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Molecular Endocrinology 19(2):327–339 Copyright © 2005 by The Endocrine Society doi: 10.1210/me.2004-0306

Progesterone Receptors Induce Cell Cycle Progression via Activation of Mitogen-Activated Protein Kinases Andrew Skildum, Emily Faivre, and Carol A. Lange Departments of Medicine (Division of Hematology, Oncology, and Transplantation) and Pharmacology, University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Progestins induce proliferation of breast cancer cells and are implicated in the development of breast cancer. The effects of progestins are mediated by progesterone receptors (PRs), although it is unclear whether proliferative effects are delivered through activities as ligand-activated transcription factors or via activation of cytoplasmic kinases. We report that progestin induces S phase entry of T47D cells stably expressing either wildtype (wt) PR-B or a transcriptionally impaired PR-B harboring a point mutation at Ser294, a liganddependent and MAPK consensus phosphorylation site (S294A). Both wt and S294A PR are capable of activating p42/p44 MAPKs and promoting proliferation. However, cells expressing wt, but not S294A PR, exhibited enhanced proliferation in response to combined epidermal growth factor and progestin. S phase progression correlated with up-regulation of cyclin D1. The PR antagonist RU486 also

induced MAPK activation, increased cyclin D1 expression, and stimulated S phase entry, which was blocked by inhibition of either p42/p44 or p38 MAPKs, whereas proliferation induced by R5020 was sensitive only to p42/p44 MAPK inhibition. MCF-7 cells stably expressing a mutant PR unable to bind c-Src and activate MAPK failed to support progestin-induced proliferation. These data suggest that PR mediate cell cycle progression primarily through activation of cytoplasmic kinases and independently of direct regulation of transcription, whereas the coordinate regulation of both aspects of PR action are required for enhanced proliferation in response to progestins in the presence of growth factors. Targeting the ability of steroid receptors to activate MAPKs may be beneficial for breast cancer patients. (Molecular Endocrinology 19: 327–339, 2005)

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progestins promote cell growth are not clear. A better understanding of the biology of steroid hormone signaling may reveal novel therapeutic targets to more effectively manage breast cancer. The effects of progesterone are mediated by progesterone receptor (PR) A and B isoforms, members of the nuclear steroid receptor superfamily of ligandactivated transcription factors. Like most family members, PR-A contains a DNA binding domain, a ligand binding domain, and two transcriptional activating motifs: the C-terminal, ligand-dependent activation function (AF)-2 and the N-terminal, more constitutively active AF-1. The more transcriptionally potent B isoform of PR (PR-B) contains an additional N-terminal AF-3. PRs have numerous sites for posttranslational modification, including phosphorylation (10–12), ubiquitination (13), and sumoylation (14–16). One phosphorylation site, Ser294 on PR-B, has been identified as contributing to transcriptional synergy between progestins and peptide growth factors, ligand-dependent PR down-regulation through the ubiquitin/proteosome pathway, and PR nuclear localization after growth factor treatment (13, 17, 18). However, the functions of other posttranslational modifications at numerous sites remain unknown. PRs may exert biological effects through several potential mechanisms. In the classical action of steroid

VARIAN STEROID HORMONES such as estrogen and progesterone are critical for mammary gland development and are implicated in the development and progression of breast cancer (1). The disruption of steroid hormone signaling pathways is a promising treatment strategy for breast cancer, and the estrogen receptor antagonist tamoxifen has proven effective at treating and preventing breast cancer (2). In vitro, both estrogen and progesterone cause proliferation of cell lines derived from human breast tumors (3–7), and postmenopausal women who take a combined estrogen and progesterone hormone replacement therapy have a higher incidence of breast cancer than women who take estrogen alone (8, 9). These observations strongly implicate progesterone in disease etiology, although the molecular mechanisms through which

First Published Online October 14, 2004 Abbreviations: AF, Activation function; Cdk, cyclin-dependent kinase; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; FBS, fetal bovine serum; MEK, MAPK kinase; PR, progesterone receptor; PRE, progestin response element; SB, p38 MAPK inhibitor SB203580; SRC, steroid receptor coactivator; U0, MEK1/2 inhibitor U0126; wt, wildtype. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 327

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hormone receptors, binding of progesterone to PR causes receptor homodimerization, nuclear translocation, and binding of PR to progestin response elements (PREs) in promoters; PR then binds transcriptional coactivators such as steroid receptor coactivator 1 (SRC-1), which recruit and activate the basal transcriptional machinery, causing increased expression of PR-regulated genes (19, 20). Liganded PR can also activate transcription of genes the promoters of which lack PREs by acting as a bridge between transcription factors and coactivators at promoters containing activator protein 1 or specificity protein 1 sites (21–23). In addition to direct genomic effects, PR can also function as a node in cytoplasmic kinase cascades. Progestin causes the activation of MAPK through direct (24) or indirect (25) activation of c-Src kinase, and activation of MAPK by expression of exogenous active MAPK kinase kinase 1 or treatment with epidermal growth factor (EGF) causes PR phosphorylation and greatly enhances ligand-dependent transcriptional activation of PR (17). Alternatively, PR may activate genes via MAPK-dependent signaling events that may not rely on the transcriptional activity of liganded PR. For example, cyclin D1, cyclin E, and p21cip1 are upregulated by progestin and growth factor treatment of breast cancer cells in a MAPK-dependent manner (6, 26). Although the promoters lack consensus PREs, transcription of cyclins D1 and E is regulated in part by Ets factors, which are phosphorylated and activated by p42/p44 MAPK (27, 28). In this way, PR may regulate gene expression indirectly by modulating kinase cascades. We speculated that the different functions of PR (as a cytoplasmic signaling molecule and as a nuclear transcription factor) may lead to different consequences on cell fate in breast cancer cells. In this report, we show that a transcriptionally impaired PR-B mutant, which is incapable of being phosphorylated on Ser294 in response to MAPK activation but is still capable of activating MAPK, causes cell cycle progression in response to synthetic progestin. However, a PR-B mutant, which is unable to activate MAPK but functions as a transcriptional activator, does not support S phase entry. These data support a model whereby the proliferative response of breast cancer cells to progestins is due to the activation of cytoplasmic signaling cascades rather than from direct transcriptional activation.

