Oncogene (2002) 21, 761 ± 768 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Estrogen-like e€ects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells

Sumi Dinda1, Amelita Sanchez1 and Virinder Moudgil*,1

1Department of Biological Sciences and Center for Biomedical Research, Oakland University, Rochester, Michigan, MI 48309, USA

T47D cells represent an estrogen-responsive human Keywords: Estrogen; thyroid hormone; p53; retinoblas- ductal carcinoma cell line which expresses detectable toma protein; tumor suppressor proteins; breast cancer levels of estrogen receptor (ER). We have previously shown that estradiol (E2) treatment of T47D cells causes an increase in the level of p53 and a concomitant Introduction phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 Di€erent hormones in¯uence the development, growth and phosphorylation state of pRb and compared the and di€erentiation of human breast tissue. The two e€ects of E2 and triiodothyronine (T3) on these steroid hormones, E2 and progesterone, impact phenomena. Cells were grown in a medium containing cellular functioning of the normal breast cells and charcoal-treated serum to deplete the levels of endogen- may continue to be active in its malignant state. It has ous steroids. Upon con¯uency, the cells were treated with been well established that nearly 30% of breast 712 77 T3 (10 to 10 M) for 24 h and the presence of p53 cancers are hormone-dependent and respond well to and pRb was detected by Western analysis. E2 treatment endocrine therapy (Clarke and Sutherland, 1990; of cells caused a 2 ± 3-fold increase in the level of p53. Horwitz, 1985, 1992). Although the relationship Presence of T3 in the medium caused a gradual increase between breast cancer and the more active form of in the level of p53 in a concentration-dependent manner. thyroid hormone, T3, is not known, T3 appears to Under the above conditions, pRb was phosphorylated stimulate lobular development and contribute to the (detected as an upshift during SDS ± PAGE) in the di€erentiation of normal breast tissue (Burke and presence of E2 and T3. Supplementation of growth McGuire, 1978). medium with T3 (1 mM) caused an increase in the rate of The products of tumor suppressor genes, p53 and proliferation of T47D cells and induced hyperpho- pRb, appear to play important roles in the incidence, sphorylation of pRb within 4 h; this e€ect was progression or management of breast cancer (Hurd et maintained for up to 12 h. When ICI 164 384 (ICI) al., 1995, 1997). pRb plays a major role in the control (1 mM), an ER antagonist, was combined with E2 (1 nM) of cell division and di€erentiation. Active form of pRb or T3 (1 mM), e€ects of hormones on cell proliferation is hypophosphorylated and present in quiescent G1 and hyperphosphorylation of pRb were blocked. Western phase cells (White, 1994; Weinberg, 1995). Tumor analysis of p53 was supplemented with its cytolocaliza- suppressor function of pRb is lost due to its hyperpho- tion by immuno-labeling using laser scanning confocal sphorylation causing it to dissociate from the mitogenic ¯uorescence microscopy, which revealed an ICI-sensitive factor E2F. Mutations in the p53 gene have been increase in the abundance of p53 in hormone-treated reported in over 50% of breast and colon cancers cells. Steroid binding analysis revealed lack of competi- (Oren, 1992). The main function of p53 is to switch o€ 3 tion by T3 for the [ H]E2 binding. These results indicate replication of damaged DNA allowing its repair, or that T3 regulates T47D cell cycle progression and induce apoptosis to disrupt cell proliferation. proliferation raising the p53 level and causing hyperpho- The T47D breast cancer cell line is ER and sphorylation of pRb by a common mechanism involving progesterone receptor (PR)-positive and expresses p53 ER and T3 receptor (T3R)-mediated pathways. with a missense mutation at codon 194 converting Oncogene (2002) 21, 761 ± 768. DOI: 10.1038/sj/onc/ leucine to phenylalanine in the protein (Nigro et al., 1205136 1989). We had previously reported that E2 regulates the expression of p53 and the state of phosphorylation of pRb in breast cancer cells (Hurd et al., 1997). There is, however, a paucity of information about the regulation of tumor suppressors or proto-oncogenes *Correspondence: VK Moudgil, Department of