Induced Oxidative DNA Damage As mentioned, antiestrogens primarily activate transcription of QR preferentially via ER␤ (16). However, expression of ER␤ in MCF7 and other breast epithelial cell lines has been rather controversial. To specifically determine the levels of ER␤ in our cell lines, we immunoblotted for ER␤ using the ER␤-7B10.7 monoclonal antibody. As expected, ER␤ is detected in both MCF7 and MDA-MB-231 cells, whereas MCF10A cells do not express the full-length form (Fig. 6A). The smaller band is similar in size to the ER-␤2(␤cx) splice variant, an inactive dominant negative repressor of ER␣ (29, 30), although specific antibodies are necessary for such determination. We also confirmed these results using a different ER␤ monoclonal antibody (14C8, Novus Biologicals, Littleton, CO) (data not shown). These results are slightly different from those reported by Fuqua et al. (17). They show a truncated ER␤ in MDA-MB-231 cells with their own monoclonal antibody. However, Girdler et al. (19) also observes full-length ER␤ in MDA-MB-231 cells using another noncommercially available monoclonal antibody. To evaluate the role of ER␤ in antiestrogen protection against E2-induced oxidative damage, we again used the self-contained tetracycline-regulated pBPSTR1 vector (28) to under- or overexpress ER␤ before E2 treatment in MCF7 cells. Figure 5 shows the reduced or increased expression of ER␤ with ER␤ antisense/sense retroviral infection. Lowering ER␤ in MCF7 cells (average decrease, 40%) raises the levels of E2-induced 8-OHdG, and tamoxifen is significantly less protective against the

Fig. 4. QR Protects against Estrogen-Induced Oxidative DNA Damage A, MCF7 cells were transiently infected with QR retroviruses (䡺) or control retroviruses (f). The cells were then treated with vehicle (Control), E2 (10⫺8 M), or the antiestrogen TOT (10⫺7 M) for 24 h and immunostained for 8-OHdG. *, Level of significance, P ⬍ 0.01 vs. vehicle-treated control cells as determined by t test. B, Same experiment, except that MCF7 cells were transiently infected with QRAS retroviruses (䡺) or control retroviruses (f). a, Level of significance P ⱕ 0.01 vs. respective control as determined by t test. b, Level of significance P ⱕ 0.05 vs. cells infected with control retroviruses with same treatment as determined by t test. C, MCF10A cells were transiently infected with QRAS retroviruses (䡺) or control retroviruses (f). *, Level of significance P ⬍ 0.01 vs. cells infected with control retroviruses with same treatment as determined by t test. Shown are the relative levels of 8-OHdG after quantification. Values are the means ⫾ SE of three adjacent fields from two or more separate experiments.

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damage (Fig. 6B). Interestingly, overexpressing ER␤ in MCF7 cells (average increase, 30%) (Fig. 5) prevents E2-induced DNA damage even without tamoxifen (Fig. 6B). The same result occurs in MCF10A cells expressing ER␤ (Fig. 6C). Estrogen Treatment Induces XPA in Breast Epithelial Cells To determine the possible downstream effects of estrogen-induced DNA damage, we measured levels of XPA, a member of the nucleotide excision repair complex, after E2 treatment. Although nucleotide excision repair is more commonly associated with bulky adduct repair, it has also recently been shown to repair certain oxidative lesions, including 8-OHdG (31–36). We were able to detect about a 2-fold significant increase in XPA with E2 (10⫺8 M) (P ⬍ 0.01) in MCF7 cells and slightly more in MCF10A cells (P ⬍ 0.05) (Fig. 7A). However, the two cell lines are not significantly different. Thus, the cells may be responding to the increase in DNA damage by activating certain repair enzymes. To determine whether induction of XPA follows 8-OHdG induction, we again performed a time-course study. XPA shows a slow induction with maximum levels occurring 24 h after E2 treatment in MCF7 cells (Fig. 7B) vs. 8-OHdG, which is significantly increased 4 h post treatment (Fig. 1C). Because reduction of ER␤ leads to increased production of oxidative DNA damage in MCF7 cells (Fig. 6), we tested what effect loss or gain of ER␤ would have on levels of XPA in the cell. Interestingly, in ER␤ antisense-expressing cells, we see a significant rise in XPA levels to those of the E2-treated cells (Fig. 7C). It should also be noted that although TOT is able to block the E2-induced XPA in control cells, TOT has no effect in ER␤ antisense cells treated with E2. Overexpression of ER␤ prevents the E2 increase in XPA. These are similar to the results obtained for 8-OHdG (Fig. 6). Taken together, this suggests that TOT requires ER␤ for protection against E2-mediated oxidative DNA damage and the resulting induction of XPA.

