Estrogen and Progesterone Regulate Radiation-Induced P53 Activity in Mammary Epithelium Through TGF-B-Dependent Pathways

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Estrogen and progesterone regulate radiation-induced p53 activity in mammary epithelium through TGF-b-dependent pathways

Klaus A Becker1,2, Shaolei Lu1,2, Ellen S Dickinson2, Karen A Dunphy1,2, Lesley Mathews1,3, Sallie Smith Schneider1,3 and D Joseph Jerry*,1,2

1Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003, USA; 2Paige Laboratory, Department of Veterinary and Animal Sciences, University of Massachusetts, 161 Holdsworth Way, Amherst, MA 01003, USA; 3Baystate Medical Center/UMass Biomedical Research Institute, Springﬁeld, MA 01099, USA

DNA damage normally induces p53 activity, but responses hormone human chorionic gonadotropin enhance radia- to ionizing radiation in the mammary epithelium vary tion-induced apoptosis in mouse mammary epithelium among developmental stages. The following studies through activation of p53 (Kuperwasser et al., 2000; examined the hormones and growth factors that regulate Sivaraman et al., 2001; Minter et al., 2002). As these radiation-responsiveness of p53 in mouse mammary hormones have also been shown to prevent carcinogen- epithelium. Immunoreactive p21/WAF1 and TUNEL induced mammary tumors (Russo et al., 1990; Sivaraman staining were used as indicators of p53 activity following et al., 1998), it was proposed that regulation of p53 exposure to ionizing radiation. In ovariectomized mice, activity may mediate the protective effects of these radiation-induced accumulation of p21/WAF1 was mini- hormones in the mammary epithelium. The absence of mal in the mammary epithelial cells (o1%). Systemic the parity-induced protection from tumors in p53- injections of estrogen and progesterone (E þ P) for 72 h deficient mammary tissues (Medina et al., 2003) provided were necessary to recover maximal expression of p21/ direct evidence that the p53 pathway mediates the WAF1 following ionizing radiation (55%). The effects of chemopreventive effects of these hormones. Consistent E þ P on radiation-induced p21/WAF1 were p53-depen- with the concept that p53 activity can modify risk, the dent as responses were absent in Trp53À/À mice. Though p53 pathway was found to be refractory to radiation- hormonal treatments stimulated increases in the propor- induced DNA apoptosis in mammary epithelium of tion of cycling cells (PCNA-positive), this was not directly nulliparous mice (Kuperwasser et al., 2000; Minter et al., correlated with p53 activity. Whole organ cultures were 2002). However, responses to radiation in mammary used to determine whether E þ P act directly upon the epithelium of nulliparous mice were further impaired in mammary gland. Treatment with E þ P was sufficient to TGF-b1-deficient mice (Ewan et al., 2002a), demonstrat- render p53 responsive to radiation, but TGF-b-neutraliz- ing a role for the TGF-b pathway in regulating p53. Like ing antibodies blocked responsiveness. In the absence of p53, expression of TGF-b in the mammary gland was E þ P, TGF-b1 alone did not alter p53 activity. These responsive to changes in hormonal stimulation (Ewan results demonstrate that estrogen and progesterone et al., 2002b). As both p53 and transforming growth together with TGF-b signaling are necessary for main- factor b (TGF-b) can influence incidence of mammary tenance of p53 activity in the mammary epithelium. tumors (Pierce Jr et al., 1995; Jerry et al., 2000), these Oncogene (2005) 24, 6345–6353. doi:10.1038/sj.onc.1208787; tumor suppressor pathways represent important regula- published online 6 June 2005 tors of both susceptibility and resistance to mammary tumors. Therefore, experiments were undertaken to Keywords: p53; estrogen; progesterone; TGF-b; chemo- determine the hormonal and growth factor requirements prevention; mammary for activation of p53 in mouse mammary epithelium. The results demonstrate that the effects of estrogen and progesterone on p53 activity are inhibited by receptor antagonists and that TGF-b is required for optimal p53 Introduction activity. The inability of TGF-b to activate p53 in the absence of E þ P supports a cooperative mechanism One full-term pregnancy reduces the risk of breast cancer involving crosstalk between these signaling pathways. by up to 50% (Rosner et al., 1994). The prophylactic effect of pregnancy can be mimicked by treatment with estrogen and progesterone in rodents (Sivaraman et al., Results 1998). These ovarian steroids as well as the placental Hormonal effects on p53 activity in vivo *Correspondence: DJ Jerry; E-mail: [email protected] Received 30 December 2004; revised 15 April 2005; accepted 