RESULTS Progestin-Induced Proliferation Is Independent of PR-B Ser294 Phosphorylation The effects of progestins on T47D breast cancer cell progression are well characterized (6, 7, 29–34). In defined culture conditions, progestins induce several rounds of cell division (29). In the absence of other hormones, multiple clones of T47D breast cancer

cells, which express the B isoform of PR (PR-B), have also been shown to respond to synthetic progestins in a biphasic manner: cells complete one round of division and then arrest in G1 phase (7, 30). We wondered how the same receptor/ligand interaction could cause biphasic effects on the cell cycle, and speculated that the different outcomes may segregate according to the functions of PR-B as a MAPK substrate and a regulator of transcription, and as an activator of cytoplasmic kinases. To test this possibility, we compared the cell cycle phase distribution of progestin-treated breast cancer cells expressing wild-type (wt) PR-B with cells expressing the S294A mutant PR-B, in which a MAPK phosphorylation site is eliminated. S294A-PR-B is transcriptionally impaired compared with wt PR-B, as measured by greatly diminished activation of exogenous PRE-driven reporter constructs and endogenous progestin responsive genes (13, 17, 35). The S294A mutant PR-B is resistant to ligand-dependent proteolysis through the ubiquitin pathway (Fig. 1A and Ref. 13), but cells that stably express S294A PR-B activate p42/44 MAPK in response to PR ligands (Fig. 1B), similar to cells containing wt PR-B, as has been reported by others (25). T47D cells stably expressing either wt PR-B or S294A-PR-B were plated and allowed to attach overnight. Before treatment, cells were deprived of serum for 2 d. At t ⫽ 0 h, 21% of cells expressing wt PR-B were in S phase; a slightly higher percentage of S294A-PR-B-expressing cells were in S phase (Fig. 1C). After treatment with the synthetic progestin R5020, cells expressing wt PR-B exhibited the typical biphasic cell cycle profile (7, 30), reaching a maximum of 39% S phase 18 h after treatment. The percentage of cells in S phase then decreased steadily, reaching a nadir of 12% at 48 h, with a corresponding increase in the percentage of cells in G1 phase (not shown). Surprisingly, the changes in cell cycle phase distribution after progestin treatment of breast cancer cells stably expressing the transcriptionally impaired S294A mutant PR-B were qualitatively similar to the biphasic effects observed in cells harboring wt PR-B. However, the peak of the percentage of cells in S phase was reduced and delayed compared with cells expressing wt PR-B: in this experiment, S294A-PR-B-expressing cells reached a maximum of 30% S phase 24 h after treatment. These cells also experienced a greater decrease in proliferation during the growth arrest phase of the progestin response, with only 8% of cells in S phase by 48 h after treatment. Because cells expressing the S294A mutant PR-B had diminished and delayed proliferation in response to progestin, we chose to examine this phase of the progestin response in more detail. Cells expressing wt or S294A PR-B were deprived of steroid hormones by culturing for 2 d in phenol red-free media supplemented with dextran/charcoal-stripped serum, and then treated for 18 h with 10 nM R5020, 100 nM of the PR transcriptional antagonist RU486, the combination

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Skildum et al. • Progestins Cause Proliferation by MAPK Activation

Fig. 1. T47D Breast Cancer Cells Stably Expressing the S294A Mutant PR-B Activate p42/44 MAPK and Exhibit Biphasic Growth Regulation Characteristic of Cells Expressing wt PR-B T47D-YB and T47D-S294A cells were starved in serumfree, phenol red-free media for 2 d and then treated with 10 nM R5020, 100 nM RU486, or ethanol vehicle. A, Cells were harvested 66 h after treatment and cellular lysates were prepared. Total PR and actin were detected by Western blot. B, Cells were harvested 10 min after treatment and levels of total and phosphorylated p42/44 MAPK were detected in lysates by Western blot. C, Cells were harvested at intervals after treatment and their cell cycle phase distribution was determined by DNA staining and flow cytometry. The percent of cells in S phase is indicated; error bars indicate 1 SD (n ⫽ 3).

of both PR ligands, or ethanol vehicle. Cells were then harvested and analyzed for cell cycle phase distribution based on DNA content (Fig. 2A). Deprivation of steroid hormones in the presence of stripped serum consistently resulted in a more robust G1 phase block, with fewer cells in S phase than when cells were cultured in serum-free media, as shown in Fig. 1. As in the previous experiment, both cell lines increased the percentage of cells in S phase after progestin treatment, although cells expressing the S294A mutant had a slightly diminished response compared with wt PR. The progestin effect was partially inhibited by cotreatment with RU486, and RU486 alone induced modest cell cycle progression in both cell lines, as has been reported in cells expressing wt PR-B (3, 7, 36, 37). We then examined the expression of key cell cycleregulatory proteins under the same conditions (Fig. 2B). In cells expressing wt PR-B, progestin treatment

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Fig. 2. T47D Breast Cancer Cells Stably Expressing the S294A PR-B Proliferate in Response to PR Ligands, But Have Altered Expression of Key Cell Cycle-Regulatory Proteins Compared with Cells Containing wt PR-B T47D-YB and T47D-S294A cells were deprived of steroid hormones for 2 d and then treated with 10 nM R5020, 100 nM RU486, both PR ligands, or ethanol vehicle (Et) for 18 h. A, Cells were harvested by trypsinization, fixed in ethanol, and stained with propidium iodide, and their cell cycle phase distribution based on DNA content was measured by flow cytometry. The percent of cells in S phase is shown; error bars indicate 1 SD (n ⫽ 3). B, Cellular lysates were prepared, separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with antibodies that recognize PR, cyclin D, cyclin E, Cdk2, p21, and actin; proteins were detected by chemiluminescence. The 36-kDa cyclin D1 isoform is predominant in these cells. C, T47D-YB and T47D-S294A cells were arrested for 2 d and treated with 10 nM R5020 or vehicle as described. Cells were harvested at intervals from 10 min to 72 h after treatment, and whole-cell lysates were immunoblotted for cyclin D.

