Hypoxia Induces Proteasome-Dependent Degradation of Estrogen Receptor {alpha} in ZR-75 Breast Cancer Cells

Matthew Stoner, Bradley Saville, Mark Wormke, Dana Dean, Robert Burghardt and Stephen Safe

Department of Veterinary Physiology and Pharmacology (M.S., M.W., S.S.), Department of Biochemistry and Biophysics (B.S.), and Department of Veterinary Anatomy and Public Health (D.D., R.B.), Texas A&M University, College Station, Texas 77843-4466

Address all correspondence and requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of estrogen receptor {alpha} (ER{alpha}) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O2) or hypoxia (1% O2 or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1{alpha} protein within 3 h after treatment, whereas ER{alpha} protein levels were dramatically decreased within 6–12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ER{alpha} mRNA; however, hypoxic conditions decreased basal and 17ß-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17ß-estradiol and hypoxia induce proteasome-dependent degradation of ER{alpha}, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HYPOXIA OR DECREASED oxygen levels occurs during neovascularization in diverse tissues, and hypoxic conditions in tumors are associated with their growth and metastasis (1, 2, 3, 4, 5, 6, 7). Cellular adaptation to hypoxia results in activation of genes involved in glucose transport and metabolism, angiogenesis and erythropoiesis, and down-regulation of fatty acid ß-oxidation pathways (1, 2, 3, 4, 5, 6, 7). Modulation of hypoxia-induced genes is regulated, in part, through differential expression of transcription factors such as hypoxia-inducible factor 1{alpha} (HIF1{alpha}), which is typically elevated in most hypoxic tissues/tumors, and several genes that are up-regulated in low oxygen conditions contain hypoxia response elements (HREs) in their respective promoters (5, 6, 8, 9, 10). Vascular endothelial growth factor (VEGF), which is also elevated under hypoxic conditions, plays a critical role in angiogenic processes that require new blood vessels in response to wound healing, ischemia, and tumor growth (11, 12, 13, 14, 15, 16). VEGF and other angiogenic factors are expressed in multiple tumor types (17, 18, 19, 20, 21, 22, 23, 24), and development of drugs and therapies that inhibit angiogenesis are promising new technologies for treating various cancers (25, 26, 27, 28, 29, 30, 31). VEGF expression is increased in many tumors and is used as a prognostic factor for several cancers (17, 18, 19, 20, 21, 22, 23, 24). For example, increased levels of VEGF are a negative prognostic factor for survival of women with breast cancer (24, 32, 33), and both hypoxia and hormonal stimulation with 17ß-estradiol (E2) increase VEGF expression in some breast cancer cell lines (34, 35, 36).

HIF1{alpha} is also detected in many tumor types, whereas the corresponding levels of this protein in normal tissues are low to nondetectable (5, 6). For example, one study reported a linear correlation between HIF1{alpha} expression and the relative vascularity of a series of brain tumors (37). For a series of mammary tumor specimens that were classified histopathologically, levels of HIF1{alpha} increased with increasing pathological stage of the tumor and were directly associated with more aggressive tumors and lower survival rates (24). Increased HIF1{alpha} levels in mammary tumors also correlated with increased VEGF and estrogen receptor {alpha} (ER{alpha}) expression (24). ER{alpha}-positive tumors are responsive to endocrine therapy and are associated with increased patient survival compared with women with more aggressive ER-negative tumors (38), and it is paradoxical that ER{alpha} levels also correlated with negative prognostic factors such as VEGF and HIF1{alpha}. The VEGF/ER{alpha} correlations could be related, in part, to the hormone inducibility of VEGF in some breast cancer cell lines; however, causal linkages between HIF1{alpha} and ER{alpha} expression have not been reported.

Recent studies in this laboratory have shown that E2 induces VEGF gene expression in ER-positive ZR-75 cells (39), and we have used this cell line to investigate the effects of growth under low oxygen conditions (1%) or cobaltous chloride on HIF1{alpha} and ER{alpha} protein levels and on hormone-dependent transactivation. Results of this study show that ZR-75 cells grown under normoxic conditions exhibit relatively high levels of ER{alpha} and low levels of HIF1{alpha} proteins, whereas expression levels of these two proteins are reversed in cells grown under hypoxia in 1% oxygen or 500 µM cobaltous chloride. Hypoxic conditions do not affect ER{alpha} mRNA levels, and studies with protease inhibitors show that degradation of ER{alpha} protein is proteasome-dependent and this is accompanied by decreased basal and hormone-induced transactivation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of CoCl2 on HIF1{alpha} Protein Levels in ZR-75 Cells
The effects of hypoxia on HIF1{alpha} and ER{alpha} protein levels were investigated in ZR-75 cells grown under different concentrations of cobaltous chloride (100, 500, and 1000 µM) for 24 h; CoCl2 is routinely used to simulate hypoxic conditions in cancer cell lines (40, 41). In cells treated with dimethylsulfoxide (DMSO) or 10 nM E2 cotreated with 100, 500, or 1000 µM CoCl2, there was a significant increase in HIF1{alpha} protein; in contrast, 500 or 1000 µM CoCl2 caused a decrease in immunoreactive ER{alpha} protein, whereas significant changes in ER{alpha} were not observed in cells treated with 100 µM CoCl2 (Fig. 1AGo). Sp1 protein levels were also decreased by 500 and 1000 µM CoCl2 in cells treated with DMSO or E2, whereas HIF1ß protein levels were decreased only in cells grown in 1000 µM CoCl2 (data not shown). These results suggest that 500 µM CoCl2 was optimal for inducing hypoxic responses in ZR-75 cells, and this concentration was used in subsequent studies. The results illustrated in Fig. 1BGo show the time-dependent effects of 500 µM CoCl2 on immunoreactive ER{alpha}, HIF1{alpha}, and Sp1 protein levels in ZR-75 cells. Decreased ER{alpha} and Sp1 levels were observed between 3 and 16 h after treatment with CoCl2, whereas HIF1{alpha} was induced between 1 and 3 h, indicating a different temporal pattern of CoCl2-mediated effects on ER{alpha}, HIF1{alpha}, and Sp1 protein. In subsequent experiments, CoCl2 treatment decreased ER{alpha} protein levels at 6 h and maximally decreased levels after 12 h (data not shown).