Biological Sciences by thyroid and steroid hormones. Although abundance Oakland University, Rochester, MI 48309-4476, USA; of c-Fos protein has been shown to be in¯uenced by E-mail: [email protected] Received 14 May 2001; revised 17 October 2001; accepted 31 thyroid hormones (Lin et al., 1999). It has been October 2001 proposed that these hormones may act via their nuclear Thyroid hormone regulation of p53 and pRb S Dinda et al 762 receptors to in¯uence proto-oncogene expression Figure 1 demonstrates the time course regulation of (Franklyn and Sheppard, 1989). E2-like e€ects of T3 p53 and pRb by T3. The level of p53 grown in stripped have been observed in a variety of experimental serum medium is generally low when compared with its systems, including the in¯uence of these hormones in level in cell grown in whole serum (Hurd et al., 1995, the healthy tissues or breast cancer cells (Franklyn and 1997). In the present studies, cells were grown in stripped Sheppard, 1989; Shao et al., 1995; Blohmer et al., 1999; serum and the level of p53 in these cells provided the Parhar et al., 2000; Qi et al., 1997; Dellovade et al., base line value. Supplementation of culture medium with 1999, 2000; Morgan et al., 2000; Barrera-Hernandez et 1 mM T3 resulted in a 220% increase in the level of p53 al., 1999). Thyroid hormone-induced cell proliferation over a period of 24 h with a maximum (400%) at 8 h in rat pituitary GC cells has been reported to be (Figure 1, upper panel). We have previously shown that mediated by cyclin/cyclin-dependent kinase (CDK) rise in p53 following E2 supplementation is an early activity (Barrera-Hernandez et al., 1999). Physiological event (Hurd et al., 1995). The pRb protein was resolved concentrations of T3 appear to enhance estrogen into a rapidly migrating lower band corresponding to a stimulation of growth of breast carcinoma, which is hypophosphorylated active proliferation suppressing blocked by the addition of ligands inducing retinoid X form (pRb), and a slower migrating upper band receptor (RXR) homodimers (Shao et al., 1995). corresponding to a hyperphosphorylated form (ppRb) Thyroid hormones have also been reported to either (Figure 1, lower panel). Cells grown in stripped serum in¯uence or interfere with the estrogen-stimulated medium exhibited mainly the hypophosphorylated pRb female reproductive behavior (Dellovade et al., 1999, (control, C). Treatment of cells with T3 caused 2000; Morgan et al., 2000). It has been proposed that hyperphosphorylation of pRb in a time-dependent T3R interacts with p53 in vivo to reverse p53-induced manner exhibiting a maximum between 4 ± 12 h (Figure suppression of promoter activity (Qi et al., 1997). A 1, lower panel). The increase in p53 level in cells treated cross talk between p53 and ER has been suggested in with T3 for 1 h could represent both transcriptional and hormone-activated signaling pathways (Bhat et al., post-transcriptional mechanisms. The latter may involve 1997). Although the published studies addressed the stabilization of p53 but such investigations were neither inter-relationships between T3 and p53 or T3-induced performed nor considered in the scope of this work. cell proliferation and estrogen in¯uences, e€orts to Figure 2 demonstrates e€ects of di€erent concentra- incorporate all these variants in a single system and tions of T3 on the level of p53 and degree of under similar experimental conditions have been phosphorylation of pRb. The levels of p53 in the lacking and were initially reported from this laboratory control (C, stripped serum) samples were low but (Dinda et al., 1998). The present study focuses on (a) detectable. Treatment of cells with increasing concen- the e€ects of E2,T3 and antiestrogen (ICI) on the trations of T3 raised the level of p53 in a gradual regulation of p53 and pRb, (b) the e€ects of E2,T3 and manner showing a maximum near 1 ± 10 nM (Figure 2, ICI on the proliferation of T47D cells, and (c) to upper panel). The p53 level in the cells treated with T3 examine the correlation, if any, between immunoloca- was equivalent to that seen in cells treated with 1 nM lization and expression of p53 with the cell prolifera- E2. When the degree of phosphorylation of pRb was tion. analysed under these conditions, pRb was observed to exist mainly in its hypophosphorylated pRb form