17 April The p53 tumor suppressor gene participates in cellular 2005; published online 6 June 2005 responses to a variety of cellular stresses and types of Hormonal regulation of p53 activity KA Becker et al 6346 DNA damage, but is essential for directing cell cycle (51%) (Figure 1a; solid bars). Effects of E þ Pon arrest and apoptosis in response to DNA double-strand radiation-induced expression of p21/WAF1 were max- breaks (Kastan et al., 1992). Therefore, ionizing radia- imal at 72 h and were maintained through 96 h (55%). tion was used to induce DNA double-strand breaks to Immunoreactive p21/WAF1 was detected in nuclei of examine the temporal effects of estrogen and progester- mammary epithelial cells throughout the ducts one on p53-dependent responses to DNA damage in (Figure 1b). This was in contrast to vehicle-treated mice ovariectomized mice. The cyclin-dependent kinase in- where radiation-induced expression of p21/WAF1 was hibitor p21/WAF1 is among the transcriptional targets observed in less than 1% of mammary epithelial cells of p53 and provides a direct indicator of p53 activity in (Figure 1c). Radiation-induced p21/WAF1 was also the mammary epithelium (Jerry et al., 1998). In undetectable in BALB/c-Trp53À/À mice treated with unirradiated, ovariectomized controls, immunoreactive E þ P (Figure 1d), demonstrating that the effects of p21/WAF1 was o1% in all treatments providing a the hormones on p21/WAF1 expression were p53- stable baseline to examine changes in p53-dependent dependent. Nuclear accumulation of p53 accompanied responses to DNA damage. Radiation-induced expres- radiation-induced expression of p21/WAF1 in mice sion of p21/WAF1 was increased progressively by treated with E þ P (Figure 1e), but was absent in mice treatment with E þ P for 24 h (3%), 48 h (26%) or 72 h treated with Vehicle (Figure 1f).

Figure 1 Hormonal regulation of radiation-induced expression of p21/WAF1 and PCNA in ovariectomized mice. (a) Ovariectomized BALB/c-Trp53 þ / þ females received daily injections of E þ P(2mg and 1 mg, respectively) for 24, 48, 72 or 96 h. Error bars represent s.e.m. Immunohistochemical detection of p21/WAF1 in the mammary epithelium of mice irradiated after 72 h treatment with E þ P(b), vehicle alone (c), or BALB/c-Trp53À/À mice treated with E þ P(d). Immunohistochemical detection of p53 in mammary gland epithelium of BALB/c-Trp53 þ / þ mice irradiated after 96 h treatment with E þ P(e) or vehicle alone (f). Immunohistochemical detection of PCNA in the mammary epithelium of BALB/c-Trp53 þ / þ mice irradiated after 96 h treatment with E þ P(g). Micrograph images represent Â 200 magniﬁcation

Oncogene Hormonal regulation of p53 activity KA Becker et al 6347 As ovarian steroids also regulate growth of the mammary gland, proliferative responses were measured. Proliferating cell nuclear antigen (PCNA) was used as an indicator of cycling cells (Figure 1a, open bars). Basal levels of PCNA-positive mammary epithelial cells were o5% in ovariectomized mice treated with Vehicle alone. The proportion of PCNA-positive cells was increased by treatment with E þ P and followed the same temporal changes observed for the p21/WAF1 responses. Neither radiation treatment nor p53 genotype affected the number of nuclei staining positive for PCNA (data not shown). As with p21/WAF1, staining for PCNA was detected throughout the ducts (Figure 1g). These results confirm that the hormonal treatments recruited cells into the cell cycle. To determine the minimal hormonal treatment required to activate p53, ovariectomized mice were treated with either estrogen or progesterone alone, or in combination for 72 h. The 72 h time point was selected as radiation-induced responses were maximal at this time. Radiation-induced expression of p21/WAF1 was detected in 50% of the ductal epithelial cells in mice receiving both E þ P, but was o1% in mice receiving the vehicle (sesame oil). Treatment with E þ P was more effective in activating p53 than either estrogen or progesterone alone (Figure 2a; Po0.01) as responsiveness to radiation was reduced in animals receiving estrogen alone (28%) or progesterone alone (9%). The p53-dependence of the responses was confirmed by the absence of p21/WAF expression in BALB/c-Trp53À/À mice treated with E þ P and radiation (as shown in Figure 2 Hormonal requirements for p53-dependent responses to Figure 1d). radiation. (a) Ovariectomized BALB/c-Trp53 þ / þ females received As treatment with E þ P resulted in similar propor- daily injections of either 2 mg estrogen alone (E), 1 mg progesterone tions of cells that were PCNA-positive (59%) and alone (P), both in combination (E þ P), or vehicle alone for 72 h. (b) radiation-responsive (50% p21/WAF1-positive) in Fig- Ovariectomized BALB/c-Trp53 þ / þ females were treated for 96 h ure 1, a relationship between cycling cells and radiation- with E þ P(2mg and 1 mg, respectively) either alone or in combination with 200 mg Mifepristone (M) or 100 mg ICI 182,780 induced p53 activity was suggested. However, the (I). Error bars represent s.e.m. individual hormone treatments revealed a dissociation between these parameters. While the proportion of PCNA-positive cells was elevated to a similar extent by treatment was reduced to 10% when mice received ICI estrogen alone or progesterone alone (47 and 38%, 182,780 together with E þ P (Figure 2b, E þ P þ I). These respectively), estrogen alone resulted in a significantly levels of p21/WAF1 were comparable to the effect of larger proportion of cells positive for p21/WAF1 progesterone alone (Figure 2a), indicating that the compared to progesterone alone (Figure 2a; 28 and estrogen receptor was necessary. The apparent lack of 9%, respectively). Therefore, recruitment of cells into the inhibition by Mifepristone alone (E þ P þ M) appears to cell cycle by E þ P is not sufficient to activate p53- contrast with the striking inhibition when both Mife- dependent responses to ionizing radiation. Estrogen and pristone and ICI 182,780 were used in combination. As progesterone generally act as signaling molecules; how- treatment with progesterone alone had only a modest ever, acute exposure to estrogen can cause formation of effect on radiation responsiveness (Figure 2a), blocking adducts in DNA, which may initiate p53 responses. To progesterone action with Mifepristone may result in a address this possibility, hormone receptor antagonists negligible effect. However, Mifepristone can also have were used to determine whether the effects of estrogen stimulatory effects on the estrogen receptor (Jeng et al., and progesterone on p53 occur through their receptors. 1993; Dibbs et al., 1995), which would serve to enhance ICI 182,780 (I) and Mifepristone (M) were used to the effect of estrogen in the E þ P þ M treatment. When inhibit the estrogen and progesterone receptors, respec- combined, Mifepristone blocks the activity of progester- tively. These receptor antagonists in combination one while ICI 182,780 blocks estrogen action as well as blocked activation of p53-dependent responses to DNA the estrogenic effect of Mifepristone resulting in the damage by E þ P (Figure 2b, E þ P þ I þ M). Treatment complete inhibition observed with E þ P þ I þ M. More- with Mifepristone did not reduce p21/WAF1 responses over, the inhibitory effects of receptor antagonists rule to ionizing radiation (Figure 2b; E þ P þ M). In contrast, out nonspecific effects of E þ P through direct DNA cells staining positive for p21/WAF1 after radiation damage.

Oncogene Hormonal regulation of p53 activity KA Becker et al 6348 The hormone receptor antagonists ICI 182,780 and ing irradiation (Figure 4a). Direct analysis of P-Smad2 Mifepristone also altered proliferative responses of expression in the epithelium by immunohistochemical estrogen and progesterone. Treatment with E þ P staining showed similar levels and patterns of expression stimulated active cycling in 60% of mammary epithelial in both vehicle-treated and E þ P-treated mice cells, and this effect was abolished by addition of both (Figure 4b). Importantly, P-Smad2 was expressed ICI 182,780 and Mifepristone (Figure 2b, primarily within the mammary epithelial cells suggesting E þ P þ I þ M). Although Mifepristone had no discern- that TGF-b acts directly on the mammary epithelium able affect on the number of cycling cells induced by and that basal levels of P-Smad2 are not limiting p53 E þ P, selective inhibition of estrogen receptor with ICI activity in mammary tissues from ovariectomized mice. 182,780 in combination with E þ P reduced the number of cells staining positive for PCNA (Figure 2b, E þ P þ I). This suggests that the estrogen receptor is Effects of hormones on whole organ cultures (WOC) required for the effects of E þ P as the effect of E þ P þ I Systemic hormone treatments can act indirectly by mirrored the response to progesterone alone (Figure 2a). inﬂuencing endocrine secretions from a variety of tissues These results suggest that E þ P enhance p53 activity such as the pituitary. Therefore, WOC were used to and proliferation via receptor-mediated pathways. determine if individual growth factors act directly on the Levels of p53 mRNA were examined to determine mammary gland to alter p53 activity. This cell culture whether the hormone treatments may alter transcription method maintains expression of both the estrogen and of the p53 gene. There were