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Figure 1. Western Blot Analysis

A, Effects of chemically induced hypoxia on HIF1{alpha}, ER{alpha}, and Sp1 proteins in ZR-75 breast cancer cells. Cells were plated overnight in phenol red-free medium supplemented with 2.5% CSS, and then treated with 0, 100, 500, or 1000 µM CoCl2 in the presence of DMSO or 10 nM E2 for 24 h. Duplicate aliquots of each treatment group are shown. B, Time course modulation of HIF1{alpha} and ER{alpha} protein levels by CoCl2. ZR-75 cells were grown in phenol red-free medium supplemented with 2.5% CSS and incubated in 21% O2, with or without 500 µM CoCl2 for the indicated times. Duplicate determinations for each treatment group are shown.

 
The effects of CoCl2 on transcription factors were also determined in gel mobility shift assays using nuclear extracts from ZR-75 cells. Nuclear extracts did not consistently form a specifically bound retarded band using a consensus [32P]HRE; however, binding studies of nuclear extract with consensus [32P]ERE (estrogen response element) or [32P]Sp1 (GC-rich) oligonucleotides gave specifically bound retarded bands. Nuclear extracts from cells treated with DMSO or E2 formed ER{alpha}-ERE complexes (lanes 2 and 3), and CoCl2 decreased intensity of these bands (lanes 4 and 5, Fig. 2AGo). In addition, retarded band intensity was decreased after competition with excess unlabeled ERE (lane 6) and supershifted with ER{alpha} antibodies (lane 7), but not IgG (lane 8). Decreased binding of nuclear extracts from hypoxia-induced cells reflected the lower levels of ER{alpha} protein (Fig. 1Go); however, although E2 also decreases ER{alpha} levels compared with controls (DMSO), the retarded band intensity was not decreased. Retarded band intensities observed after incubation of [32P]Sp1 with nuclear extracts from cells treated with DMSO (lane 2), E2 (lane 3), DMSO + CoCl2 (lane 4), E2 + CoCl2 (lane 5), or DMSO + IgG (lane 9) were all similar. Sp1- and Sp3-DNA complexes (as indicated) were supershifted by Sp1 (lane 7) and Sp3 (lane 8) antibodies as previously described, and the retarded band intensity was decreased after coincubation with excess unlabeled Sp1 oligonucleotide (lane 6, Fig. 2BGo). These results suggest that the slightly decreased levels of immunoreactive Sp1 protein in whole-cell extracts after treatment with CoCl2 did not affect binding of nuclear extracts to [32P]Sp1, in contrast to the decreased DNA binding of ER{alpha} in these same extracts. CoCl2 alone added to nuclear extracts did not affect ER{alpha} or Sp1 binding to radiolabeled [32P]ERE or [32P]Sp1, respectively.



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Figure 2. EMSA

A, Nuclear extracts of DMSO- and 10 nM E2-treated ZR-75 cells cultured for 24 h under 21% O2, in the absence or presence of 500 µM CoCl2, were obtained as described in Materials and Methods. ER{alpha} forms specific complexes with [32P]ERE under normoxic conditions (lanes 2 and 3) but not under hypoxic conditions (lanes 4 and 5). Excess unlabeled ERE oligonucleotide competes the ERE band (lane 6) and the ER{alpha}-DNA complex is specifically supershifted (S.S.) with monoclonal antibody against ER{alpha} (lane 7); negative control, normal mouse IgG, does not have an effect (lane 8). B, The same extracts used for the ERE EMSA in Fig. 1AGo were used in Sp1/Sp3-DNA EMSA. Nuclear extracts from both DMSO- and 10 nM E2-treated ZR-75 cells bound [32P]Sp1 with similar affinity (lanes 2–5). Excess unlabeled Sp1 oligonucleotide depleted all bands (lane 6). Antibodies against Sp1 and Sp3 proteins specifically shifted Sp1-DNA and Sp3-DNA complexes (lanes 7 and 8, respectively). Normal rabbit IgG did not affect shifted band patterns (lane 9).

 
Mechanism of Hypoxia-Induced Degradation of ER{alpha} Protein
The effects of DMSO (Fig. 3AGo) or 10 nM E2 on ER{alpha} mRNA levels were determined in ZR-75 cells grown under conditions of normoxia (21% O2), hypoxia (1% O2), or 500 µM CoCl2 for 24 h. These experiments were carried out in quadruplicate, and the results showed that ER{alpha} mRNA levels were unchanged in all treatment groups; similar results were observed for 28S RNA (Fig. 3AGo). In contrast, hypoxic conditions (CoCl2 and 1% O2) significantly up-regulated GAPDH mRNA levels, and this corresponded to induced GADPH mRNA levels observed in rat alveolar epithelial cells grown under hypoxic conditions (42). These results suggest that hypoxia-induced down-regulation of ER{alpha} protein was not related to altered transcriptional regulation.