caused PR down-regulation, whereas PR was stabilized in the S294A mutant-expressing cells. Progestin caused an increase in cyclin D1, cyclin E, cyclindependent kinase (Cdk)2, and p21 relative to actin controls in cells that harbor wt PR-B, consistent with the roles of these proteins in promoting cell cycle progression (38–41). Whereas the up-regulation of cy-

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clin E, Cdk2, and p21 was blocked by RU486 in cells with wt PR-B, cyclin D1 expression was slightly but consistently increased in RU486-treated cells (see also Fig. 4B). Surprisingly, whereas the changes in cell cycleregulatory proteins roughly mirrored the changes in cell cycle in cells that express wt PR-B, cells that express the S294A mutant appeared to have blunted regulation of these G13 S regulators in response to R5020 treatment. In wtPR-B-expressing cells, R5020 caused a 2.4-fold increase in cyclin D1 protein relative to ethanol controls as measured by densitometric analysis of Western blot bands. In S294A-PR-B-expressing cells, R5020 induced only a 1.4-fold increase. Cyclin E, Cdk2, and p21 levels in S294A PR-B-expressing cells were insensitive to PR ligand treatment. However, basal expression of cyclin D1, cyclin E, and p21 in S294A PR-B-expressing cells were increased relative to cells expressing wtPR-B, approximately equaling the levels observed in R5020treated wt PR-B cells, whereas the two cell lines had similar expressions of actin. For example, basal cyclin D1 levels were approximately 40% higher in S294A-PR-Bexpressing cells relative to cells expressing wtPR-B. Thus, although S294A PR-B-expressing cells progress Fig. 4. The PR Antagonist RU486 Induces Phosphorylation of PR-B and the Expression of Cell Cycle-Regulatory Proteins Independently of Other Hormonal Signals A, T47D-YB cells were treated for 2 d in phenol red-free media containing charcoal-stripped serum and then treated with 10 nM R5020 alone and in combination with 100 nM RU486, ethanol vehicle, or 5% FBS. Cells were harvested at the indicated times after treatment, and total PR, cyclin D, cyclin A, and actin were detected in whole-cell lysates by Western blot. B, T47D-YB cells were starved in serum-free, phenol red-free medium for 2 d, and then treated with 100 nM RU486 alone or in combination with 5% FBS, 20 ng/ml heregulin ␤1 (HRG), or 10 nM R5020 for 18 h. Whole-cell lysates were prepared and immunoblotted with antibodies recognizing total PR, PR phosphorylated on S294, cyclin D, p21, and actin.

Fig. 3. Progestin and Growth Factors Do Not Cooperate to Induce Cell Cycle Progression in Cells Expressing S294A PR-B Relative to Cells Expressing wt PR-B T47D-YB and T47D-S294A cells were arrested for 2 d in serum-free, phenol red-free media and then treated with 10 nM R5020, 10 ng/ml EGF, both hormones, or vehicle for 24 h. A, The cell cycle phase distribution of ethanol-fixed cells was determined by DNA staining and flow cytometry. The percent S phase is shown; error bars indicate 1 SD (n ⫽ 3). The table below the graph shows the P values from two-tailed, unpaired t tests comparing the hormone treatments within each cell line. B, Whole-cell lysates were immunoblotted with antibodies to total PR, cyclin D, p21, and actin.

through the cell cycle in response to PR ligands (Fig. 2A), levels of cell cycle-regulatory proteins are not appreciably changed (Fig. 2B). Cell cycle progression in both cell lines correlated with increased cyclin D1 protein levels. To confirm that cyclin D1 protein is regulated by progestin despite the presence of a transcriptionally impaired mutant PR, cells stably expressing either wild type or S294A PR-B were arrested for 2 d and treated with R5020 or vehicle as described. Cells were harvested at intervals from 10 min to 72 h after treatment, and cyclin D1 protein levels were detected in wholecell lysates by Western blot. Cyclin D1 was induced by R5020 in both cell lines. Although the cyclin D1 induction at 6 h was attenuated in cells expressing the S294A mutant, at 18 h and thereafter, cyclin D1 levels were similar in both cell lines. Interestingly, cyclin D1 protein was sustained above basal levels for 72 h, well into the growth-inhibitory phase of R5020’s biphasic growth pattern (Fig. 1C and Refs. 7 and 30).

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Enhanced Proliferation Induced by EGF and Progestin Requires PR-B Ser294

Cyclin D1 and Cell Cycle Progression Are Induced by Progestin and Antiprogestin

The combination of PR ligands and peptide growth factors synergistically activates wt but not S294A PR-B transcription (17). We wondered whether this transcriptional synergy had consequences on cell growth. To test this idea, T47D cells stably expressing either wt PR-B or S294A PR-B were cultured in serum-free, phenol red-free media for 2 d, and then treated with the synthetic progestin R5020, EGF, both hormones, or vehicle control for 24 h in the absence of serum. Cells were then harvested and their cell cycle phase distribution was determined as described (Fig. 3A). Consistent with the results of the experiments described in Fig. 1, R5020 caused G1 to S progression in both cell lines in the absence of growth factor treatment. Cells stably expressing wt PR-B had more cells in S phase after cotreatment with R5020 and EGF than either treatment alone. In contrast, treatment with both R5020 and EGF did not cause a significant increase in the percentage of S294A-PR-B-expressing cells in S phase relative to either treatment alone. Both cell lines were weakly stimulated by EGF alone. Interestingly, cells stably expressing S294A PR-B underwent slightly increased proliferation when treated with either R5020 or EGF alone relative to cells containing wt PR-B, suggesting that wt PR-B may serve to limit cell cycle progression in the presence of multiple mitogens. We then compared the expression of cell cycleregulatory proteins in similarly treated cells. In cells that express wt PR-B, cyclin D1 expression was induced by R5020, and to a lesser degree by EGF. Consistent with the proliferative response observed in Fig. 3A, cotreatment of wt PR-B-containing cells with R5020 and EGF enhanced cyclin D1 expression, causing a 7.2-fold increase in cyclin D1 protein levels relative to ethanol controls, compared with only a 3.5-fold increase after R5020 treatment alone, as measured by densitometry. In contrast, cells that express S294A PR-B had higher basal levels of cyclin D1 in the vehicle-treated samples (Fig. 2B), and cotreatment with R5020 and EGF did not result in significant elevation of cyclin D1 levels above that induced by R5020 alone (1.7-fold vs. 1.4-fold increase from ethanol-treated controls, respectively). Interestingly, although EGF did cause G1 to S phase transition in S294A cells, cyclin D1 levels were lower in EGF-treated cells than in vehicle-treated controls. Levels of the cyclin-dependent kinase inhibitor p21 were insensitive to the different treatments and may reflect the very high levels of p21 present in serumstarved cells. These data suggest that wt PR-B may function to blunt proliferative responses in the absence of both steroid and growth factor treatment, and that this subtle regulation of growth is lost in the S294A-PR-B mutant.