no differences in steady- progesterone receptors and preserves the ductal archi- state levels of p53 mRNA (P>0.05) among unirradiated tecture that is necessary to recapitulate hormonal tissues (Figure 3). As observed previously (Minter et al., regulation of p53 observed in vivo (Minter et al., 2002), p53 protein was undetectable in unirradiated 2002). As growth and development of the mammary tissues. These results suggest that estrogen and proges- gland is regulated by combined effects of mammogenic terone initiate a cascade of events that develop over a and lactogenic hormones and growth factors, these 72 h period, which serve to enhance the ability of p53 to factors were tested for their ability to enhance radiation detect and respond to DNA damage induced by ionizing responsiveness of p53 in the presence of E þ P. TUNEL radiation, but do not increase steady-state levels of p53 mRNA. As TGF-b1 is required for radiation-responsiveness in the mammary epithelium (Ewan et al., 2002a), the effects of E þ P may be due to alterations in TGF-b signaling. Levels of phosphorylated Smad2 (P-Smad2) provide an indicator of signaling through the TGF-b receptor. Smad2 was selected because it can regulate the activity of p53 (Cordenonsi et al., 2003). Western blot analysis showed detectable levels of P-Smad2 in ovariectomized mice and levels were unchanged follow-

Figure 4 Effects of hormonal stimulation of P-Smad2. Ovariecto- mized mice were treated with estrogen and progesterone as described in Figure 1. (a) The lymph nodes were removed from whole mammary glands, then homogenized and analysed for P- Smad2 by Western blot. Actin was used as an internal control to normalize for variations in loading. (b) Immunohistochemical Figure 3 Effects of hormonal stimulation on basal levels of p53. staining for P-Smad2 was performed on sections of formaldehyde- Ovariectomized mice were treated with estrogen and/or progester- fixed, paraffin-embedded mammary tissues. Immunocomplexes one as described in Figure 1. Total RNA was extracted from were detected with polymer-linked horseradish peroxidase visua- unirradiated tissues and analysed by Northern blot hybridization lized with DAB (brown staining) and counterstained with Mayer’s and quantified the digital light units (dlu) using a phosphorimager. hematoxylin. Positive-staining nuclei were prevalent in the luminal Mean signal intensities for three replicate samples are reported. epithelial cells. Occasional staining was also detected in the stromal Error bars represent s.e.m. cells

Oncogene Hormonal regulation of p53 activity KA Becker et al 6349 was used to detect apoptosis because growth regulators demonstrate that the enhancement of p53 activity in the such as TGF-b can induce p21/WAF1 directly. In basal mammary epithelium is not a generalized effect of media (Control), irradiation resulted in minimal apop- growth factor stimulation, but is speciﬁc for E þ P. tosis (1%; Figure 5a, c). The addition of estrogen and While basal levels of signaling through the TGF-b progesterone increased TUNEL-positive nuclei of mam- receptors (as determined by P-Smad2; Figure 4) were mary epithelial cells to 6% (Control þ E þ P; Figure 5a, unaltered by treatment with E þ P in vivo, it remained d). Neither epidermal growth factor (EGF), prolactin possible that TGF-b may participate in the effects of (PRL), nor hydrocortisone (HC) provided any enhance- E þ P on p53 activity. To examine this possibility, WOC ment of p53 activity compared to E þ P (Figure 5a). Tri- were treated with pan-speciﬁc anti-TGF-b antibodies to iodothyronine (T3) induced a modest increase (Po0.01) deplete endogenous TGF-b. Antibodies against TGF-b in the proportion of TUNEL-positive cells, possibly blocked the effects of E þ P(Po0.01) on radiation- through metabolic effects within the gland. These data induced apoptosis (Figure 5b, e; E þ P þ anti-TGFb),

Figure 5 Growth factor requirements for radiation-induced apoptosis in whole organ cultures. (a) Whole organ cultures were maintained in either basal media alone (Control) or supplemented with estrogen and progesterone (E þ P). Additional factors were added to the E þ P-containing media to examine possible additive effects. All cultures were maintained for 96 h. Apoptotic responses to ionizing radiation were determined using the TUNEL method. Concentrations of hormones were 1 ng/ml 17b-estradiol, 1 mg/ml progesterone, 5 ng/ml EGF, 100 ng/ml ovine PRL, 2 mg/ml hydrocortisone, or 8 mg/ml tri-iodothyronine. (b) Whole organ cultures were incubated with either basal media alone (Control), basal media containing estrogen and progesterone (Control þ E þ P), function- blocking antibodies against TGF-b (E þ P þ anti-TGFb), anti-IgG antibodies as a nonspeciﬁc control (E þ P þ anti-IgG). Additional tissues were incubated with basal media with E þ P supplemented with TGF-b1 or an equal volume of diluent (E þ P þ 4mM HCl ¼ 10 mlof4mM HCl, 1 mg/ml added to 3 ml media). The effect of TGF-b1 without E þ P was also tested (TGFb alone). Tissues were maintained for 96 h. Apoptotic responses to ionizing radiation were determined using the TUNEL method. Concentrations of hormones were 1 ng/ml 17b-estradiol, 1 mg/ml progesterone, 25 mg/ml anti-TGF-b,25mg/ml anti-IgG, 10 ng/ml TGF-b1. Error bars represent s.e.m. Representative images of TUNEL results are shown for Control media (c), E þ P(d), E þ P þ anti-TGF-b (e), E þ P þ TGF-b1(f), and h TGFb1(g). Micrograph images represent Â 200 magniﬁcation

Oncogene Hormonal regulation of p53 activity KA Becker et al 6350 demonstrating that TGF-b is necessary for activation of positive and p21/WAF1-positive cells following irradia- p53 by E þ P. This was specific for TGF-b because tion (Figure 1) were consistent with expectations. isotype control antibodies against IgG had no effect However, this relationship between cell cycle and (Figure 5b; E þ P þ anti-IgG). Conversely, addition of radiation-responsiveness appears to be overly simplistic exogenous TGF-b1 enhanced radiation-induced apop- as mitogenic growth factors (EGF, prolactin) failed to tosis (Figure 5b, f; TGFb1). As TGF-b1 requires enhance p53 activity (Figure 5a). Therefore, activation reconstitution in acidified buffer (4 mM HCl; 1 mg/ml of p53 appears to be a specific response to E þ P rather BSA), separate cultures were maintained in equal than a general effect of proliferation. Furthermore, the concentrations of the buffer alone. The buffer control relationship between the fraction of cycling cells and the showed no enhancement of apoptosis compared to the fraction of cells that exhibit p53-dependent responses to treatment with E þ P alone (Figure 5b; E þ P þ 4mM ionizing radiation was disrupted in mice treated with HCl vs E þ P-control). The proapoptotic effects of progesterone alone. In mice treated with P-alone, 38% E þ P þ TGF-b treatments following ionizing radiation of cells were cycling (PCNA-positive) while only 9% were p53-dependent as TUNEL was o1% in mammary exhibited nuclear accumulation of p21/WAF1 following tissues from BALB/c-Trp53À/À mice. Though TGF-b is ionizing radiation. These results suggest that, though required for activation of p53 by E þ P, treatment with progesterone is a potent mitogen in the mammary gland, TGF-b1 alone had no effect on p53 responsiveness proliferation is not stringently coupled to enhanced (Figure 5b, g; þ TGFb1 alone). genomic surveillance through p53. This may underscore the tumor-promoting effects attributed to progesterone observed in epidemiologic studies (Schairer et al., 2000) Discussion as well as in mice (Lanari et al., 2001). Although progesterone alone is a relatively modest carcinogen, it The absence of radiation-responsiveness in mammary promotes rapid onset of aneuploidy followed by tumor epithelium of ovariectomized mice reveals the important formation in p53-deficient mammary epithelium (Goep- role for estrogen and progesterone in maintaining fert et al., 2000). The effect of progesterone has been the ability of p53 to sense and respond to DNA damage. attributed to inappropriate activation of genes involved As estrogen and progesterone are mitogenic in the in separation of sister chromatids together with the loss mammary epithelium, enhancing genome surveillance of checkpoint function due to the absence of p53 (Pati via p53 prior to replication would provide an important et al., 2004). These observations suggest that prolifera- ‘gatekeeper’ function and mitigate the risk associated tion and activation of p53 are separable pathways and with proliferation. Indeed, cells in the S phase are subject to hormonal control. more sensitive to radiation-induced DNA damage The downstream effectors of estrogen and progester- (Bedford and Dewey, 2002). Therefore, the close one that sensitize p53 and enhance responses to correlation between PCNA-positive cells (a marker of radiation-induced DNA damage were also investigated. cycling cells) and p53 responsiveness (measured by p21/ Barcellos-Hoff and co-workers (Ewan et al., 2002a) WAF1 expression) to ionizing radiation was expected. observed low levels of radiation-induced apoptosis in However, radiation-induced accumulation of p53 and nulliparous mice (ovary-intact) that were similar to p21/WAF1 is not strictly linked to cells undergoing reports by our lab (Minter et al., 2002). TGF-b1 was mitosis. Mice treated with either E-alone or E þ P implicated as a