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Figure 3. Effects of Hypoxia on ER{alpha} mRNA and Protein Levels

A, Northern blot analysis. ZR-75 cells were seeded in phenol red-free medium supplemented with 2.5% CSS, cotreated for 24 h with DMSO or E2 in the presence of 21% O2, 500 µM CoCl2, or 1% O2. Total RNA (7 µg) was transferred to a nylon membrane and probed for ER{alpha} mRNA, and then stripped and reprobed for GAPDH mRNA (control for hypoxic induction of gene expression) and 28S rRNA (RNA loading control). Representative experiments are shown and data are expressed as mean ± SE for quadruplicate determinations in each treatment group. Statistically significant differences (P < 0.05) relative to the 21% O2-DMSO treatment group are indicated (*). B, Western blot analysis of effects of proteasome and protease inhibitors on hypoxia-induced degradation of ER{alpha} protein. Cotreatments were for 24 h with DMSO or 10 nM E2, in the presence of 21% O2, 500 µM CoCl2, or 1% O2, with or without proteasome inhibitor MG132 (10 µM) or protease inhibitor calpain inhibitor II (CAL2) (10 µM). Vehicle for inhibitors was DMSO. Levels of Sp1 protein are indicated as a loading control. Statistically significant decreases (P < 0.05) relative to 21% O2 treatment with DMSO (*) or relative to 21% O2 treatment with E2 (**) (21% O2 and 500 µM CoCl2, n = 2; 1% O2, n = 1) are indicated. Similar effects of MG132 and CAL2 were observed in cells grown under hypoxia for shorter time periods (data not shown). C, ER{alpha} pulse chase. Pulse chase was performed as described in Materials and Methods. ZR-75 cells were metabolically labeled with [35S]methionine for 2 h and chased with phenol red-free medium (without serum) for the indicated times in the presence of DMSO, 500 µM CoCl2, or 10 nM E2. [35S]ER{alpha} was concentrated by immunoprecipitation onto protein A/G-conjugated agarose beads using a monoclonal antibody that recognizes ER{alpha}. Immunoprecipitates were collected by centrifugation, washed extensively in 1x PBS, boiled, and analyzed by SDS-PAGE. [35S]ER{alpha} levels are graphed as a percent of the zero time point intensity value. (* denotes nonspecific bands.)

 
We therefore further investigated the effects of hypoxia (1% O2 or 500 µM CoCl2) on expression of ER{alpha} protein (Fig. 3Go, B and C) and have also included results obtained for HIF1{alpha} protein in the same experiments. The data obtained for the comparative effects of normoxia and hypoxia (CoCl2) on ER{alpha} were determined in duplicate, and significant (P < 0.05) decreases related to the 21% O2 (normoxia) treatment with DMSO (*) or E2 (**) are indicated. Results obtained using cells grown under 1% O2 (Fig. 3CGo) were comparable to CoCl2-induced responses illustrated in Fig. 3BGo (500 µM CoCl2). In cells grown under normoxia, HIF1{alpha} protein levels are low due to highly active proteasome degradation of this protein, and this was observed in ZR-75 cells cultured in 21% O2 (Fig. 3BGo) and treated with DMSO (lane 1), 10 µM of the protease inhibitor, calpain inhibitor II (CAL2, lane 3), 10 nM E2 (lane 4), or 10 nM E2 plus CAL2 (lane 6) for 24 h. However, after treatment with the proteasome inhibitor MG132 (10 µM) plus DMSO (lane 2) or E2 (lane 5), there was an increase in HIF1{alpha} protein, and this was consistent with inhibition of ongoing proteasome degradation of HIF1{alpha} in cells grown under normoxia. In contrast, under normoxic conditions, ER{alpha} protein levels were unchanged in ZR-75 cells treated with DMSO alone or in the presence of MG132 or CAL2 (lanes 1–3); treatment with E2 decreased ER{alpha} levels (lane 4), and this was also observed after cotreatment with E2 plus CAL2, whereas MG132 restored levels of ER{alpha} protein (lane 5). The results are consistent with previous reports on E2-dependent degradation of ER{alpha} through activation of proteasomes (43, 44, 45, 46).

The results in Fig. 3BGo further compare the effects of 500 µM CoCl2 (lanes 7–12) and 1% O2 (lanes 13–18) on HIF1{alpha} and ER{alpha} protein in cells treated with DMSO or E2; an identical pattern of responses was observed in cells grown under both CoCl2 and oxygen-deprived conditions. HIF1{alpha} protein levels were increased in all treatment groups in cells grown in CoCl2 or under 1% O2 (lanes 7–18), and MG132 further enhanced HIF1{alpha} protein levels (lanes 8, 11, 14, and 17). Sp1 protein levels did not change with treatment. This indicates that hypoxia/CoCl2-induced HIF1{alpha} protein is still regulated, in part, by ongoing proteasome degradation. The results also show that hormonal treatment does not affect levels of HIF1{alpha} protein in ZR-75 cells. In cells treated with DMSO (lanes 7–9 and 13–15), it was apparent that hypoxia/CoCl2 caused a decrease in ER{alpha} protein, and this was reversed by the proteasome inhibitor MG132 (lanes 8 and 14) but not by the protease inhibitor CAL2 (lanes 9 and 15). A similar pattern was observed in hormone-treated cells in which both E2 and CoCl2/hypoxia induced proteasome-dependent degradation of ER{alpha} (lanes 10 and 16), which was inhibited by MG132 (lanes 11 and 17) but not by CAL2 (lanes 12 and 18). These data demonstrate that hypoxia activates proteasome-dependent degradation of ER{alpha} protein in ZR-75 cells, whereas levels of HIF1{alpha} protein are increased. In contrast, E2 activated proteasome-dependent degradation of ER{alpha} in ZR-75 cells under conditions of normoxia and hypoxia but did not affect HIF1{alpha} protein under these same conditions.

The results in Fig. 3CGo summarize a pulse-chase experiment using [35S]ER{alpha} in ZR-75 cells treated with DMSO (control), 10 nM E2, or 500 µM CoCl2 for 1 and 3 h. Degradation of ER{alpha} in control or CoCl2-treated cells was similar after 1 h; however, after 3 h, degradation of [35S]ER{alpha} by hypoxia was greater than observed in control cells. In contrast, more than 50% of [35S]ER{alpha} was degraded in ZR-75 cells treated with E2, and this was consistent with hormone activation of the proteasome pathway as previously reported (43, 44, 45, 46, 47). In a separate study, CoCl2-induced degradation of [35S]ER{alpha} over a period of 1 or 4 h was completely reversed by the proteasome inhibitor MG132 (data not shown). Thus, hypoxia induces the rate of [35S]ER{alpha} degradation by activation of proteasomes; however, this does not exclude an effect of CoCl2 on ER{alpha} protein stability.