DISCUSSION Four important conclusions can be drawn from these studies: 1) physiological concentrations of E2 lead to oxidative damage in MCF7 cells, MDA-MB-231 cells,

Fig. 5. Retroviral Infected Breast Cells Top, MCF7 cells treated with vehicle (Control) or TOT (10⫺7 M) for 24 h and immunostained for QR (1:100). Middle, first

and second rows, QRs or QRAS retrovirus-infected MCF7 cells immunostained for QR (1:100). Third and fourth rows, ER␤AS retrovirus-infected MCF7 cells immunostained for ER␤ (1:100). Bottom, QRAS retrovirus-infected MCF10A cells immunostained for QR (1:100) and ER␤S retrovirus-infected MCF10A cells immunostained for ER␤ (1:100). Control retroviruses were obtained from cells transfected with the pBPSTR1 vector alone.

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Fig. 6. ER␤ Protects against Estrogen-Induced Oxidative DNA Damage A, Western blot analysis for ER␤ in MCF10A, MDA-MB231, and MCF7 cells. Recombinant ER␤ was loaded as a control. Ponceau S stain shows equal protein loading across lanes. B, MCF7 cells were transiently infected with ER␤S retroviruses (u), ER␤AS retroviruses (䡺), or control retroviruses (f). The cells were then treated with vehicle (Control), E2 (10⫺8 M), or the antiestrogen TOT (10⫺7 M) for 24 h and immunostained for 8-OHdG. Shown are the relative levels of 8-OHdG after quantification. a, Level of significance P ⬍ 0.05 vs. vehicle-treated cells; b, P ⬍ 0.05 vs. E2 alone as determined by t test. C, MCF10A cells were transiently infected with ER␤S retroviruses (䡺) or control retroviruses (f). The cells were then treated with vehicle (Control), E2 (10⫺8 M) ⫾ the antiestrogen TOT (10⫺7 M) for 24 h and immunostained for 8-OHdG. Shown are the relative levels of 8-OHdG after quantification. *, P ⬍ 0.05 vs. control E2 as determined by t test. Values are the means ⫾ SE of three adjacent fields from two or more separate experiments.

and MCF10A cells; 2) antiestrogens can protect against such damage; 3) QR and ER␤ also protect against such damage, and ER␤ plays an essential role in mediating antiestrogen protection; and 4) the E2-