Because RU486 caused a weak proliferative response in cells expressing either mutant or wt PR-B (Fig. 2) and activated MAPK in both cell lines (Fig. 1B), we wondered whether the transcriptional antagonist could promote cell cycle progression by similar regulatory pathways, i.e. MAPK-induced regulation of cell cycle molecules. To examine the regulation of cyclin D1 as a correlate to cellular proliferation, breast cancer cells that express wt PR-B were arrested for 2 d in phenol red-free media supplemented with stripped serum and then treated with either ethanol vehicle or 10 nM R5020 alone and in combination with 100 nM RU486 in the absence of serum. As a positive control for proliferation, arrested cells were treated with 5% fetal bovine serum (FBS). Cells were harvested at the indicated times after treatment, and PR-B, cyclin D1, cyclin A, and actin were detected in cellular lysates via Western blot (Fig. 4A). R5020 treatment caused downregulation of PR-B, which was partially blocked by cotreatment with RU486. Cyclin D1 levels increased 6 h after R5020 treatment alone and also increased, but to a lesser extent, in the presence of both R5020 and RU486. By 24 h post treatment, cyclin D1 levels were similar in cells treated with agonist alone and agonist plus antagonist, suggesting that the transcriptional activity of PR is not required for cyclin D1 upregulation in response to progestins, or that cyclin D1 is responsive to the partial agonist effects of RU486. In contrast to cyclin D1 regulation by progestins, cyclin A, a marker of progression to S phase (42, 43), increased at 24 h after R5020 treatment, and its expression was largely antagonized by RU486. Treatment of PR-B-expressing cells with FBS caused no detectable shift in electrophoretic mobility of PR-B and only a slight increase in cyclin D1 levels, although cyclin A was induced to levels observed in R5020-treated cells, suggesting that cyclin D1 regulation is highly responsive to treatment with either PR ligand in breast cancer cells. In cells stably expressing wt PR-B that have not been exposed to PR agonists, growth factor treatment alone has only a modest effect on cell cycle entry (44), but growth factors enhance the proliferative effects of R5020 (Fig. 3). To characterize the effects of the PR-B transcriptional antagonist RU486 on cell cycle-regulatory proteins, including cyclin D1, in the presence and absence of other serum factors, breast cancer cells that express wt PR-B were starved for 2 d in phenol red-free, serum-free media, and then treated for 18 h with RU486 alone and in combination with FBS, heregulin-␤1, and R5020. Protein levels were then determined in whole-cell lysates by Western blot (Fig. 4B). RU486 alone and in combination with other treatments caused a characteristic decrease in PR-B electrophoretic mobility consistent with a change in its phosphorylation status (10). The antagonist caused specific phosphorylation of PR-B at the Ser294 MAPK

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site, as has been reported by others (45), consistent with the activation of MAPK by RU486 observed in Fig. 1. FBS and heregulin alone caused a slight increase in phosphorylation at this site. Levels of cyclin D1 and p21 were also increased after RU486 treatment, similar to the levels observed with R5020 treatment at this time point (18 h). PR Ligand-Dependent Proliferation Is MAPK Dependent Because RU486 blocks PR-B transcriptional activity at reporters containing consensus PREs (46–49), we speculated that the effects of both the PR agonist R5020 and the antagonist RU486 on promoting cell cycle progression may be due to PR-dependent activation of cytoplasmic kinases by both these agents (Fig. 1B). MAPK family members may then alter the expression of growth-promoting genes such as cyclin D1 in the absence of direct PR-regulated transcription (Figs. 2 and 4). To test this possibility, we deprived breast cancer cells that express wt PR-B of serum for 2 d in phenol red-free media and then treated the cells with R5020, RU486, both PR ligands, or ethanol vehicle for 18 h in the presence of the p38 MAPK inhibitor SB203580 (SB), the MAPK kinase (MEK)1/2 inhibitor U0126 (U0), or dimethylsulfoxide (DMSO) vehicle (Fig. 5A). Consistent with our previous experiments, in the absence of kinase inhibitors, R5020 treatment caused an increase in the percentage of cells in S phase, which was partially antagonized by RU486, and RU486 caused a small but significant and reproducible increase in the percentage of cells in S phase. Treatment with the p38 inhibitor SB blocked the increase in proliferation caused by RU486 without blocking the ability of R5020 to stimulate S phase entry. In serumfree conditions, the MEK1/2 inhibitor U0 made cells completely refractive to proliferation caused by PR ligands. U0 and SB have no effect on R5020-stimulated PR transcriptional activity at PRE-driven promoters in the absence of added growth factors or activated MAPKs (17). To confirm the specificity of the kinase inhibitors under these serum-free conditions, cells were deprived of serum and treated as described above for 6 h, a time point that corresponds with robust induction of cyclin D1 protein (Fig. 4A). Cells were harvested, and levels of phosphorylated p38 and p42/44 MAPKs and total cyclin D were detected in cellular lysates via Western blot (Fig. 5B). R5020 caused an increase in phosphorylated, active p38 and p42/44 MAPKs 6 h after treatment. Treatment with U0 eliminated phosphorylated p42/44 without blocking the induction of phosphorylated p38, whereas SB blocked the induction of phosphorylated p38 without affecting the activation of p42/44. R5020 treatment caused an increase in cyclin D1 level, and, consistent with the cell cycle progression data in Fig. 5A, this increase was appreciably blocked by inhibition of p42/44 MAPKs and, to a lesser degree, by p38 MAPK inhibition (Fig.