The effects of hypoxia on transactivation of hormone-responsive genes/reporter genes was also investigated in ZR-75 cells. pS2 gene expression is induced by E2 in MCF-7 cells; however, the results in Fig. 4AGo show that basal pS2 mRNA levels were relatively high in ZR-75 cells, and treatment with 10 nM E2 for 24 h significantly increased transcript levels (Fig. 4AGo). However, in ZR-75 cells grown in 500 µM CoCl2, there was a decrease in pS2 mRNA levels in DMSO- and E2-treated groups. These results are in direct contrast to the effects of hypoxia (CoCl2 or 1% O2) on ER{alpha} mRNA, GADPH mRNA, or 28S rRNA, which were either unchanged or increased (Fig. 3AGo). ZR-75 cells were transfected with p3xERELuc, and hormone inducibility and the effects of hypoxia were determined in the absence or presence of cotransfected ER{alpha} expression plasmid (47). The results in Fig. 4BGo show that E2 increased luciferase activity under normoxic conditions; however, there was a more than 80% decrease in luciferase activity in cells treated with DMSO or 10 nM E2 and grown in 500 µM CoCl2. In a parallel experiment, cells were maintained in 21% O2 and transfected with p3xERELuc and 5 ng ER{alpha} expression plasmid, and treatment with 10 nM E2 induced a 7.7-fold increase in luciferase activity. Hypoxic conditions decreased luciferase activity in cells treated with DMSO and E2 but did not decrease E2 inducibility. Hypoxia did not affect transactivation in ZR-75 cells transfected with pVEGF6, a construct containing a GC-rich proximal region of the VEGF gene promoter (Fig. 4CGo) (39). In contrast, hypoxia increased activity in cells transfected with pVEGF1, which contains a hypoxia response element in the distal region of the VEGF promoter. These results suggest that hypoxia-induced down-regulation of ER{alpha} decreases both basal and inducible expression of an E2-responsive gene (pS2) and ERE-dependent reporter gene; however, E2 responsiveness (e.g. fold-induction) is retained. In contrast, transactivation was not affected in cells transfected with a GC-rich construct (pVEGF6) (Fig. 4CGo).



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Figure 4. Northern Blot Analysis

A, Effect of hypoxia on a classical estrogen-induced gene, PS2. Total RNA (15 µg) was obtained from ZR-75 cells that were first seeded for 1 d in phenol red-free medium with 2.5% CSS, 1 d in medium without serum, and then treated for 24 h with DMSO or 10 nM E2 in the presence of 21% O2 or 500 µM CoCl2. The membrane was probed for pS2 gene expression and then stripped and reprobed for 28S rRNA to verify even loading. Results are representative of triplicate determinations for each treatment group. B, Transient transfection assay. Effect of hypoxia on p(ERE)3-Luc in ZR-75 cells. Cells in 12-well plates were transfected with 500 ng of p(ERE)3-Luc (plasmid containing three tandem copies of a consensus ERE upstream of the luciferase gene) and 250 ng ß-galactosidase expression plasmid for normalization of transfection efficiency, with or without cotransfected pCDNA3.1-hER{alpha} expression plasmid (5 ng). Cells were transfected for 6 h and exposed to 21% O2 (normoxia) or 500 µM CoCl2 (hypoxia) for 20–24 h before cotreatment with DMSO or 10 nM E2 for an additional 12–24 h. Luciferase activity is normalized to ß-galactosidase activity. C, VEGF gene promoter-luciferase transient transfection. ZR-75 cells were transiently transfected with 500 ng pVEGF1 or pVEGF6, 250 ng ER{alpha} expression plasmid, and 250 ng ß-galactosidase expression plasmid and placed in normoxia (21% O2) or hypoxia (1% O2) for 24–48 h. Results are expressed as mean ± SE [*, P < 0.05 (n = 3)]. A similar profile was observed for activities that were not normalized for ß-galactosidase.

 
Figure 5Go illustrates immunostaining of ER{alpha} in ZR-75 cells grown under normoxic and hypoxic conditions. Under normoxia, ER{alpha} staining in untreated cells was intensely nuclear with a diffuse cytoplasmic signal (Fig. 5CGo), whereas after treatment with 10 nM E2, there was a decrease in nuclear ER{alpha} protein (Fig. 5DGo). Cells grown under CoCl2 in the absence (Fig. 5EGo) or presence of E2 (Fig. 5FGo) exhibited lower nuclear staining of ER{alpha} protein, and this is consistent with the Western blots (Fig. 3BGo) demonstrating that hypoxia induces ER{alpha} protein degradation. Results obtained with different antibodies recognizing the hinge region of ER{alpha} also indicated that CoCl2 induced degradation of nuclear ER{alpha} protein; however, staining of membrane-associated ER{alpha} was essentially unchanged by hypoxia (data not shown). In ZR-75 cells stained only with the secondary antibody (Fig. 5Go, G–J), there was a low-intensity background, nonspecific cytoplasmic staining in all treatment groups. Thus, immunostaining and Western blot analysis of ZR-75 cell extracts are consistent with hypoxia-induced degradation of nuclear ER{alpha} protein.



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Figure 5. Immunofluorescence of ER{alpha} Protein in ZR-75 Cells