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induced DNA damage results in the downstream induction of the DNA repair enzyme XPA. Our proposed model for how this occurs is shown in Fig. 8. It has been demonstrated extensively that estrogen exposure is linked to the overall risk of breast cancer in one’s lifetime. Most studies have focused on ER and the transcriptional regulation of mitogenic genes that could be oncogenic directly or indirectly through growth factors. Recently, there have been studies showing that estrogen itself may be a genotoxic mutagen capable of causing chromosomal mutations in animals and cell culture (3, 4, 37), leading to tumorigenesis in various animal models (1, 3). Estrogen also causes MCF10 breast epithelial cells to take on a transformed phenotype independently of the ER (4). Specifically, the catecholestrogens 2-OH-E2 and 4-OH-E2, which are formed in a cell-specific manner by cytochrome P-450-catalyzed hydroxylation, are considered to be mutagenic (21). This is perhaps through the oxidation of catecholestrogens to semiquinone radicals, and subsequently to quinones. Redox cycling between quinones and unstable semiquinones causes hydroxyl radical formation that can lead to hydroxylated nucleotide bases (e.g. 8-OHdG) and mutagenic DNA damage if not repaired (2, 3, 8). Also, the quinone-catecholestrogens can bind to DNA forming DNA adducts (37). Thus, initiation may be due not only to ER-mediated proliferation, but also to DNA damage caused by a combination of estrogen metabolism and preexisting lesions. Once initiated, the ER may then confer a selective advantage to these premalignant cells. QR may be linked to estrogenmediated DNA damage by reducing the reactive quinone-catecholestrogen to the hydroxy-catecholestrogen allowing for conjugation and excretion. Thus, we chose to monitor whether QR could protect against estrogen-mediated oxidative DNA damage. We used 8-OHdG as a marker for oxidative damage because it is one of the most common oxidized bases and has demonstrated mutagenic potential (22). 8-OHdG lesions result in mutational frequencies of 1–5% (mainly G:C to T:A transitions) (38). 8-OHdG may also be a prognostic marker in that both normal and malignant breast tissue from breast cancer patients was shown to have higher levels of 8-OHdG than control subjects (39, 40). Before we examined the role of QR, we first needed to show that physiological concentrations of E2 induce damage in breast cells. This was important because previous studies in breast cells have used higher concentrations of estrogen (4, 20, 21). Also, all of the animal models have been developed using pharmacological doses of E2 to obtain tumors in a short period of time (3). At physiological concentrations, we are able to detect a dose-dependent increase in 8-OHdG over control, providing additional validity to the animal models. For reference, plasma concentrations of E2 in the average female range between 10⫺10 and 10⫺9 M (41). However, it should be noted that local concentrations of E2 and its metabolites in the breast may be

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Fig. 8. Proposed Model for Antiestrogen Protection against E2-Induced Oxidative DNA Damage Estrogen may be metabolized in breast cells to catecholestrogens. These compounds may then be further oxidized to catecholestrogen-semiquinones (CE-SQ) and catecholestrogen-quinones (CE-Q). DNA damage may occur through reactive oxygen species generated from redox cycling between the CE-SQ and CE-Q or from direct reaction of the CE-Q with DNA forming DNA adducts. If not repaired, this DNA damage could lead to tumor initiation. QR protects against oxidative damage by directly reducing the CE-Q back to the hydroxy-catecholestrogen. Conjugation though methylation, glucuronidation, or sulfation then occurs, allowing for detoxification and excretion. Antiestrogens protect against E2-induced oxidative damage by binding to the ER␤ and mediating transcriptional activation of the QR promoter EpRE regulatory region.

Fig. 7. Estrogen Induces XPA in Breast Epithelial Cells A, MCF7 (f) or MCF10A (䡺) cells grown on coverslips were treated with vehicle (Control), E2 (10⫺8 M), or the antiestrogen TOT (10⫺7 M) for 24 h. The cells were then immunostained for XPA and quantified. Values are the means ⫾ SE of three adjacent fields from three or more separate experiments. *, Level of significance P ⬍ 0.05; **, level of significance P ⬍ 0.01 vs. respective vehicle-treated cells as determined by t test. B, Time-course analysis of XPA induction. C, MCF7 cells were transiently infected with ER␤S retroviruses (u), ER␤AS retroviruses (䡺), or control retroviruses (f). The cells were then treated with vehicle (Control), E2 (10⫺8 M), and/or the antiestrogen TOT (10⫺7 M) for 24 h and immunostained for 8-OHdG. Shown are the relative levels of 8-OHdG after quantification. Values are the means ⫾ SE of three adjacent fields from two or more separate experiments. a, Level of significance P ⬍ 0.05 vs. respective vehicle-treated cells; b, P ⱕ 0.05 vs. E2 alone; c, level of significance P ⬍ 0.05 vs. cells infected with control retroviruses with same treatment as determined by t test.

significantly higher due to production by aromatase and metabolism by the CYP1A/1B enzymes (3). To verify an estrogen metabolism-mediated effect, we treated MCF7 cells with ␣-naphthoflavone, an inhibitor of CYP1A and 1B that prevents catecholestrogen formation. As expected, ␣-naphthoflavone reverses the E2-induced damage. This is consistent with the hamster model, in which ␣-naphthoflavone completely suppresses E2-induced kidney carcinogenesis (42). Furthermore, our studies show that 4-OH-E2 is the only major catecholestrogen capable of producing the oxidative damage, because 2-OH-E2 has no effect in our system. These data support other in vitro and in vivo studies showing 4-OH-E2 to be the more mutagenic and carcinogenic metabolite (1, 3, 37, 43). This may be due to an increased ability of the 4-OH-E2 metabolite to consume oxygen and generate DNA strand breaks in the presence of Cu(II) (43) or the decreased levels of inactivating conjugation compared with 2-OH-E2 (6). Unfortunately, we do not observe as much damage from 4-OH-E2 as theoretically expected on the basis of rates of estrogen metabolism in MCF7 cells. Thus, we cannot make a direct comparison between E2- and 4-OH-E2-induced damage. Several possible reasons for this discrepancy exist, including autooxidation of applied 4-OH-E2 in the media before it reaches the cell. Moreover, because of redox cycling of the catecholestrogens, there may not

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be a 1:1 correspondence between 8-OHdG formed. Although we cannot rule out an ER-mediated effect in E2-induced damage, the increase in 8-OHdG was not dependent on ER-mediated proliferation because both MCF10A and MDA-MB-231 cells also incur damage, neither of which proliferate in response to E2 (25, 26). Additionally, this oxidative damage occurs in nontumorigenic MCF10A cells, suggesting that damage is not specific to cancer cells. Interestingly, we found that 8-OHdG accumulates much slower after E2 treatment in MCF710A cells compared with MCF7 cells. We hypothesize that this may be due to decreased E2 metabolism in MCF10A cells. Not only is the rate of E2 metabolism much slower in MCF10A cells, but they also contain lower amounts of CYP1B1 mRNA (7). In 1998, the antiestrogen tamoxifen became the first drug to be approved for the reduction of risk for breast cancer, showing about a 50% reduction in both noninvasive and invasive breast cancer (12). Tamoxifen has traditionally been thought to protect against breast cancer by blocking ER-mediated transcription. However, it is also possible that tamoxifen protects cells against tumor-promoting events by inducing detoxification enzymes such as QR (15, 16). We did see inhibition of E2-induced 8-OHdG with pharmacological concentrations of TOT, RAL, and ICI-182,780 but only in cells containing ER, confirming a probable ERmediated effect. However, we do not know whether the protective effect of tamoxifen is through QR alone or other phase II detoxification genes that may also be regulated by ER at the EpRE. This is currently being pursued in our laboratory. Although tamoxifen does not induce damage in ERcontaining MCF7 cells, there was a modest but significant induction of 8-OHdG in MCF10A cells. This is a controversial topic because one study reported 8-OHdG formation by tamoxifen derivatives, although this was an in vitro reaction (44). However, another study has shown that tamoxifen can reduce oxidative damage to DNA in vitro and in mouse epidermis (45). In another similar study, estrogen was found to elevate superoxide anion and hydride radicals in mice uteri, whereas tamoxifen and ICI-182,780 decreased radical formation (46). Studies wherein preisolated DNA is analyzed using in vitro methods should be interpreted with caution. Cell compartmentation and environment (e.g. metals) are critical to oxidative damage effects. It is also possible that tamoxifen may cause oxidative damage in ER-negative cells, while being protective in ER-positive cells through induction of detoxification enzymes such as QR. As expected from previous reporter assays showing ER␤ to be the active isoform on the EpRE (16), ER␤ is sufficient for conferring antiestrogen protection in MDA-MB-231 cells that contain only ER␤. Further confirming the protective effects of ER␤, antisense inhibition of ER␤ leads to elevated levels of 8-OHdG in MCF7 cells, and tamoxifen was significantly less protective against E2-induced 8-OHdG. Unexpectedly, by overexpressing ER␤ in MCF10A or MCF7 cells, we