Results

Regulation of p53 and pRb by T3: Time course and concentration dependency

Trace amounts of endogenous steroids, including E2, are routinely removed from media by treatment of serum with charcoal suspension (stripped) in order to monitor the e€ects of exogenously added ligands (Hurd et al., 1995). Recently, our laboratory has shown the levels of p53 and the degree of phosphorylation of pRb are maximal at a 24 h treatment of T47D cells cultured in media containing stripped serum supplemented with E2 (1 nM) (Hurd et al., 1997, 1999). The hyperphosphoryla- tion of pRb occurred between 12 and 24 h in T47D cells. Figure 1 Regulation of p53 and pRb by T3: Time course. Cells In this study, we have compared the e€ects of 1 nM E2 were plated into medium containing 5% serum for 2 days and then and T3 within a broad range of hormone concentrations withdrawn from E2 stimuli by culturing them for 7 days in medium (10712 ±1077 M) on the levels of p53 and pRb. Equal containing charcoal-treated serum. Cells grown in stripped serum number of cells were plated in whole serum (containing were then treated with 1 mM concentration of T3 for 0 ± 24 h as represented in the ®gure. The control is represented by C (grown in endogenous E2) for 2 days and then maintained for 6 charcoal-treated serum) and had no hormone treatment. High salt days in stripped serum containing media. The cells were protein extracts were prepared and quantity of p53 and pRb were treated with the hormones for 24 h. determined by SDS ± PAGE (8%) and Western analysis

Oncogene Thyroid hormone regulation of p53 and pRb S Dinda et al 763

Figure 2 Regulation of p53 and pRb by T3: Concentration dependency. Cells were plated into medium containing 5% serum Figure 3 E€ects of E ,T, and antiestrogen (ICI) on the for 2 days and then withdrawn from E stimuli by culturing them 2 3 2 phosphorylation of pRb and induction of p53. Cells were plated for 7 days in medium containing charcoal-treated serum. The into medium containing 5% serum for 2 days and then withdrawn control is represented by C (grown in charcoal-treated serum) and from E stimuli by culturing them for 7 days in medium had no hormone treatment. The cells were then treated for 24 h 2 containing charcoal-treated serum. C represents control grown with E (1 nM) and T concentrations (10712 ±1077 M). High salt 2 3 in strip serum and received no hormone treatment. Cells were protein extracts were resolved by SDS ± PAGE (8%), transferred treated with E (1 nM), T (1 mM), and combinations of E (1 nM) to a PVDF protein blotting membrane and probed with primary 2 3 2 or T (1 mM) plus ICI (1 mM). High salt protein extracts were antibodies; puri®ed anti-human pRb clone G3-245 (PharMingen), 3 prepared and the quantity of p53 and pRb were determined by anti-p53 monoclonal antibody (Transduction) and secondary SDS ± PAGE (8%) and Western analysis HRP-conjugated anti-mouse IgG. Bands were visualized by enhanced chemiluminescence (ECL)