Cells were seeded on glass slides and treated for 24 h, and ER{alpha} protein was detected as described in Materials and Methods. A, 21% O2 and DMSO (phase contrast, normoxia control); B, 21% O2 and 10 nM E2 (phase contrast); C, 21% O2 and DMSO (hypoxia control); D, 21% O2 and 10 nM E2; E, CoCl2 and DMSO; F, CoCl2 and 10 nM E2. Panels G–J are respective negative controls for each treatment group in which the primary antibody was omitted and only the secondary antibody was used. Note: Background staining with the secondary antibody is nonnuclear and accounts for some of the nonspecific cytosolic staining in panels C–F. Field width of each panel is 240 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell and tissue functions maintained under hypoxic conditions are accompanied by decreased oxidative metabolism and an increase in pathways that have reduced oxygen requirements (1, 2, 3). Cellular adaptation to hypoxic conditions is accompanied by extensive modulation of gene expression and up-regulation of genes that are important for angiogenesis, erythropoiesis, and glycolysis. Many genes that are critical for hypoxia-induced responses are regulated by the HIF1({alpha} and ß) complex through interactions with HREs in their respective 5'-promoter regions (4, 5, 6, 8, 9, 10). HIF1ß (or Arnt) is highly expressed in most cell types; comparable levels of HIF1ß are observed in cells grown under hypoxia or normoxia, and this was also observed in ZR-75 cells (data not shown). In contrast, growth of cells (including ZR-75 cells) under hypoxia results in a rapid increase in HIF1{alpha} protein, and this is due to multiple factors including decreased ubiquitination of HIF1{alpha} and decreased proteasome-dependent degradation of this protein (9). A recent study also reported that hypoxia-induced HIF1{alpha} gene protein expression in Hep3B cells is also dependent on induction of a small GTPase Rac1 (48), and hypoxia activates and deactivates other kinase pathways and their downstream nuclear transcription factors (49, 50, 51, 52, 53, 54). Discher et al. (55) also reported that hypoxia-induced ß-enolase and pyruvate kinase M in myocytes was HIF1{alpha} independent and due to increased Sp1/Sp3 ratios related to increased degradation of Sp3 (but not Sp1) protein under hypoxic conditions.

Research in this laboratory has been focused on mechanisms of inhibitory cross-talk between the aryl hydrocarbon receptor (AhR) and ER{alpha}-mediated signaling pathways in breast cancer cells (56, 57, 58, 59, 60, 61, 62, 63, 64, 65). In addition, we are also developing selective AhR modulators (SAhRMs) for treatment of breast cancer (56, 57), and SAhRMs induce proteasome-dependent degradation of both AhR and ER{alpha} proteins in breast cancer cells (43). The effects of hypoxia on ER{alpha}-mediated signaling in breast cancer cells have not previously been investigated; however, it was recently reported that both E2 and hypoxia induced VEGF expression in MCF-7 cells, and a combination of E2 plus hypoxia on VEGF protein levels (whole-cell extracts) was approximately additive (34). Hypoxia also enhanced transcriptional activation and protein stability of the urokinase plasminogen activator receptor in MCF-7 cells, whereas rpL32, a ribosomal protein, was unaffected (54).

ZR-75 cells are E2 responsive and, in this study, we have investigated the effects of hypoxia (1% O2 or different concentrations of CoCl2) on ER{alpha} protein/mRNA levels and E2 responsiveness in transactivation assays. The results show that hypoxia induces a time-dependent decrease in ER{alpha} protein (Fig. 1Go) but not mRNA (Fig. 3AGo); Sp1 protein levels are only slightly decreased, whereas hypoxia induced HIF1{alpha} protein as commonly observed in many cells/tissues (4, 6, 8, 9, 10). Analysis of nuclear extracts from cells grown under normoxia or hypoxia showed that hypoxia also decreased formation of nuclear ER-ERE complexes (Fig. 2Go) but did not affect nuclear Sp1 (or Sp3) binding as previously reported in C2C12 myocytes (55). Hypoxia-induced up-regulation of HIF1{alpha} is observed within 3 h, whereas ER{alpha} is decreased within 6–12 h (data not shown) and is nearly undetectable at 16–24 h. Huss et al. (66) recently reported that hypoxia inhibited peroxisome proliferator-activated receptor {alpha}/retinoic acid X receptor (RXR)-mediated gene expression in cardiac myocytes, and this response was due to degradation of RXR but not peroxisome proliferator-activated receptor {alpha} protein. Hypoxia-induced degradation of ER{alpha} is also accompanied by decreased basal and inducible gene/reporter gene expression (Fig. 4Go), and these results show that another member of the nuclear receptor superfamily is regulated by hypoxia. However, there were important differences between hypoxia-induced down-regulation of ER{alpha} and RXR; the former protein is significantly degraded within 6–12 h in ZR-75 cells, whereas significant degradation of RXR protein is observed only after 24–48 h in cardiac myocytes (66). Hypoxia-induced degradation of ER{alpha} is reversed by proteasome inhibitors (Fig. 3Go), whereas the mechanism of RXR degradation is unknown and probably involves an indirect mechanism due to the time lag required for a significant drop in levels of RXR (66).

Previous studies show that estrogens and ligands that bind the AhR also induce proteasome-dependent degradation of ER{alpha} (43, 44, 45, 46), and results of this study have now identified a third pathway (i.e. hypoxia) that also activates degradation of ER{alpha} through the proteasome pathway. It was also apparent in short-term (1–3 h) pulse-chase experiments (Fig. 3CGo) that both E2 and hypoxia enhanced the rate of [35S]ER{alpha} degradation; however, the loss of radiolabeled hormone receptor was more pronounced in cells treated with E2. In breast cancer cells treated with E2, decreased ER{alpha} levels (e.g. Fig. 3BGo, lane 4) are associated with increased transactivation, whereas cotreatment with E2 plus the proteasome inhibitor MG132 increases ER{alpha} levels but decreases hormone responsiveness (43, 44). This apparent inverse relationship between ER{alpha} levels and E2 responsiveness may be due to recruitment of coactivator complexes that initially enhance transactivation and then later target ER{alpha} for degradation. LXXLL motifs in coactivators not only facilitate interactions with ER but are also sites for ubiquitination (67). Cotreatment of breast cancer cells with E2 and AhR agonist or hypoxia markedly decrease ER{alpha} levels (43, 44, 45, 46, 57) (Figs. 1Go and 3Go) and for AhR agonists and SAhRMs, this is accompanied by significantly decreased E2 responsiveness (i.e. fold induction) for various genes or reporter genes in transfection studies (56, 57, 58, 59, 60, 61, 62, 63, 64, 65). In contrast, hypoxia treatment decreased both basal and E2-inducible expression of pS2 mRNA and luciferase activity in cells transfected with p3xERELuc; however, E2-responsiveness (fold-inducibility) was increased (Fig. 4Go) in cells cotransfected with ER{alpha}. Hypoxia did not affect transactivation from a GC-rich promoter (Fig. 4CGo), and this is consistent with the minimal effects of hypoxia on Sp1 protein levels (Figs. 1Go and 3Go) and DNA binding (Fig. 2Go).