Bianco et al. • QR Inhibits E2-Induced DNA Damage

saw that E2-induced damage is prevented. This may be due to low levels of ligand-independent activation of the overexpressed ER␤ at the EpRE, which we also observe in our transfection reporter assays (data not shown). It is also possible that ER␤ has an additional innate protective role independent of ligand activation. To more directly assess the role of QR in E2-induced oxidative stress, we modulated the levels of QR activity in MCF7 cells and then measured the levels of 8-OHdG. Simply overexpressing QR inhibits E2induced 8-OHdG. In contrast, reduction of QR activity by antisense inhibition leads to an increase in control and E2-induced 8-OHdG levels. However, 8-OHdG is not elevated in TOT-treated QR antisense (QRAS) cells, perhaps because of compensation by TOT-induced QR gene transcription. Further support for this hypothesis comes from the fact that TOT-treated QRAS cells are susceptible to damage in MCF10A cells that have very low ER and would not be able to compensate. Although the QR inhibitor dicoumarol increases basal levels of 8-OHdG significantly, it unexpectedly has no effect with E2 or TOT (data not shown). We hypothesize that this is due to additional nonspecific effects of dicoumarol in our system, because dicoumarol inhibits a number of enzymes (47). This may also explain why 2(3)-t-butyl-4-hydroxyanisol and dicoumarol, which stimulate or inhibit QR, respectively, both lowered tumor incidence by estrogen in the Syrian hamster model (48). Finally, we measured levels of the DNA repair enzyme XPA after E2 and found an increase subsequent to the increase in 8-OHdG, suggesting that the cells are responding by increased repair. Further support for increased repair comes from the rapid decrease in 8-OHdG levels between 24 and 36 h post treatment, which occurs promptly after XPA has reached full induction at 24 h. This repair readiness may also explain why 8-OHdG does not reaccumulate when fresh estrogen is added after 36 h. We also found a significant rise in XPA in cells depleted of ER␤, again suggesting that ER␤ plays a protective role against DNA damage and the resulting induction of repair enzymes. This is important because the role of ER␤ in breast cancer is not well established, although it may play a protective role against breast cancer (49, 50). The fact that there is no further increase in XPA with E2 in ER␤AS cells relative to untreated cells suggests to us that there is already a maximal induction of XPA in these cells. Although the induction of XPA is rather modest, the induction is similar to what is observed after 20 h treatment with cisplatin in an ovarian carcinoma cell line (51). XPA is involved in nucleotide excision repair of bulky nucleotide lesions and perhaps oxidative DNA damage such as 8-OHdG (31–36). Thus, XPA may be involved in the repair of both oxidative damage and/or DNA adducts caused by estrogen. Although tamoxifen significantly protects against breast cancer, there are also several drawbacks to tamoxifen prophylactic use. These side effects have included strokes, pulmonary embolisms, and an in-

Bianco et al. • QR Inhibits E2-Induced DNA Damage

creased risk of endometrial cancer (12, 52). RAL also appears to protect against breast cancer and estrogen-induced DNA damage, and it may have fewer side effects, although thromboembolic disease is still a problem (14). We believe the research presented here supports two additional prophylactic strategies. First, drugs aimed at reducing extrahepatic catecholestrogen formation (specifically 4-OH-E2) may be effective in blocking the harmful by-products of catecholestrogen metabolism, while still maintaining the beneficial effects of estrogen. An additional therapy that is currently being pursued is the induction of QR and other phase II detoxification enzymes. Oltipraz is one such drug that is currently in clinical trials (53). We propose that this therapy would be protective against estrogeninduced DNA damage in the breast and possibly other estrogen target tissues that form catecholestrogens, as well as certain chemically induced cancers.