(Figure 2, lower panel; C, control). Presence of 0.01 ± loss of the upper band of pRb. Similarly, ICI was able 10 nM T3 in the culture medium raised the level of to downshift the T3-induced ppRb form to its pRb phosphorylated pRb with an optimum e€ect seen at hypophoshorylated form. 712 10 nM T3. The darker/heavier lower band at 10 M The Western analyses in the studies were supple- T3 represents a more strongly hypophosphorylated mented by image analysis by confocal microscopy pRb species, and could also indicate inhibition of involving immuno¯uorescence localization of cellular phosphorylation of pRb. Although possible, it is p53. Results in Figure 4 demonstrate that p53 is 712 unlikely that T3 at 10 M concentration activates a exclusively localized in the T47D cell nuclei under all phosphatase. In the absence of any known signi®cant conditions of testing. Treatment of cells with either e€ects of other hormones at 10712 M, we believe hormone increased the intensity of nuclear staining of further exhaustive analysis of e€ects of low T3 p53 in T47D cells. When cells were exposed to both concentrations is warranted and is in progress. Clearly, hormones simultaneously, the degree of immuno¯uor- results of Figures 1 and 2 indicate T3 induces changes escence was only marginally greater than when e€ects in T47D cells, in the context of p53 and pRb/ppRb of hormones were tested separately. Again, ICI levels, that are similar to those brought about by E2. blocked the extent of immuno¯uorescence generated by the treatment of cells with both the ligands. The nuclear staining of p53 indicates that E ,T and Coordinate regulation of pRb phosphorylation and up- 2 3 antiestrogens do not a€ect the localization of p53 in regulation p53 by hormones and antihormones T47D cells (under the conditions of testing), but rather In order to determine the mechanism of T3 and E2 the accumulation of nuclear p53. The density of actions in T47D cells which contribute to the observed nuclear ¯uorescence correlated well with the protein e€ects on p53 and pRb, the in¯uence of full ER levels determined by Western analysis. antagonist, ICI was examined. T47D cells were cultured for 6 days in stripped serum and then treated Effects of T3 on the proliferation of T47D cells with E2 (1 nM) and T3 (1 mM) in the absence and presence of ICI (1 mM). We have previously shown the We next assessed the e€ects of E2,T3 and the level of p53 decreases in cells grown in stripped serum antiestrogens on the T47D cell proliferation in order compared with when grown in whole serum (Hurd et to correlate this with pRb hyperphosphorylation and al., 1995, 1997, 1999). Figure 3, (upper panel) shows cellular level of p53. Cells were treated with ligands as supplementation of culture medium with E2 and T3 described earlier and in Methods. T47D cell prolifera- resulted in an increase in the level of p53 expression. tion was determined by cell counting (Figure 5). The potent antiestrogen ICI blocked both the E2 and Culturing cells in stripped serum reduced the cell T3-induced e€ects on p53 levels. Treatment with E2 population by 46% (compared to controls in stripped and T3 also caused pRb hyperphosphorylation (Figure versus whole serum) (not shown) suggesting that 3, lower panel), which is associated with the cell charcoal treatment removed factor(s) required for proliferation. ICI (1 mM) was able to block the E2- maximal T47D cell proliferation (Hurd et al., 1995, induced phosphorylation as indicated by the signi®cant 1997; Iwasaki et al., 1999). It is known charcoal

Oncogene Thyroid hormone regulation of p53 and pRb S Dinda et al 764

Figure 4 Cytolocalization of p53 in T47D cells by immuno¯uorescence. Cells in triplicate were plated in 12-well growth (30 000 cells/well) on cover slips cultured for 2 days in whole serum and then treated with 1 nM E2, 100 nM 4 hydroxy-tamoxifen (OHT), 1 mM ICI (ICI) or 1 mM tamoxifen (TAM) or cultured in stripped serum supplemented with 1 nM E2 plus antiestrogens. Fresh media and ligands were replaced at 2 day intervals. p53 localization was determined by Immuno-labeling and laser scanning confocal ¯uorescence microscopy

treatment removes T3/T4 as well as steroids in addition to a variety of other hormones and growth factors. E2 addition increased the cell count consistent with the increased p53 expression during the E2-induced pro- liferative state (Hurd et al., 1995, 1997; Iwasaki et al., 1999). The addition of E2 and T3 caused a signi®cant increase (104 and 577%, respectively) in the cell count. The E2 e€ect on proliferation re¯ects our earlier ®ndings (Hurd et al., 1995, 1997; Iwasaki et al., 1999) and those expected from thyroprival cells exposed to E2. The combination of E2 and T3 did not increase the cell number signi®cantly compared to E and T , individually suggesting absence of a Figure 5 E€ects of E2,T3, and antiestrogens on the proliferation 2 3 of T47D cancer cells. Cells (30 000) were plated in a 12-well plate synergistic mechanism with regard to cell proliferation, for 2 days in medium containing 5% bovine serum. The cells were and perhaps re¯ecting a physiological e€ect. The withdrawn from E2 stimuli by culturing for 6 days in medium antiestrogen, ICI, inhibited E2 and T3-dependent cell containing stripped serum and the ligands. The medium and the proliferation. Since ICI is also capable of inhibiting ligands were replaced at 2 day intervals. After 7 days in the presence of E ,T, and the antiestrogens, the cell number was T R in the absence of added E which could be 2 3 3 2 determined by Coulter Counter (model #Z2). Error bars represent explained on the basis of bar 5 in Figure 5. Whether standard deviations of triplicate cell counts ICI blocks some of the primary genomic e€ects of T3 in T47D cells is not known. It was not the focus of this work to directly link p53 and cell proliferation. The increase in cell proliferation was studied more as a represented in Figure 6. The extent of binding of a mark of estrogen action than its association with wild particular ligand to ER was indicated by the 3 or mutated p53. Both forms of p53 could be induced, comparative displacement of [ H]E2. The decrease in yet the increase in the mutated form would be non- height of individual bars in the ®gure re¯ects 3 functional. T47D cells express a mutated p53 with a competition for the [ H]E2 binding by E2 and T3.E2, 3 missence mutation at codon 194 (Nigro et al., 1989). and not T3, competed for [ H]E2 binding suggesting the Shih et al. (2001) have shown that T4 causes ser-15 existence of independent mechanisms inducing compar- phosphorylation of p53. Whether p53 phosphorylation able changes in the level of p53 and the state of was also in¯uenced by treatment of T47D cells by E2 phosphorylation of pRb. and T3 will be a target for future studies.