Thus, there are at least three pathways that lead to proteasome-dependent degradation of ER{alpha}, i.e. treatment with E2 (43, 44, 45, 46, 57, 67), hypoxia (this study), and AhR agonists (43, 57); however, their effects on hormone-induced transactivation are variable. Current studies in this laboratory are focused on the mechanistic differences between E2-, hypoxia-, and AhR-mediated proteasome-dependent degradation of ER{alpha} and their combined impact on critical hormone-regulated genes required for breast cancer cell growth and metastasis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Lines, Chemicals, and Biochemicals
The ZR-75 human breast cancer cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA) and was cultured in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO; JRH Biosciences, Lenexa, KS; or Atlanta Biologicals, Inc., Norcross, GA), 2.2 g/liter sodium bicarbonate, 4.8 g/liter HEPES, 4.5 g/liter dextrose, 0.22 g/liter sodium pyruvate. DMSO, E2, and CoCl2 were obtained from Sigma. The potent cell-permeable proteasome inhibitor MG132 and an inhibitor of calcium-dependent neutral cysteine proteases, calpain inhibitor II (CAL2), were obtained from Calbiochem-Novagen Corp. (San Diego, CA). Cells cultured under normoxic conditions were maintained in 37 C incubators with humidified 5% CO2-95% air. For hypoxia experiments, cells were placed in a modular incubator flushed with a gas mix containing 94% nitrogen, 5% carbon dioxide, and 1% oxygen. Exactly equal temperatures were maintained among all treatment groups by sealing and placing the modular incubator inside of the same incubator used for normoxia conditions. The hypoxia-mimicking agent CoCl2 (Sigma) was extensively used in time course and multiple treatment studies to facilitate access to multiple treatment groups carried out in duplicate for quantitative analysis of data. In previous studies, cobalt, nickel, and manganese salts induced erythropoietin gene expression in human hepatoma cell line, Hep3B, and coincubation under hypoxic conditions did not produce additive effects. Furthermore, inhibition of heme synthesis blocked hypoxia as well as cobalt-induced erythropoietin gene expression. These results suggested that the divalent cobalt cations replace the ferrous heme iron and make heme proteins unable to bind oxygen (68). In addition, parallel treatments with 1% O2 and CoCl2 were determined to confirm comparable patterns of response.

Oligonucleotides and Plasmids
Oligonucleotides and PCR primers were synthesized by Genosys/Sigma (The Woodlands, TX), the laboratory of Dr. James Derr (Department of Veterinary Pathobiology, Texas A&M University), or by Integrated DNA Technologies (IDT) (Coralville, IA). Consensus and mutant Sp1 oligonucleotides were designed according to published sequences by Promega Corp. (Madison, WI) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) catalogs. A consensus ERE probe used in EMSA competition experiments was the sense strand 5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3', annealed to its complementary sequence.

Transient Transfection Assays
The pVEGF1 and pVEGF6 constructs contain -2018 to +50 and -66 to +54 VEGF promoter insert as previously described (39). p(ERE)3-Luc reporter plasmid, pCDNA3.1-His-LacZ expression plasmid (Invitrogen, Carlsbad, CA) (for normalization of transfection efficiency) and pCDNA3.1-hER{alpha} expression plasmid (original construct kindly provided by Dr. Ming-Jer Tsai, Baylor College of Medicine, Houston, TX) were transiently transfected into ZR-75 cells using Fugene6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany), essentially according to the manufacturer’s protocol, except that transfection cocktails were prepared in 1x PBS. pCDNA3 or pCDNA3.1 (Invitrogen) empty vectors were transfected to maintain DNA mass balance among different transfection groups. After transfection for 6–12 h in medium supplemented with 2.5% charcoal-stripped serum, cells were left in normoxia or pretreated with 500 µM CoCl2 for 20–24 h, and then treated with DMSO (solvent control) or 10 nM E2 for another 12–24 h. Cells were washed three times with 1x PBS and harvested by scraping in 1x lysis buffer (Promega Corp.), and soluble protein was extracted by one cycle of freezing/thawing the cells. Thirty-microliter aliquots of transfected cell lysates were assayed for luciferase activity using Luciferase Assay Reagent (Promega Corp.) and ß-galactosidase activity using Tropix Galacto-Light Plus Assay System (Tropix, Bedford, MA) in a Packard Lumicount microwell plate reader (Packard Instrument Co., Downers Grove, IL). Luciferase activity was normalized to ß-galactosidase activity to obtain transfection data.

Northern Blot Analysis
ZR-75 cells were cultured in phenol red-free medium with 2.5% dextran-coated charcoal stripped-serum for 1 d with or without 500 µM CoCl2 and then treated with DMSO or 10 nM E2 for 24 h. In some experiments, CoCl2 was added in cotreatment. Total RNA was obtained with RNAzol B (Tel-Test, Friendswood, TX), according to the manufacturer’s protocol. RNA concentrations were measured by UV 260/280 nm absorption ratio. For Northern blot analysis, 7–15 µg of total RNA per lane was resolved on a gel containing 1.2% agarose, and 20 mM Na2PO4, 2 mM trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid at pH 6.8, and 3% formaldehyde.

Primers used to amplify a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe were: 558 bp GAPDH(F) 5'-AAT CCC ATC ACC ATC TTC CA-3' and 558 bp GAPDH(R) 5'-GTC ATC ATA TTT GGC AGG TT-3'. Primers used to amplify a pS2 cDNA probe of 210 bp were: 210 bp PS2 (F) 5'-GCG AAG CTT GGC CAC CAT GGA GAA CAA GG-3' and 210 bp PS2 (R) 5'-GCG GAT CCA CGA ACG GTG TCG TCG AA-3'. An antisense oligonucleotide specific for 28S rRNA was: 5'-AAC GAT CAG AGT AGT GGT ATT TCA CC-3'.