regulator of p53 activity in these studies resulted in 10 and 31% of mammary epithelial cells as radiation-induced apoptosis was impaired in TGF- being BrdU-positive (Said et al., 1997). Using Ki-67 as a b1-deficient mice (Ewan et al., 2002a). Furthermore, marker, we observed a similar fraction of cells in the S ovarian steroids enhance the levels of active TGF-b1 phase in the E þ P treated mice (28%, data not shown). (Ewan et al., 2002b), suggesting that the effects of E þ P As the proportion of cells that exhibit nuclear accumu- on p53 activity may be mediated by TGF-b. The TGF-b lation of p21/WAF1 following ionizing radiation receptors transduce their signals by phosphorylation of exceeds the fraction of cells that are in the S phase, several Smads (Massague, 1998), thus changes in the the effect of estrogen and progesterone cannot be level of P-Smads provide an indicator of TGF-b attributed solely to the increased proliferative fraction signaling. In these studies, P-Smad2 was analysed of cells. Indeed, the proportion of p21/WAF1-positive because it has been shown to augment p53 activity cells in the mammary epithelium was nearly two-times (Cordenonsi et al., 2003). Levels of P-Smad2 were the expected S-phase fraction. These results are con- unchanged in mammary tissue following treatment of sistent with those in the small intestine where the mice with E þ P, suggesting that P-Smad2 levels were fraction of p21/WAF1-positive cells exceeded the S- not rate-limiting for p53 responses (Figure 4). However, phase fraction (incorporation of 3H-labeled thymidine) function-blocking antibodies against TGF-b abrogated by nearly two-fold (Wilson et al., 1998). This result the effects of E þ P on p53 in mammary epithelial cells would be expected as p53 has a major role in G1 in WOC. Although TGF-b was a required co-factor, it checkpoint function mediated by p21/WAF1. As PCNA was not sufficient to render p53 responsive to ionizing levels are elevated in cells that are progressing through radiation in the absence of E þ P (Figure 5b). Therefore, G1 as well as cells that are undergoing active DNA crosstalk between intracellular signals induced by synthesis (Celis and Celis, 1985; Iatropoulos and estrogen and progesterone and TGF-b is essential for Williams, 1996), the similar proportions of PCNA- proper regulation of p53 activity in the mammary

Oncogene Hormonal regulation of p53 activity KA Becker et al 6351 epithelium. As p53 has been shown to be required for 2000). Alternatively, TGF-b may modify signaling pregnancy-induced prophylaxis of mammary tumors through the receptors for estrogen and progesterone. (Medina and Kittrell, 2003), these data suggest that Smads have been shown to bind ERa (Matsuda et al., TGF-b would also be essential for the protective effect 2001; Yamamoto et al., 2002; Wu et al., 2003) and may of pregnancy. redirect the signaling to enhance transcription of specific The mechanisms by which estrogen, progesterone and genes that sensitize p53. TGF-b act in the mammary epithelium to sensitize p53 While such a signaling cascade could be completed are of intense interest as they represent targets for within a few hours following hormonal treatments, chemopreventive therapies for breast cancer. The it does not provide an answer to why maximal receptors for estrogen, progesterone and TGF-b are p53-dependent responses were delayed until 72 h after coexpressed in a subpopulation of mammary epithelial initiation of estrogen and progesterone treatments. The cells in rodents and humans (Clarke et al., 1997; Russo requirement of 72 h to elicit maximal p53-dependent et al., 1999; Barcellos-Hoff, 2003; Anderson and Clarke, responses suggests the involvement of multiple tran- 2004). Receptor antagonists inhibited the effects of scriptional targets downstream of estrogen and proges- estrogen and progesterone ruling out direct DNA terone receptors as well as sequential changes in the damage (alkylation and epoxidation) by these steroids mammary gland. Gene expression profiling by micro- as a mechanism for increased p53 activity (Figure 2). array will likely identify additional components that Neither is a direct transcriptional mechanism suggested regulate p53 activity providing novel targets for because E þ P do not alter levels of p53 mRNA in the chemoprevention. mammary gland (Figure 3). Therefore, an indirect pathway is favored in which stability and activity of the p53 protein is regulated by either post-translational Materials and methods modification or interaction with co-factors. The activity of p53 protein is subject to regulation by a large cast of Hormonal treatment of mice enzymes that phosphorylate, acetylate, sumoylate and