Discussion Interaction of E2 and T3 with estrogen receptor (ER): Comparative steroid binding analysis The clinical signi®cance of the in¯uence of thyroid 3 T47D cells were treated with 1 nM [ H]E2 in the hormone in breast cancer has not been established presence and absence of T3 (1 nM and 1 mM)as with certainty (Cerbon et al., 1981). It has been

Oncogene Thyroid hormone regulation of p53 and pRb S Dinda et al 765 Furthermore, the kinetics of p53 expression relative to pRb phosphorylation suggests that this occurs during G1 phase of the cycle where p53 can attenuate pRb phosphorylation. We have observed that ICI blocked the E2 and T3 induced expression of p53 and hyperphosphorylation of pRb in T47D breast cancer cells. These data may imply an ER-mediated mechanism for T3-induced p53 Figure 6 Interaction of various concentrations of E2 and T3 expressions and hyperphosphorylation of pRb. Steroid with estrogen receptor (ER): Competitive steroid binding analysis. 3 binding analysis has shown that T does not compete T47D cells were incubated with [ H]E2 in the absence and 3 presence of competing ligands. Media was then brought to a 1 nM with E2 for binding to ER in T47D cells. E2 and T3 3 concentration with respect to [ H]E2 alone (C) or plus 1 nM E2, have been reported to compete for ER binding in 1nM T3, and 1 mM T3. Cells were placed in the incubator for 2 h, MCF-7 cells (Nogueria and Brentani, 1996). In the protein extracts were prepared, aliquots of the sample were 3 absence of T3R, the thyroid hormone response element incubated with [ H]E2 in the absence and presence of radioinert steroids. The extent of steroid binding was determined by (TRE) can act as an imperfect estrogen response Dextran-coated charcoal ligand binding assay, which involved element (ERE) and mediate E2-dependent activation 3 removal of unbound or free [ H]E2 by the charcoal suspension. of transcription (Dellovade et al., 1995). These data 3 The macromolecule-bound [ H]E2 was quanti®ed by scintillation suggest the possibility of existence of complex interac- counter tions between estrogen and thyroid hormones and their in¯uence on gene expression. Alternatively, T3-bound T3R may stimulate ERE function in T47D cells suggested that hypothyroidism is a risk factor for the eliciting E2-like e€ects. The latter observation is development of breast cancer (Alvarado-Pisani et al., supported by the assumption that ER and T3R bind 1986). The main aim of the present study was to to parts of the same hormone-response elements of compare the e€ects of E2 and T3 on the regulation of target genes (Dellovade et al., 1995). p53 and pRb in breast cancer cells. The p53 and pRb We propose a hypothesis to explain the observed are key factors regulating proliferation of breast cancer estrogen-like e€ects of thyroid hormone. T3R and ER cells. We had previously demonstrated that culturing bind to di€erent parts of the same hormone response T47D cells in stripped serum decreases the level of p53 elements of a target gene and compete with each other by 90%, and a supplementation with a physiological for inducing a response. By this mechanism, ER and concentration of E2 (0.1 ± 1 nM) was sucient to T3R interactions would have stimulatory or inhibitory maintain high levels of p53 (Hurd et al., 1995). A e€ects depending on the absence or presence of ligands, physiological concentration of E2 was sucient to and the consequences of protein-DNA interactions stimulate both p53 expression and pRb phosphoryla- (Dellovade et al., 1995; Graupner et al., 1991). It has tion. We have shown that T3 treatment increased the been proposed that E2-like e€ects of thyroid hormone level of p53 and caused hyperphosphorylation of pRb. are possibly in¯uenced by events which occur sub- The proliferative e€ects of E2 on the breast epithelial sequent to the process of steroid binding (Dellovade et cells are well documented and appear to be mediated al., 1999, 2000). The noted E2-like proliferative e€ects by the cyclin D1-cdk 2/4-Rb pathway (Dulic et al., of T3 in our study have revealed an unexplored 1994; El-Deiry et al., 1993; Wa®k et al., 1994). It has potential of thyroid hormones in breast cancer patients been reported that p53-dependent transactivation of undergoing T3 treatment. Further work in this area the cdk inhibitor p21WAF1/CIP1 attenuates pRb phos- should help provide insight into the mode of action of phorylation and thus cell cycle progression from G1 to T3 in T47D cells as well as its potential clinical S phase of the cell cycle (Xiong et al., 1993). Although relevance in breast cancer. the role of T3 in the progression of breast cancer is not In summary, we propose a uni®ed model of the established, E2 and T3-dependent p53 expression could regulation of p53 and pRb by E2 and T3 in breast in¯uence breast epithelial cell proliferation. Mutation, cancer cells (Figure 7). Accordingly, E2 binding to ER loss or insucient expression of p53 during breast cell leads to the formation of steroid-receptor complex that proliferation could theoretically contribute to breast interacts with EREs in the T47D cell genome. Since cancer development by default of p53-mediated cell ER is a nuclear transcription factor, and E2 is a known arrest or apoptosis. It is not clear whether the levels of regulator of gene expression, a number of hormone- p53 are regulated by E2 and T3 or if indirect signals sensitive genes are known to be in¯uenced by hormone emitted by proliferating cells, in general, regulate p53. treatment of T47D cells. We have previously shown The rate of proliferation of T47D cells and the levels that E2 stimulates c-Myc in T47D and MCF-7 cell lines of p53 decline to a minimal state in cells grown in (Shao et al., 1995). A speci®c E2-dependent ER- culture medium containing charcoal-stripped serum. mediated c-Myc species was induced, which was Supplementation of media with exogenous E2 or T3 sensitive to full ER antagonist, ICI. We believe, among enhances, but does not recover the maximum rate of other possibilities, the e€ects of E2 are mediated via c- proliferation of cells grown in medium containing Myc expression since E2 was shown to regulate the P1 whole serum. E2 and T3 induce p53 to near maximal promoter of the p53 gene which contains a c-Myc levels which is independent of the proliferation state. responsive element (Reisman et al., 1988). The