To make GAPDH and pS2 cDNA probes, ZR-75 total RNA was reverse transcribed at 42 C for 25 min using oligo d(T) primer (Promega Corp.), and a PCR product was subsequently amplified from the RT cDNA using 2 mM MgCl2, 1 µM of each gene-specific primer, 1 mM deoxynucleoside triphosphates, and 2.5 U Taq DNA polymerase (Promega Corp.) using 30 cycles (95 C, 30 sec; 58 C, 30 sec; 72 C, 30 sec). After amplification in a PCR Express thermal cycler (Hybaid US, Franklin, MA), samples were loaded on a 2% agarose gel containing ethidium bromide. Electrophoresis was performed at 80 V in 1x TAE buffer (40 mM Tris-HCl, 1 mM EDTA, 20 mM acetic acid, pH 8.0) for 1 h, and the gel was photographed by UV transillumination using Polaroid film. GAPDH (558 bp) and PS2 (210 bp) cDNA bands were excised from the gel and purified using the Qiaquick gel-slice extraction kit (QIAGEN Inc., Valencia, CA). In addition, a 2.1-kb ER{alpha} cDNA insert was excised from pCDNA3.1-hER{alpha} by EcoRI digest and recovery from an agarose gel. GAPDH, pS2, and ER{alpha} probes were then labeled using the random-primed labeling kit (Roche Molecular Biochemicals). 28S rRNA antisense DNA probe was end labeled using T4 polynucleotide kinase (Promega Corp.) and [{gamma}32P]ATP (NEN Life Science Products-DuPont, Boston, MA). Approximately 1 million cpm/ml hybridization solution (250 mM sodium phosphate, 7% sodium dodecyl sulfate, pH 7.2) was used to detect RNA transferred to Zeta-Probe-charged nylon membrane (Bio-Rad Laboratories, Inc., Hercules, CA) by capillary electrophoresis. Equal RNA loading was verified by probing for 28S rRNA. Membranes were exposed to phosphor-storage screens, and the screens were scanned on a STORM densitometer (Molecular Dynamics, Inc., Sunnyvale, CA).

Preparation of ZR-75 Cell Nuclear Extracts
Cells were cultured in 150-mm diameter culture dishes in medium without phenol red, supplemented with 2.5% charcoal-stripped serum. DMSO or 10 nM E2 was added to cells for 24 h before washing three times in 1x PBS and scraping in 1 ml 1x lysis buffer (low salt buffer) (Promega Corp.). Cells were allowed to swell on ice for 15 min and centrifuged at 14,000 x g for 1 min at room temperature, and a pellet containing crude nuclei was isolated. The supernatant was removed, and the pellet was washed three times more in 1 ml of 1x lysis buffer. After the last wash, approximately five pellet volumes of 1x lysis buffer supplemented with 500 mM KCl (high-salt buffer) were added to the pellet and further incubated on ice for 30 min to 1 h with frequent vortexing. Nuclei in high-salt buffer were centrifuged at 14,000 x g for 1 min at room temperature, and aliquots of nuclear proteins in supernatant were stored at -80 C for use in EMSA.

EMSAs
Five picomoles of double-stranded consensus ERE oligonucleotide were 5'-end labeled using T4 kinase (Promega Corp.) and [{gamma}-32P]-ATP (NEN Life Science Products-DuPont). A 20- to 30-µl EMSA reaction mixture contained approximately 75–150 mM KCl (a balance of low-salt and high-salt buffers used in the nuclear extraction protocol), 5 µg of crude nuclear protein, 1 µg poly(dI-dC) (Roche Molecular Biochemicals), with or without unlabeled competitor oligoucleotide, and 10 fmol labeled probe. Reactions were incubated for 15 min on ice, and antibodies to Sp1, Sp3, and hER{alpha} proteins (Santa Cruz Biotechnology, Inc.) were sometimes added to reaction mixes immediately after probe and incubated an additional 15 min on ice. Protein-DNA complexes were resolved by 5% PAGE, in 1x TBE (0.09 M Tris-base, 0.09 M boric acid, 2 mM EDTA, pH 8.3), at 120 V at room temperature for 2.5 h. Specific DNA-protein and antibody supershifted complexes were observed as more slowly migrating complexes in the gel. Dried gels were exposed to phospho-storage screens, and the screens were scanned on a STORM densitometer (Molecular Dynamics, Inc.).

Western Immunoblot Analysis
ZR-75 cells were seeded into six-well (4 x 105 to 6 x 105 cells per well) or 12-well plates (1 x 105 to 2 x 105 cells per well) in phenol red-free culture medium supplemented with 2.5% charcoal-stripped serum. Cells were exposed to normoxia (21% O2), physiological hypoxia (1% O2), or chemically induced hypoxia (500 µM CoCl2) in the presence of DMSO or 10 nM E2 for varying times. Cells were harvested with 1x Laemmli’s loading buffer (50 mM Tris-HCl, pH 6.8; 2% sodium dodecyl sulfate; 0.1% bromophenol blue; 10% glycerol; 10% ß-mercaptoethanol) and boiled for 5 min. Equal volumes of samples were loaded on a 7.5% sodium dodecyl sulfate-polyacrylamide gel and were resolved by electrophoresis at 150–180 V for 3–4 h at room temperature. Total cell numbers per well did not vary significantly across treatment groups, as treatments were for 24 h or less (data not shown). Separated proteins were transferred (transfer buffer: 48 mM Tris-HCl, 29 mM glycine, and 0.025% sodium dodecyl sulfate) to polyvinylidine difluoride membrane (Bio-Rad Laboratories, Inc.), and specific proteins on the membrane were detected with polyclonal antibodies against ER{alpha} (1:1000 dilution), HIF1{alpha} (1:200 dilution), and Sp1 (1:2000 dilution; Santa Cruz Biotechnology, Inc.), followed by incubation with horseradish peroxidase-conjugated antirabbit secondary antibody (1:5000 dilution; Santa Cruz Biotechnology, Inc.). Blots were exposed to enhanced chemiluminescent substrate (NEN Life Science Products-DuPont) and placed on Kodak X-O-Mat autoradiography film (Eastman Kodak Co., Rochester, NY). Protein band intensities were scanned on a JX-330 scanner (SHARP Corp., Mahwah, NJ) using Adobe Photoshop 3.0 (Adobe Systems Inc., Palo Alto, CA).