Fig. 5. Proliferation in Response to PR Ligands Is Mediated by Cytoplasmic Kinases A, T47D-YB cells were starved for 2 d in serum-free, phenol red-free medium and then treated with 100 nM RU486, 10 nM R5020, both PR ligands, or ethanol vehicle in the presence of the p38 MAPK inhibitor SB, the p42/44 MAPK inhibitor U0, or DMSO vehicle. Cells were harvested 18 h after treatment, and their cell cycle phase distribution was determined by DNA staining and flow cytometry. The percent S phase is shown; error bars indicate 1 SD. The boxed numbers indicate the P values of unpaired, two-tailed Student’s t tests comparing the percent S phase in ethanol vs. RU486-treated samples. B, Cells were arrested as in Fig. 4A and then treated with R5020 vehicle for 6 h in the presence of SB, U0, or DMSO vehicle. Active, phosphorylated p42/44 and p38 MAPK and total cyclin D1 were detected by Western blot. C, T47D-YB cells were cotransfected with plasmids encoding Renilla luciferase under control of a constitutive promoter and firefly luciferase under control of a 1093-bp fragment of the cyclin D1 promoter. Cells were then starved and treated with 10 nM R5020 or ethanol vehicle in the presence of SB, U0, or DMSO vehicle for 7 h, and luciferase levels in cellular lysates were detected on a luminometer. The average intensity of firefly luciferase relative to Renilla luciferase is shown; error bars indicate 1 SD (n ⫽ 3).

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Skildum et al. • Progestins Cause Proliferation by MAPK Activation

5B). Latent activation of MAPKs by progestins does not occur in PR null T47D cells (data not shown). Cyclin D1 protein levels are regulated at multiple levels including transcription (50), message stability (51, 52), and protein degradation (53, 54). To test whether the synthetic progestin R5020 regulates cyclin D1 at the level of transcription, wt PR-B-expressing cells were transfected with a reporter plasmid encoding firefly luciferase under control of the proximal 1093 bp of the cyclin D1 promoter; to control for transfection efficiency, a second plasmid encoding sea pansy (Renilla) luciferase under control of a constitutive promoter was cotransfected. Cells were then starved in serum-free, phenol red-free media, pretreated with U0 or DMSO vehicle for 30 min, and then treated with R5020 or ethanol vehicle for 7 h. The relative abundance of firefly and sea pansy luciferase was then measured on a luminometer. Consistent with cyclin D1 protein expression, the cyclin D1 promoter was activated in response to R5020 treatment; the normalized firefly luciferase level was 1.7-fold higher in R5020-treated cells relative to ethanol-treated cells (Fig. 5C). Whereas p42/p44 MAPK inhibition by U0126 had no effect on basal activity of the cyclin D1 promoter, U0 blocked the increase in cyclin D1 promoter activity in response to R5020. These experiments reveal a close correlation between cyclin D1 expression and PR ligand-dependent proliferation. Taken together, these data suggest that cell cycle progression caused by RU486 is dependent on the activities of p38 and p42/p44 MAPKs, whereas the more robust proliferation caused by the transcriptional activator R5020 requires p42/p44 but not p38 activities. To confirm our findings that cytoplasmic kinase activation through the extranuclear actions of PR is important for breast cancer cells to progress through G1 to S phase in response to progestins (Fig. 5), we made use of a mutant PR-B (PR-BmPro) described by Boonyaratanakornkit et al. (24) in which an SH3 domaininteracting polyproline region in the PR N terminus is disrupted. Unlike wt PR-B, the mPro mutant does not bind c-Src and fails to induce rapid activation of downstream kinases in response to PR ligands (24). To confirm the expression of functional PR in this model system, serum-starved parental MCF-7 cells and cells that stably express either wt PR-B or PR-BmPro were treated with R5020, RU486, both PR ligands, or ethanol vehicle for 18 h in serum-free and phenol red-free media; R5020 and vehicle-treated T47D-YB cells were included for comparison. Whole-cell lysates were prepared and total PR was detected by Western blotting (Fig. 6A). PR-B was detected in MCF-7 cells engineered to stably express either wt or mPro PR-B, and the PR-B protein exhibited the characteristic shift in electrophoretic mobility consistent with phosphorylation after treatment with PR ligands. However, the level of PR-B protein was much less in the MCF-7 cells relative to the T47D-YB model: the upper and lower panels of Fig. 6A are short (1 min) and long (⬎20 min) exposures of the same blot. Furthermore, both wt and

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Fig. 6. Cells Expressing a Mutant PR Incapable of Binding and Activating Src Kinase Do Not Proliferate in Response to Progestins MCF-7 cells engineered to express wt PR-B or PR-BmPro were plated in parallel with parental MCF-7 cells and cultured for 2 d in serum-free, phenol red-free media. A, Cells were treated with 10 nM R5020, 100 nM RU486, both ligands, or ethanol vehicle for 18 h. Whole-cell lysates were prepared and immunoblotted for total PR and actin. Similarly treated T47D-YB cells were included for comparison. B, Cells were treated with 10 nM R5020 or ethanol vehicle and harvested at intervals after treatment. Cell cycle phase distributions were determined as described. The percentage of cells in S phase is shown; error bars indicate 1 SD (n ⫽ 3). This experiment was repeated with similar results.