Oncogene Thyroid hormone regulation of p53 and pRb S Dinda et al 766

Figure 7 A hypothetical model for explaining regulation of p53 and pRb by E2 and T3 in T47D breast cancer cells. ER, estrogen receptor; ERE, estrogen response elements; P, phosphorylation; pRb, retinoblastoma protein; T3R, thyroid hormone receptor

estrogen-like e€ects of T3 appear to be mediated via after further experimentation, it will necessitate re- T3R interaction with ERE to stimulate gene expression evaluation of our model (Figure 7). since T3 does not appear to compete for the E2 binding to ER (Figure 6). This postulation is now being tested in the laboratory. The tumor suppressor activity of wild type p53 is Materials and methods expressed via its ability to cause DNA repair, induce apoptosis or synthesis of inhibitor of cyclin-cyclin- Materials dependent kinase, p21. The latter blocks cell division. ICI 164 384 was received from Zeneca Pharmaceuticals These functions are compromised in cells containing (Cheshire, UK). Anti-human pRb AG clone G-3-245 was mutated p53, which can lead to cell proliferation. In its purchased from Pharmingen (San Diego, CA, USA). p53 active tumor suppressor form, pRb exists as the antibody was from Transduction Laboratories (Lexington, 3 hypophosphorylated entity. Results of our earlier KY). [2,4- H] estradiol (40 Ci/mmol) was purchased from studies (Hurd et al., 1995, 1997, 1999) and reported Moravek Biochemicals (Brea, CA, USA). Estradiol, triio- here (Figures 1 ± 3) have shown E causes hyperpho- dothyronine, phenylmethylsulfonyl ¯uoride (PMSF), insulin, 2 penicillin, streptomycin, amphotericin B solution, and sphorylation of pRb and increasing cell proliferation. activated charcoal were purchased from Sigma Chemical The exact nature of the kinase(s) involved in the Co. (St. Louis, MO, USA). Leupeptin, pepstatin-A, and estrogen-induced hyperphosphorylation is not known chymostatin were from Peninsula Laboratories (Belmont, and is a target of our future studies. A number of CA, USA). Dextran T-70 was from Pharmacia Biotech Inc. additional possibilities exist as to the role of hormones (Uppsala, Sweden). Ethylhexadecyldimethyl ammonium in the regulation of tumor suppressors and proto- bromide was from Eastman Kodak Company (Rochester, NY, USA). Horseradish peroxidase was from Boehringer oncogenes. T3/T4 could complex p53 with MAPK and with TR (Shih et al., 2001). An interaction between TR Mannheim (Indianapolis, IN, USA). Biodpy 581/591 and p53 was suggested earlier (Bhat et al., 1997; Qi et phalloidin was from Molecular Probes (Eugene, OR, al., 1997, Yap et al., 1996). The complexing of TR by USA), antimouse IgG conjugated with FITC and Cy3 from Jackson Immuno Research Laboratories, Inc. (West Grove, p53 could then decrease the transcriptional activity of PA, USA). p53. Phosphorylation of ER by MAPK has been shown to result in serine phosphorylation of the latter Cell culture and ligand treatment (Kato et al., 1995). Thus it is possible ER and T3R may interact in T47D cells in the presence of T3 and E2 T47D cells were obtained from American type culture to in¯uence p53 activity. Should this become probable collection (ATCC) (Rockville, MD, USA). Cells were