MATERIALS AND METHODS Chemicals and Materials Cell culture media was purchased from Life Technologies, Inc. (Grand Island, NY). Calf serum was from HyClone Laboratories, Inc. (Logan, UT), and fetal calf serum from Atlanta Biologicals (Norcross, GA). 17␤-Estradiol, 2-OH-E2, 4-OHE2, dicoumarol, ␣-naphthoflavone, NAC, and the antiestrogens TOT and RAL were obtained from Sigma (St. Louis, MO). The antiestrogen ICI-182,780 was obtained from Tocris (Ballwin, MO). Plasmid Construction To make pBPSTR1-QR (sense or antisense), QR cDNA was released from pMT2-QR (54) by NcoI/AflIII digestion, blunted, and inserted into BamHI-digested and blunted pBPSTR1 vector (28). To make pBPSTR1-ER␤ (sense or antisense), ER␤ cDNA was released from Flag-ER␤⫺BSII-SK⫹ (16) by NcoI/HindIII digestion, blunted, and inserted into BamHIdigested and blunted pBPSTR1 vector. Tissue Culture and Retroviral-Mediated Transfection Breast epithelial cells (MCF7, MDA-MB-231, and MCF10A) and PA317 amphotropic packaging cells were obtained from American Type Culture Collection (Manassas, VA) and maintained according to their recommended protocols. Before the experiments, breast epithelial cells were depleted of estrogen by growth in improved MEM minus phenol red containing 5% charcoal dextran-treated calf serum for 5 d before experiments. Retroviruses were made by transfecting PA317 cells with the pBPSTR1 plasmid alone or pBPSTR1 containing fulllength QR or ER␤ in the sense or antisense orientation. Breast epithelial cell lines were infected with retroviruscontaining supernatants in the presence or absence of 3 ␮g/ml tetracycline. The self-contained, tetracycline-regulated retroviral vector pBPSTR1 contains both the response unit, composed of tetracycline resistance operon regulatory elements (tetO) within a minimal cytomegalovirus promoter, and the regulator unit, encoding the tTA protein (the tetracycline repressor fused to the transactivator protein VP16) (28). Gene expression is inhibited by tetracycline, which binds the transactivator protein tTA, causing it to dissociate from the tetO