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mPro PR-B stably expressed in MCF-7 cells were not completely degraded after 18 h ligand treatment, although the mPro mutant appeared to be somewhat more sensitive than wt PR to ligand-dependent downregulation. PR-B was not detected in parental MCF-7 cells under these conditions. Because our previous experiments suggested that proliferative signaling through the PR depended on the activation of cytoplasmic kinases, we predicted that breast cancer cells that stably express PR-BmPro would have an attenuated proliferative response to progestin treatment relative to cells expressing wt PR-B. MCF-7 cells engineered to stably express either wt PR-B or PR-BmPro were plated in parallel with parental MCF-7 cells and arrested for 2 d in phenol red-free, serum-free media. Cells were then treated with R5020 or ethanol vehicle in the absence of serum, and cells were collected and fixed at intervals after treatment. The cell cycle phase distribution at each time point was determined by flow cytometry as described. The average percent S phase at each time point is shown in Fig. 6B. Parental MCF-7 cells had 30% of cells in S phase at the beginning of the time course and were insensitive to R5020 treatment: the fraction of cells in S phase decreased gradually to about 20% in progestin- and vehicle-treated samples. R5020 treatment of MCF-7 cells expressing wt PR-B caused a characteristic biphasic growth pattern (7, 30), which was qualitatively similar to that observed in T47D cells that express PR-B (see Fig. 1 and Ref. 7). This result indicates that the progestin-induced biphasic proliferative response observed in Fig. 1 is not specific to the T47D-YB clone used, but rather is reflective of PR-specific regulation of cell cycle events (7, 30). However, the magnitude of the early proliferative phase and the later inhibitory phase are attenuated compared with the T47D model, perhaps owing to the lower levels of PR expressed in the MCF-7 system. In contrast to the biphasic growth observed in cells with wt PR-B, cells with PR-BmPro failed to proliferate in response to R5020: approximately 23% of cells were in S phase before treatment, and the percentage in S phase decreased gradually after progestin treatment to 15% at 48 h. At the 18-h time point, only R5020-treated MCF-7 cells that express wtPR-B demonstrated a statistically significant increase in the percentage of cells in S phase relative to ethanoltreated controls (P values from a two-tailed Student’s t test comparing the percentage of cells in S phase between R5020 and ethanol-treated cells were 0.934, 0.00129, and 0.0177 in MCF-7, MCF-7-wtPR-B, and MCF-7-PR-BmPro cells, respectively). Indeed, similar to parental MCF-7 cells, PR-BmPro cells exhibited a gradual growth arrest, characteristic of cells that are dependent on estrogen for proliferation in the absence of alternative mitogenic stimuli such as progestin. We conclude that PR that cannot activate cytoplasmic kinases fail to enter S phase in response to progestin treatment and undergo cell cycle arrest upon prolonged serum deprivation in the absence of steroid

hormones, as do PR-negative cells. Our results suggest that progestin-induced S phase entry (18–24 h after treatment) is mediated by Src-dependent activation of MAPK, whereas the growth-inhibitory phase (⬎48 h after treatment) observed in this system and reported by others (7, 30) is likely facilitated by transcriptional up-regulation of p21 and p27 cell cycle inhibitors (7) or by the activation of additional protein kinases, including Cdk2 (12), phosphatidylinositol 3-kinase/Akt (55), or p38 (26, 56). Exploration of these possibilities awaits a more detailed molecular understanding of PR action at non-PRE-containing promoters and its newly discovered ability to coordinately activate multiple kinase pathways.

DISCUSSION Originally described as ligand-activated transcription factors, recent studies suggest that steroid hormone receptors are much more complex and multifunctional signaling molecules critical for development and implicated in disease. The idea that steroid hormone receptors can translate discrete hormonal signals to coordinated cellular outcomes is consistent with the complex regulation of cell division, remodeling, and death during the various phases of breast development. In this report, we show that the growth-promoting effects of progestins are due to PR’s function as an activator of cytoplasmic kinase cascades rather than its direct activation of transcription. Direct activation of MAPK by steroid hormone receptors provides a mechanism by which cancer cells may receive proliferative signals despite therapeutic blockade of steroid hormone pathways (57, 58). In support of this idea, the overactivation of growth factor signaling pathways correlates with advanced tumor grade and with the acquisition of steroid hormone independence in breast cancer (59–61). Mutation of the consensus MAPK site at Ser294 to a nonphosphorylatable residue results in stabilization of the PR protein and impairs PRE-driven transcription in response to PR ligands and activation of growth factor signaling pathways (13, 17); S294A-PR-B also exhibit greatly reduced expression of endogenous progesterone regulated genes, including c-myc, insulin receptor substrate 1, and p21 (17, 35). Similarly, stable expression of S294A mutant PR-B in breast cancer cells resulted in altered expression of cyclin E and p21 without qualitatively changing the proliferative response of cells to PR ligands, whereas both cell cycle progression and cyclin D1 up-regulation were delayed and somewhat attenuated compared with cells expressing wt PR-B (Figs. 1C, 2, and 3). Furthermore, whereas combined progestin and EGF treatment caused induction of cyclin D1 and enhanced S phase entry in cells containing wt PR-B, S294A PRB-expressing cells had higher basal levels of cyclin D1, and combined treatment with both progestin and EGF

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Skildum et al. • Progestins Cause Proliferation by MAPK Activation

caused no further increase in cyclin D1 levels or cell cycle progression relative to R5020 treatment alone (Fig. 3). These data suggest that phosphorylation of PR-B at Ser294 provides a mechanism for fine tuning of the cellular response to PR ligands via positive feedback or feed-forward, depending on what other hormones are present, thereby maximizing transcriptional responses and increasing the proliferative response in the presence of both steroid hormone and peptide growth factor signaling. PR-B phosphorylation by MAPKs and/or Cdk2 during cell cycle progression may explain, in part, the priming effects of growth factors on PR action (7, 56, 62). We show that EGF treatment alone caused cell cycle progression in T47D-YB cells (Fig. 3), in contrast to previous reports (7, 44) that indicated that EGF caused no proliferation in progesterone-naı¨ve T47D cells. Because PR can function as a node in cytoplasmic kinase cascades independently of ligand, the proliferation we observe after EGF treatment may reflect increased levels of PR present in serum-starved T47D-YB cells, in which PR-B is stably expressed from a constitutive CMV-driven promoter, compared with T47D cells, in which endogenous PR is expressed from its native, estrogen-responsive promoter. Alternatively, the lack of EGF-mediated proliferation observed by others may be due to an estrogenic effect of the pH indicator phenol red present in culture media, which may alter some aspect of EGF-dependent proliferation (63). The experiments described in this work were conducted in phenol red-free media. Interestingly, although RU486 partially blocked the induction of cyclin D1, cyclin E, and p21 when cells containing wt PR-B were treated in stripped serum (Fig. 2B), in the absence of serum RU486 failed to block the increase in cyclin D1 in response to R5020 treatment (Fig. 4B). These data suggest that the transcriptional antagonist is able to signal to downstream cell cycle-regulatory proteins through the PR, and that the output of these signals depends on their hormonal context. Cooperation of PR with multiple hormonal signals suggests a mechanism by which PR can mediate graduated outcomes necessary during the different phases of breast development, when proliferation, differentiation, and death of breast cells must be tightly coordinated. We observed a modest increase in proliferation of wt PR-B expressing cells after treatment with the PR antagonist RU486, as has been reported by others (Fig. 2 and Ref. 36). RU486-induced proliferation was completely blocked by small molecule inhibitors of p38 MAPK and MEK1/2 (Fig. 5). These data suggest that RU486 signals cell cycle entry through activation of kinase cascades, and that both p38 and p42/44 MAPKs are required for RU486-mediated growth. In contrast, proliferation in response to R5020 was insensitive to inhibition of p38 MAPK but remained sensitive to p42/p44 inhibition, suggesting that PR agonists can cause cell cycle progression through ac-