Oncogene Thyroid hormone regulation of p53 and pRb S Dinda et al 767 routinely cultured at 378C in an incubator in RPMI 1640 antibodies, anti-p53 monoclonal antibody (Transduction media with L-glutamine supplemented with 5% heat Laboratories) or a 1 : 500 dilution of puri®ed anti-human inactivated fetal bovine serum (FBS) or single stripped fetal pRb, clone G3-245 (PharMingen). The membrane was then bovine serum (SSFBS), 25 mM HEPES, 24 mM sodium washed for 30 min with three changes of TBS-tween, bicarbonate, 0.5% 1006non-essential amino acids, 100 reblocked for 30 min with TBS-tween plus 5% Carnation units/ml penicillin, 0.1 mg/ml streptomycin, 0.25 mg/ml instant non-fat dry milk and then incubated in the same amphotericin B, and 0.14 IU/ml insulin. Stock solutions of solution plus a 1 : 1000 dilution of HRP-conjugated anti- the ligand were prepared in ethanol to a 1000-fold higher mouse IgG H+L chain (Boehringer Mannheim). After a ®nal concentration than the ®nal concentrations during cell wash, p53 speci®c bands were visualized by the enhanced treatment. Aliquots (10 ml) of ligand were added directly to chemiluminescence (ECL) method according to the instruc- 10 ml of media. Controls contained identical quantities of tions from Amersham. ethanol vehicle as cells treated with ligands. Media and ligands were added fresh at 2-day intervals. For proliferation Immunolabeling and laser scanning confocal fluorescence studies, cells were passed into triplicate Corning 12-well microscopy dishes. After treatments with E2,T3 (ICN Pharmaceuticals Inc., Costa Mesa, CA, USA) or the antiestrogen, cell number Cells were plated in triplicate in 12-well growth plates (30 000 was determined by Coulter counter model Z2 as previously cells/well) on cover slips prior to treatment with E2 or the described (Hurd et al., 1997, Kodali et al., 1994). antiestrogens for 7 day treatment periods. Subsequent incubations were performed at 238C. The cells were ®xed on cover slips for 10 min with 1% formalin in PBS, air dried Binding of [3H]E in T47D cell extract 2 with ice-cold acetone: methanol (50 : 50) for 3 min and then T47D cells were grown to con¯uency in SSFBS. Media was washed 3X with PBS. Staining procedures were performed in 3 brought to a 1 nM concentration of [ H]E2 alone or plus a humidi®ed chamber at 238C. Nonspeci®c binding of IgG 1 mM and 1 nM of E2, and with di€erent concentrations of T3 was suppressed by incubating cells for 20 min in 10% goat (1, 10, 100, 1000 nM). Cells were placed in an incubator for serum. Cells were then incubated with primary anti-p53 Ab 2 h prior to preparation of protein extracts. An equal volume (Signal Transduction) at a 1 : 150 dilution for 2 h, washed 4X of Dextran-coated charcoal solution (0.5% charcoal, 0.05% with PBS and then incubated with secondary Ab, anti-mouse Dextran T-70 in 10 mM Tris, 1 mM EDTA, pH 7.4) was IgG conjugated to FITC (Sigma) at 1 : 150 dilution and then added to 150 ml of protein extract, the solution vortexed and washed 56 with PBS and mounted in Fluoromount-G incubated on ice for 5 min. Charcoal suspension was pelleted according to instruction from Electron Microscopy Sciences by centrifugation at 3000 g for 5 min at 48C. The radio- (FT. Washington, PA 19034). The distribution of three- activity