þ / þ ribosylate the p53 protein (Ljungman, 2000; Jerry et al., For the in vivo treatments, virgin BALB/c-Trp53 mice were ovariectomized at 8–9 weeks of age. Hormonal treatments were 2002; Bode and Dong, 2004). These post-translational delayed for 1 week following ovariectomy to allow endocrine changes in p53 can facilitate interactions with co-factors status to reach baseline. The animals were injected intraper- that stabilize the active form of p53 (Waterman et al., itoneally with 2 mg 17-b-estradiol (E), 1 mg progesterone (P), 1998; Stavridi et al., 2001). Heritable breast cancers 200 mg Mifepristone (M), 100 mg ICI 182,780 or 100 ml sesame provide clues to which of these many candidates may be oil (Vehicle). The E, P, M, and sesame oil were purchased from most relevant. Heritable mutations in ATM and CHK2 Sigma (St Louis, MO, USA). The ICI 182,780 was purchased genes have been identified in some familial breast cancer from Tocris (Ellisville, MO, USA). The treatments were patients (Meijers-Heijboer et al., 2002; Thorstenson administered by intraperitoneal injection in 50–100 ml of sesame et al., 2003) demonstrating their significance in regulat- oil and repeated daily either singly or in combination for up to ing susceptibility to breast cancer. Both ATM and 96 h. A minimum of four mice was used for each treatment group. At the end of the treatment period, the animals were CHK2 proteins play critical roles in sensing DNA subjected to 5 Gy whole-body ionizing radiation using a 137Cs damage and phosphorylate human p53. Therefore, source. Tissues were harvested 6 h postirradiation. Unirra- steroid hormones are likely to alter the balance of these diated wild-type BALB/c females were used to determine enzymes resulting in stabilization and activation of the baseline p53 activity in response to treatments. The treatments p53 protein. This stabilization is only apparent in were replicated in age-matched BALB/c-Trp53À/À females to response to DNA damage as hormone treatment alone assure that effects were p53-dependent. The fourth mammary (without irradiation) does not result in accumulation of glands were fixed in 10% neutral-buffered formalin and were active p53 or p21/WAF1 proteins. This feature is embedded in paraffin. All animals were maintained in important because if exposure to estrogen and proges- accordance with procedures approved by the Institutional terone simply increased p53 activity, it would prevent Animal Care and Use Committee. regrowth of the mammary gland during subsequent pregnancies. Instead, estrogen and progesterone can Whole organ cultures promote mammary gland development, but also en- For WOC, fourth mammary glands from unprimed, 8–9 week hance genome surveillance by p53 to prevent oncogenic old virgin BALB/c-Trp53 þ / þ and BALB/c-Trp53À/À mice were changes. removed aseptically, cut in half, then floated on siliconized lens Alternative pathways are also emerging. The estrogen paper in 35-mm tissue culture dishes containing 3 ml of receptor alpha (ERa) can bind in a ternary complex with medium. Each tissue culture dish contained the mammary HDM2 and p53 proteins leading to stabilization of p53; gland halves of four individual mice. Basal medium consisted however, this effect was observed only in overexpression of serum-free, phenol red-free DMEM/F12 buffered with systems and was ligand-independent (Liu et al., 2000). HEPES and NaHCO3, plus 5 mg/ml bovine insulin (GibcoBRL Life Technologies, Carlsbad, CA, USA). Hormone-supple- The p53 protein can also bind ERa and alter transcrip- mented media was prepared by adding E þ P (17-b-estradiol, tional responses to estrogen (Liu et al., 1999). Likewise, 1 ng/ml; progesterone, 1 mg/ml) to the basal media. Additional the DNA binding domain of the progesterone receptor growth factors were tested: epidermal growth factor (5 ng/ml, was found to form complexes with DNA-PK in cellular Upstate Biotechnology Inc., Saranac Lake, NY, USA), ovine extracts that can phosphorylate p53 (Sartorius et al., prolactin (100 ng/ml, NIDDK, NIH), tri-iodothyronine (8 mg/