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tivation of p42/p44 MAPKs in the absence of other growth factor inputs. Clearly, progestins can act as mitogens (29), and cyclin D1 is a key integrator of mitogenic signals (64, 65). Interestingly, the developmental phenotype of cyclin D1 knockout mice is similar to PR knockouts: both fail to undergo pregnancy-dependent lobuloalveolar proliferation (66–68), suggesting that these two proteins cooperate to control proliferation during breast development and that corruption of their function could result in the development of malignancy. Cyclin D1 is the regulatory partner of Cdk4 and Cdk6 and serves to integrate growth signals with the machinery of the cell cycle, and cyclin D1 is frequently amplified and/or overexpressed in human malignancies (64, 65). Cyclin D1 protein levels are regulated at multiple levels, including transcription (50, 69), mRNA stability (51, 52), and protein degradation (53, 54). The cyclin D1 promoter lacks canonical steroid response elements, and transcriptional regulation of cyclin D1 in breast cancer cells by steroid hormones depends on Akt and Erk activation (70–72). We find that the synthetic progestin R5020 activates the cyclin D1 promoter in a p42/p44 MAPK-dependent manner (Fig. 5C). Consistent with this idea, PR-BmPro, which is unable to mediate c-Src-dependent MAPK activation, fails to support proliferation in response to progestin (Fig. 6). We were unable to detect changes in cyclin D1 protein or promoter activity in response to PR ligands in these cells (data not shown), perhaps owing to their low levels of PR-B expression compared with the T47D model system (Fig. 6A). However, the uterus provides a precedent for steroid hormones altering cell proliferation by affecting the subcellular localization of cyclin D1 without changes in the overall cyclin D1 protein level (73); progestins may regulate the cell cycle in MCF-7 cells stably expressing low levels of wtPR-B by a similar mechanism. The mPro mutant PR-B is reported to cause a delay in progestin-mediated initiation of Xenopus oocyte germinal vesicle breakdown and inhibition of proliferation of immortalized breast epithelial cells relative to cells expressing wt PR-B (24). Thus, the role of PRinduced MAPK activation may be to coordinate cell cycle progression with global changes in gene expression in hormonally regulated tissues. Our experiments suggest that PR ligands promote proliferation in human breast cancer cells primarily through PR-dependent activation of the p42/p44 MAPK cascade and regulation of cyclin D1. Progestins are reported to both promote and inhibit the growth of breast cancer cells in vitro (7, 29, 30, 32, 74), although cotreatment with peptide growth factors, probably a more accurate reflection of the in vivo environment of breast cells, can change cells’ response to PR ligands, making progestins highly proliferative agents (56, 62). Thus, PR may be an important but underappreciated therapeutic target in the treatment of breast cancer (75). The recent data from clinical trials examining hormone replacement therapy

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showing that combined estrogen plus progestin increased breast cancer risk relative to estrogen alone provide support for the need to pursue the development of PR antagonists for use in the treatment of hormone-sensitive breast cancer. However, the PR antagonist RU486 may be a less desirable therapeutic, because it activates MAPK cascades, ultimately inducing cyclin D1 expression and increased proliferation. Our data suggest that a hypothetical drug that specifically blocks PR’s activation of kinases may be more effective than existing PR ligands that are designed to primarily interfere with PR’s role as a transcription factor. Alternatively, combination therapies that include PR and ER antagonists in combination with existing potent MEK inhibitors may hold clinical promise.

were resuspended in 0.4 ml fluorescence-activated cell sorting buffer (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4, 14.7 mM KH2PO4, 1 mg/ml ribonuclease A, 0.5 mM EDTA, 0.1% Triton X-100, 0.2 mg/ml propidium iodide, and 10% FBS), pipetted several times to ensure a uniform single-cell suspension, and transferred into a polystyrene tube fitted with a filter cap (Falcon no. 35–2235, Becton Dickinson and Co., Franklin Lakes, NJ). Cells were incubated at 4 C in the dark for 30 min before analysis. Cells were analyzed on a fluorescence-activated cell sorting Caliber flow cytometer (Becton Dickinson), and data were collected using CellQuest Pro software (Becton Dickinson). Cells were gated on forward and side scatter to eliminate debris and on the width vs. area of the red fluorescent voltage pulse to eliminate cell aggregates. The area of the red fluorescence voltage pulse for the gated cells is proportional to its DNA content, and the cell cycle profile for each sample was estimated using ModFit LT software (Verity Software House, Topsham, ME). A minimum of 10,000 gated cells were analyzed for each sample, and triplicate parallel cultures were analyzed for each treatment.