in 150 ml of supernatant was determined by dimensional ¯uorescent structures were analysed using a Bio- scintillation counting. Rad 600 laser scanning Confocal microscope and photo- graphed with a Nikon Optiphot microscope. Fluorescence images were collected under a 406 objective sequentially Extraction of proteins from T47D cells using a Krypton argon (488 nm) laser beam for excitation Media was removed by aspiration, the cells were washed and a broad pass (514 ± 545 nm) emission ®lter. COSMOS with 10 ml ice-cold Hanks balanced salt solution (HBSS), software (Bio-Rad) was used for the collection of images. The and the cells were then scraped into 500 ml of extraction image dimensions were approximately 5126512 pixels with bu€er (20 mM HEPES, 50 mM NaF, 10 mM Na2MoO4, 8-bits of resolution. Software `Confocal assistant' was used 8mM Na2HPO4,2mM NaH2PO4,40mM NaCl, 1 mM for pseudocolor restitution. Fluorescence images were EDTA, 10% glycerol, 1 mM leupeptin, 30 mg/ml pepstatin- obtained by pseudocolor image restitution from gray scale A, 30 mg/ml chymostatin and 0.3 mM PMSF). Cells were images. burst by the freeze/thaw method via immersion in liquid nitrogen for 20 s and thawed on ice. This was repeated three times. After the ®nal thaw, the cell suspension was brought Abbreviations to a ®nal ionic strength of 0.5 by addition of 4.0 M NaCl, M ATCC, American type culture collection; E ,17-b estradiol; 20 m HEPES, pH 7.4 and incubated on ice for 1 h with 2 M ECL, enhanced chemiluminescence; ERE, estrogen response gentle resuspension of the pellet. A high speed supernatant element; FBS, fetal bovine serum; ER, estrogen receptor; (HSS) of the extract was prepared via centrifugation of the HBSS,Hanks'balancedsaltsolution;HSS,highspeed burst cells at 35 000 r.p.m. for 45 min at 48C. HSS was supernatant; ICI, ICI 164 384; OHT, 4-hydroxy tamoxifen; frozen in liquid nitrogen and stored at 7808C until further p53, 53-kDa tumor suppressor protein; PBS, phosphate- use. bu€ered saline; PMSF, phenylmethylsulfonyl ¯uoride; PVDF, polyvinylidene ¯uoride; PR, progesterone receptor; SDS ± PAGE and Western blot analysis pRb, retinoblastoma protein; ppRb, phosphorylated pRb; Rb, retinoblastoma; SDS ± PAGE, sodium dodecyl sulfate High speed supernatants were denatured and 75 mg of total polyacrylamide gel electrophoresis; SSFBS, single stripped protein/lane [quanti®ed by the Bio-Rad Laboratories (Her- fetal bovine serum; T , triiodothyronine; TAM, tamoxifen; cules, CA, USA) protein assay kit] were resolved on an 8% 3 T R, thyroid hormone receptor; TRE, thyroid hormone polyacrylamide gel under denaturing conditions. Proteins 3 response element. were transferred to Immobilon PVDF membrane (Millipore) at 30 Volts for 14 h or 60 Volts for 7 h in a Tris glycine bu€er system containing 0.025% SDS and 15% methanol using a Bio-Rad trans blot cell with tap water cooling. Acknowledgments Membranes were blocked for 1 h in TBS-tween (0.1%) plus The studies were supported in part by National Institutes 5% Carnation instant non-fat dry milk, then probed for 1 h of Health, and the Research Excellence Fund, Center for in the same solution containing a 1 : 500 dilution of primary Biomedical Research, Oakland University.

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