[35S]-Methionine Pulse-Chase Assay
ZR-75 cells were seeded to 80% confluency in 10-cm diameter culture dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2.2 g/liter sodium bicarbonate, and an antibiotic/antimycotic solution. Cells were allowed to attach overnight and then were washed twice with 1x PBS before the addition of serum-, phenol red-, and methionine-free medium. After incubation for 1 h in methionine-deficient medium, a metabolic labeling-grade mixture of [35S]methionine and cysteine (Moravek Biochemicals, Inc., Brea, CA) was added to a final activity of 0.1 mCi/ml medium, and cells were pulse labeled for 2 h. After [35S]methionine labeling, cells were washed twice with 1x PBS, and then phenol red-free complete medium (without serum) was added to the cells; a zero time-point (0 h) sample was harvested, and remaining plates of cells were treated with appropriate chemicals and harvested at different times. Cells were harvested in ice-cold 1x lysis buffer (Promega Corp.) supplemented with 500 mM KCl, incubated on ice for 1 h, and centrifuged to collect whole-cell extracts. Two-hundred microliters of whole-cell extract were diluted with 800 µl 1x lysis buffer (without KCl), and 20 µl protein A/G agarose conjugate beads (Santa Cruz Biotechnology, Inc.) and 2 µg mouse monoclonal ER{alpha} antibody (D-12) (Santa Cruz Biotechnology, Inc.) were added, and tubes were placed on an orbital rocker at 4 C for 12 h. Labeled ER{alpha} immunoprecipitates were collected by centrifugation at 14,000 x g, and the pellets were washed with 1x PBS. Washing and pelleting were repeated three times, and then beads were boiled for 5 min in 1x Laemmli’s buffer, and eluted proteins were analyzed by SDS-PAGE. [35S]-labeled ER{alpha} in the gels was examined by exposure to phosphor-storage screens and scanning on a STORM PhosphorImager (Molecular Dynamics, Inc.). Alternatively, dried gels were exposed to x-ray film, the films were scanned, and specific ER{alpha} bands were quantified by densitometry and background subtraction using ZeroD software (Scanalytics, Sunnyvale, CA). Data are presented as the percentage of the band intensity of [35S]-met ER{alpha} levels in the untreated 0 h control. Graphed values represent a single experiment representative of two independent experiments.

ER{alpha} Immunofluorescent Microscopy
Fifty-thousand ZR-75 cells were seeded in each well of a Lab-Tek four-well chamber slide (Nunc, Naperville, IL) in RPMI 1640 medium used for cell maintenance. Cells were allowed to attach to glass for 1 d, and then medium was changed to phenol red-free DMEM/F12 supplemented with 2.5% charcoal-stripped serum (CSS), and duplicate treatments of DMSO or 10 nM E2 in the absence or presence of CoCl2 were carried out for 24 h. After treatment, wells were removed and the slides washed in PBS for 10 min. Slides were then placed in -20 C methanol for 20 min and air dried at room temperature for 30 min. Dried slides were permeabilized and rehydrated in PBS supplemented with 0.3% Tween-20 (PBS-Tween) (Sigma) for 10 min. Blocking was carried out at room temperature for 1 h using goat serum diluted 1:20 in PBS-Tween. Goat serum was used because secondary antibodies for this experiment were produced in the goat. After blocking, cells were washed 10 min with PBS-Tween. ER{alpha} antibody H-184 (Santa Cruz Biotechnology, Inc.) was diluted 1:200 in antibody dilution buffer (stock solution: 100 ml PBS-Tween, 1 g BSA, 45 ml glycerol, pH 8.0) and added to the cells overnight at 4 C. Primary antibody was washed from cells by rinsing in PBS-Tween (three times, 10 min), and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA) diluted 1:200 in antibody dilution buffer was placed on the cells for 1 h at room temperature. As a negative control, primary antibody was omitted from duplicate slides for each treatment group, and these slides were incubated with secondary antibody alone. Finally, slides were extensively washed in PBS-Tween, blotted dry, covered with Prolong Antifade mounting solution (Molecular Probes, Inc., Eugene, OR), and sealed with a coverglass. Immunoflourescence of ER{alpha} protein was observed with a Zeiss Photomicroscope III (Carl Zeiss, Thornwood, NY) with a UV bulb. Multiple images from each treatment group were captured with a digital camera and transferred to Adobe Photoshop.

Statistical Analysis
Experiments were repeated at least two times, and representative results are shown as mean ± SE for three or four replicates per treatment group. Data were subjected to ANOVA, and Fisher’s protected least significant difference demonstrated statistical differences between treatment groups. Treatments were considered statistically significantly different from controls if P < 0.05.


    ACKNOWLEDGMENTS
 


    FOOTNOTES
 
This work was supported by NIH Grants ES-04176 and ES-09106 and the Texas Agricultural Experiment Station. S.S. is a Sid Kyle Professor of Toxicology.

Abbreviations: AhR, Aryl hydrocarbon receptor; CAL2, calpain inhibitor II; CSS, charcoal-stripped serum; DMSO, dimethylsulfoxide; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIF1{alpha}, hypoxia-inducible factor 1{alpha}; HRE, hypoxia response element; RXR, retinoid X receptor; SAhRM, selective AhR modulator; VEGF, vascular endothelial growth factor.

Received for publication December 19, 2001. Accepted for publication June 11, 2002.


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 DISCUSSION
 MATERIALS AND METHODS
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