MATERIALS AND METHODS

Western Blotting

Cell Lines and Culture Conditions

Cells were plated (250,000 per 100 mm dish) and treated as described above. Cells were harvested by scraping into icecold PBS and pelleted by centrifugation, and all but a thin meniscus of PBS was aspirated from atop the cell pellet. Cell pellets were then stored over liquid nitrogen until analysis. On the day of analysis, cell pellets were thawed over ice and then suspended in lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 1 mM EDTA; 2.5 mM EGTA, pH 8.0; 0.1% Tween-20; 10% glycerol; 1 mM dithiothreitol; 10 mM ␤-glycerophosphate; 1 mM NaF; 1 mM NaVO4; 0.1 mM phenylmethylsulfonyl fluoride; 10 ␮g/ml leupeptin; 2 ␮g/ml aprotinin). Lysates were pipetted to mix and incubated on ice for 30 min. Lysates were then cleared by centrifugation at 10,000 ⫻ g for 7 min, and supernatants were transferred to clean tubes. The protein concentration of each sample was determined by performing a Bradford assay. Samples were diluted with lysis buffer to equalize protein concentrations, and then boiled in sodium dodecyl sulfate with dithiothreitol. Lysate equivalent to 30 ␮g of protein was separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). Membranes were probed with primary antibodies recognizing cyclin D (06–137, Upstate Biotechnology, Inc., Lake Placid, NY), cyclin E (M20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Cdk2 (M2, Santa Cruz Biotechnology), total PR (Ab-8 cocktail, NeoMarkers), PR phosphorylated on Ser294 (Ab12, NeoMarkers), p21waf1 (c19, Santa Cruz Biotechnology), total p38 (9212, Cell Signaling Technology, Beverly, MA), p38 phosphorylated on Thr180 and Tyr182 (9211S, Cell Signaling Technology), total p42/p44 MAPK (9102, Cell Signaling Technology), p42/p44 phosphorylated on Thr202 and Tyr204 (9101S, Cell Signaling Technology), and actin (AC40, Sigma). Secondary antibodies conjugated to horseradish peroxidase (Amersham Life Sciences, Arlington Heights, IL) and chemiluminescent detection (SuperSignal West Pico, Pierce Chemical Co., Rockford, IL) were used to visualize target proteins. Although the Upstate Biotechnology antibody recognizes all cyclin D isoforms, the 36-kDa cyclin D1 isoform is predominant in T47D and MCF-7 breast cancer cells. Densitometric quantitation of immunoreactive bands normalized to smooth muscle actin was carried out using FluorChem 2.0 software (Alpha Innotech Corp., San Leandro, CA).

T47D-YB and T47D-S294A cells have been described previously (13, 76) and were routinely cultured in MEM (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (BioFluids, Inc., Rockville, MD), penicillin and streptomycin, insulin, nonessential amino acids, and G418. MCF-7-wt PR and MCF-7-PRmPro have been previously described (24); they were cultured in incomplete MEM (BioFluids, Inc.) with 5% FBS, penicillin and streptomycin (Life Technologies), and 0.5 mg/ml zeocin (Invitrogen, San Diego, CA); parental MCF-7 cells were cultured in identical media lacking zeocin. All cells were cultured in a humidified incubator at 37 C and 5% CO2 and split approximately every 4 d to maintain subconfluency. Hormone Treatments For all experiments, cells were plated in their respective growth media and allowed to attach overnight. Media were then removed, cells were washed twice with PBS, and phenol red-free media, with or without charcoal/dextran stripped serum (HyClone Laboratories, Inc., Logan, UT), were added. Cells were maintained in steroid hormone-free conditions for 2 d, and then cells were washed with PBS and media were replaced with phenol red-free media, with or without charcoal/dextran stripped serum, containing either 10 nM R5020 (Sigma Chemical Co., St. Louis, MO), 100 nM RU486 (Sigma), both PR ligands, or ethanol vehicle. Cells were harvested at the indicated times after treatment. In the experiments shown in Fig. 5, the MEK inhibitor U0126 and the p38 MAPK inhibitor SB20358 (both from Calbiochem, La Jolla, CA) were added to cells at 20 nM 0.5 h before treatment with PR ligands. Cell Cycle Analysis Cells (100,000 per well) were plated in six-well plates and deprived of steroid hormones for 2 d and then treated as described above. Cells were harvested by trypsinization, pelleted by centrifugation, and washed once in PBS; media and washes were retained with the adherent cells. The cells were resuspended in 1 ml PBS containing 10% FBS and pipetted several times to ensure a uniform single-cell suspension. Ice-cold 80% ethanol (10 ml) was then added to the cell suspensions. Samples were stored at ⫺20 C until the day of analysis. On the day of analysis, samples were pelleted by centrifugation and washed once with cold PBS. The cell pellets

Cyclin D1 Luciferase Assay To study the regulation of cyclin D1 transcription by progestin, cells were plated and allowed to attach overnight and then transfected with plasmids encoding firefly luciferase

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Skildum et al. • Progestins Cause Proliferation by MAPK Activation

under the control of a 1093-bp fragment of the human cyclin D1 promoter (plasmid kindly provided by Bob Sclafani) and sea pansy luciferase under control of a constitutive promoter; plasmids were introduced with the FuGene 6 reagent (Roche Clinical Laboratories, Indianapolis, IN). Cells were starved 1 d after transfection in serum-free, phenol red-free media overnight, and then cells were treated with 10 nM R5020 or ethanol vehicle with either 20 nM U0126 or DMSO vehicle for 7 h. Cells were then harvested, and lysates were analyzed for firefly and sea pansy luciferase using the Dual Luciferase System (Promega Corp.) according to the manufacturer’s directions.

Mol Endocrinol, February 2005, 19(2):327–339 337

9.

10.

Acknowledgments

11.

We thank Kathryn B. Horwitz [University of Colorado Health Sciences Center (UCHSC)] for providing T47D breast cancer cells stably expressing the wt B-isoform of PR (T47D-YB cells); Robert Sclafani (UCHSC) for the cyclin D1 promoter-luciferase reporter plasmids. We also thank Drs. Dean Edwards and Viroj Boonyaratanakornkit (UCHSC) for providing MCF-7 cells stably expressing wt and mPro mutant PR-B and for helpful discussions.

12.

13. Received July 29, 2004. Accepted October 5, 2004. Address all correspondence and requests for reprints to: Carol A. Lange, Departments of Medicine (Division of Hematology, Oncology, and Transplantation) and Pharmacology, University of Minnesota Cancer Center, MMC 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455. E-mail: [email protected]. This work was supported by National Institutes of Health Grant R01-DK053825 (to C.A.L.).

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