Oncogene Hormonal regulation of p53 activity KA Becker et al 6352 ml, Sigma), hydrocortisone (2 mg/ml, Sigma). Human TGF-b1 In total, 150 mg of each sample were loaded on 10% (R&D Systems, Minneapolis, MN, USA) was reconstituted in polyacrylamide gels containing SDS and were separated by 4mM HCl (pH 5.7) containing 1 mg/ml BSA to prepare a 1 mg/ electrophoresis under reducing conditions. The proteins were ml stock solution of active growth factor, which was added to transferred to PVDF membranes (Immobilon-P, Millipore, media to yield a final concentration of 10 ng/ml. To control for Bedford, MA, USA), then incubated in blocking buffer. nonspecific effects, tissues were incubated in an equal volume Phospho-Smad2 was detected using affinity-purified polyclo- of 4 mM HCl; BSA solution added to basal media. Pan-specific nal antibodies directed against phospho-Ser465/467 (1 : 1000, anti-TGF-b antibodies (25 mg/ml, R&D Systems) were added Cell Signaling) followed by peroxidase-linked anti-rabbit IgG to inhibit TGF-b activity in the WOC. Rabbit anti-mouse IgG antibodies from donkey (1 : 5000, Amersham Biosciences, (25 mg/ml) was used to control for nonspecific effects of Little Chalfon, Buckinghamshire, UK). The antibody com- immunoglobulins. WOC were maintained for 96 h in 5% plexes were detected using chemiluminescent reagent (Amer- CO2 : 95% air with media changed daily. After 96 h, the sham Biosciences). The blots were stripped using a reducing cultures were irradiated (5 Gy), then returned to the incubator buffer (0.7% b-mercaptoethanol; 2% SDS; 6.25 mM Tris, pH for 6 h. Control cultures were handled identically, but not 6.8) then incubated with goat antiactin antibodies (1 : 500; #I- placed in the chamber of the irradiation. The treatments were 19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) replicated in age-matched BALB/c-Trp53À/À females to assure followed by detection with secondary antibodies (1 : 5000 that effects were p53-dependent. The tissues harvested 6 h rabbit anti-goat IgG, Sigma) as described above. postirradiation were fixed, and were embedded in paraffin. Northern blot hybridization Immunohistochemical staining Tissues for RNA were snap-frozen in liquid nitrogen, then Rabbit polyclonal antibodies were used for immunohistochem- stored at À701C until processing. RNA was extracted by ical detection of p21/WAF1 (1 : 50, Oncogene Research homogenizing the tissues in UltraSpec (BioTecx Laboratories, Products, San Diego, CA, USA) and p53 (1 : 200, Novacastra, Houston, TX, USA), 1 ml/100 mg of tissue. RNA was isolated Newcastle, UK). For immunohistochemical detection of using a modified Chomczynski method (Chomczynski and PCNA, the mouse monoclonal antibody PC10 (1 : 50, BD Sacchi, 1987), according to the manufacturer’s directions. In Pharmingen, San Diego, CA, USA) was used in combination total, 10 mg of total RNA was separated on a 1.2% agarose– with the M.O.M. Detection Kit (Vector Laboratories, Burlin- formaldehyde gel, transferred onto a nylon membrane game, CA, USA), according to the manufacturer’s directions. (Zetabind; CUNO, Meriden, CT, USA) and hybridized with Tissue sections (4 mm thick) were subjected to microwave a 32P-labeled cDNA probes. Hybridization signals were antigen retrieval. Immunolabeled complexes were detected quantified with a phosphorimager (Cyclone, Parkard 0 using Vector ABC Elite kits (Vector Laboratories) and 3,3 - Bioscience, Downers Grove, IL, USA). UV-shadowing was diaminobenzidine (Sigma). Tissue sections were counterstained used to assure equal loading (Thurston and Saffer, 1989). with methyl green. Apoptosis was detected using the TUNEL method using the FragEL DNA Fragmentation Kit according to the manufacturer’s procedures (EMD Biosciences, La Jolla, Acknowledgements CA, USA). The responses were quantified by counting 1200 We thank Amy Roberts for providing p53-deficient mice for consecutive cells in each section using the Bioquant Imaging these experiments. Brooke Pazik provided technical assistance Software System (Bioquant Image Analysis Corp, Nashville, with immunohistology. We thank Michael Sutherland TN, USA). Detection of phospho-Smad2 was performed using from the Statistical Consulting Center for his assistance. We affinity-purified rabbit polyclonal antibodies (1 : 100, anti- also thank Lauren Withington, Lawrence Sandi, Kathryn Ser465/467, Cell Signaling, Beverly, MA, USA) with a Levaseure, and Jennifer Cohen, undergraduates who polymer-based detection system (Acuity, Signet Laboratories, participated in this work. This work was supported by grants Dedham, MA, USA) using horseradish peroxidase and DAB to DJJ from the National Institutes of Health (CA87531, counterstained with Mayer’s hematoxylin. CA095164), the Massachusetts Department of Public Health (43088PPP1017), and the Charlotte Geyer Foundation with additional support from the Cooperative State Research Western blot Extension, Education Service, US Department of Agriculture, Flash-frozen tissues were homogenized in RIPA buffer using Massachusetts Agricultural Experiment Station and Depart- 1 ml buffer/100 mg tissue. The protein concentration was ment of Veterinary and Animal Sciences under Project Nos. determined using BCA reagent (Pierce, Rockford, IL, USA). MAS00821 and NC-1010.

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