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minimal cytomegalovirus promoter. Changes in protein expression were verified by immunofluorescence staining. Changes in protein levels of QR and ER␤ were also verified in MCF7 cells by Western blot analysis (data not shown). Immunofluorescence Staining of Breast Cells Cells grown on coverslips were fixed in 4% paraformaldehyde. After blocking with 5% normal goat serum, samples were incubated with polyclonal rabbit QR antibody [1:100 dilution (55)], 14C8 monoclonal ER␤ antibody (1:100 dilution; Novus Biologicals), or polyclonal ER␤ antibody (kindly provided by the laboratory of Benita S. Katzenellenbogen, University of Illinois, Champaign-Urbana, IL) and goat, antirabbit IgG Alexa 488 or antimouse IgG Alexa 594 fluorescent secondary antibody (Molecular Probes, Inc., Eugene, OR). As a negative control, cells were immunostained with nonspecific rabbit IgG or with secondary antibody alone. Semiquantitative analysis was performed on a Macintosh computer using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). Mean fluorescence intensity of 15–20 cells from each experiment was measured as previously referenced by our laboratory and others (by using the luminosity channel on the histogram function) and averaged with background subtracted out from each field (56, 57). The analysis was performed a second time using the Zeiss KS300 Imaging System (Carl Zeiss Inc., Thornwood, NY) quantitation program with similar results. Western Blot Analyses Whole-cell extracts were prepared from breast epithelial cells using mammalian protein extraction reagent (Pierce Chemical, Rockford, IL). Fifty micrograms of protein extract were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred electrophoretically onto nitrocellulose membranes. Recombinant human ER␤ (PanVera Corp., Madison, WI) was included as a positive control. Blots were incubated with anti-ER␤ (long form) monoclonal antibody 7B10.7 (1:200 dilution; GeneTex, San Antonio, TX) and goat antimouse IgG secondary antibody (1:3000 dilution) for detection by chemiluminescence (ECL, Amersham Biosciences, Inc., Piscataway, NJ). Immunocytochemistry for 8-OHdG/XPA in Breast Cells Cells grown on coverslips were fixed in methacarn (methanolchloroform-acetic acid, 6:3:1) for 1 h at room temperature. Endogenous peroxidase activity in the cells was eliminated by a 30-min incubation with 3% H2O2 in methanol, and nonspecific binding sites were blocked in a 15-min incubation with 10% normal goat serum in Tris-buffered saline [150 mM Tris-HCl and 150 mM NaCl (pH 7.6)]. The cells were then pretreated with proteinase-K [20 ␮g/ml in PBS (pH 7.4)] for 15 min at room temperature (Sigma, St. Louis, MO). To detect oxidized nucleosides, we used the anti-8-oxo-dG monoclonal antibody 1F7 (1:100; Trevigen, Gaithersburg, MD). As a negative control, cells were incubated without the primary antibody. Immunostaining was developed by the peroxidaseantiperoxidase procedure. The 8-OHdG antibody also recognizes 8-hydroxyguanine (27), so to confirm that the oxidative damage was mainly to DNA, some slides were preincubated after proteinase-K treatment with DNase I (10 U/␮l in PBS for 1 h at 37 C) (Roche Diagnostics, Indianapolis, IN) or RNase (5 ␮g/ml in PBS for 1 h at 37 C) (Promega Corp., Madison, WI). For XPA staining, the XPA Ab-1 monoclonal antibody (1:50; Neomarkers, Fremont, CA) was used after the same protocol as used for 8-OHdG.

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Relative Quantification of 8-OHdG/XPA Immunoreactivity was evaluated by measuring OD as described (23, 24). The OD was assessed using a Zeiss Axiocam digital camera (Carl Zeiss, Inc.) with a KS300 Imaging System quantitation program. The OD of manually outlined cells was measured. Five cells in three adjacent fields were measured, and the background OD was subtracted from each. Each experiment was performed two or more times, and results were measured under the same optical and light conditions. Also, an electronic shading correction was used to compensate for any unevenness that might be present in the illumination. Statistical analysis was performed using the Student’s t test.

Acknowledgments We thank Zvezdana Kubat, Sandra Siedlak, and Xiaoyan Sun for technical assistance. We also thank Dr. Steven A. Reeves (Massachusetts General Hospital) for the pBPSTR1 retroviral vector and Dr. Anil Jaiswal (Baylor College of Medicine) for the pMT2-QR vector.

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Received November 18, 2002. Accepted April 7, 2003. Address all correspondence and requests for reprints to: Dr. Monica M. Montano, Ph.D., Case Western Reserve University School of Medicine, Department of Pharmacology, H. G. Wood Building W307, 2109 Adelbert Road, Cleveland, Ohio 44106. E-mail: [email protected]. This work was supported by NIH Grant CA80959 (to M.M.M.) and Institutional National Research Service Award Predoctoral Fellowships T32 GM08056 and 5T32 CA59366-10 (to N.R.B.).

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