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Endocrinology Vol. 142, No. 4 1497-1505
Copyright © 2001 by The Endocrine Society

ARTICLES

Constitutive Expression of the Steroid Sulfatase Gene Supports the Growth of MCF-7 Human Breast Cancer Cells in Vitro and in Vivo1

Mattie R. James, Todd C. Skaar, Richard Y. Lee, Alexander MacPherson, James A. Zwiebel, Balwant S. Ahluwalia, Franklin Ampy and Robert Clarke

Vincent T. Lombardi Cancer Center, Georgetown University School of Medicine (M.R.J., T.C.S., R.Y.L., A.M.), Washington, D.C. 20007; National Cancer Institute, National Institutes of Health (J.A.Z.), Bethesda, Maryland 20892; and Department of Genetics and Human Genetics, Howard University (B.S.A., F.A., M.R.J.), Washington, D.C. 20059

Address all correspondence and requests for reprints to: Robert Clarke, Ph.D., D.Sc., Room W405A, Research Building, Vincent T. Lombardi Cancer Center, Georgetown University School of Medicine, 3970 Reservoir Road NW, Washington, D.C. 20007. E-mail: clarker{at}gunet.georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many human breast tumors are driven by high intratumor concentrations of 17ß-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [3H]estrone sulfate to [3H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein·h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein·h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein·h).

17ß-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G2/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17ß-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17ß-estradiol sulfate appears more effective than 17ß-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS ARE THE most important etiological factors in the growth and development of many breast carcinomas in both pre- and postmenopausal women. Breast tumors from postmenopausal women contain high levels of 17ß-estradiol despite the presence of low plasma 17ß-estradiol concentrations (1, 2, 3). Although breast tumors can clearly accumulate serum 17ß-estradiol to concentrations higher than those seen in serum (3, 4, 5), 17ß-estradiol is a relatively minor serum estrogen in postmenopausal women. It is now widely accepted that breast tumors can synthesize 17ß- estradiol from adrenal androgen precursors. This occurs through the aromatization of androstenedione to estrone by aromatase, followed by the conversion of estrone to 17ß-estradiol by 17ß-hydroxysteroid dehydrogenase type 1 (6). However, breast cancer cells also express both steroid sulfotransferase (7, 8, 9) and steroid sulfatase (STS) activities (10, 11). The latter potentially obviate the need to synthesize significant amounts of estrone within some tumors. STS can release estrone from estrone sulfate, which is peripherally synthesized in adipose tissues.

Inhibition of aromatase provides significant benefit to many breast cancer patients (12, 13), establishing its importance in the production of estrogens. Nonetheless, as with other endocrine therapies, a significant proportion of tumors that express estrogen receptors fail aromatase therapy. Inhibition of aromatase activity does not readily discriminate between inhibition of peripheral and intratumoral aromatase and sulfatase activities, because it also should reduce both circulating and intratumor concentrations of estrone.

Although estrone sulfate is the predominant serum estrogen in postmenopausal women, the primary intratumor estrogen is 17ß-estradiol (14, 15). Estrogen sulfates have been considered biologically inactive compounds, and the contribution of serum estrone sulfate to intratumor estrogens remains controversial. Sulfated steroids are not believed to penetrate cell membranes easily because of their polarity (8); the sulfate moiety at the C3 position essentially eliminates their ability to recognize estrogen receptors (16). Nonetheless, estrogenic effects in response to sulfated estrogens have been demonstrated, and desulfation probably occurs rapidly as the estrogen sulfates penetrate the cell membrane (8).

Data from clinical studies indirectly support the importance of serum estrogen sulfates. Two first generation aromatase inhibitors, aminoglutethimide and testololactone (17), reduce aromatase activity to comparable levels, but the clinical response rate to testololactone is much lower (18). However, in addition to its inhibition of aromatase activity, aminoglutethimide significantly increases estrone sulfate clearance (19, 20), an effect not seen with testololactone (17, 18). These data suggest that estrogen sulfates may contribute to intratumor 17ß-estradiol concentrations in some human breast tumors.

Evidence that either aromatase or sulfatase expression is useful as a predictive/prognostic marker in breast cancer remains contradictory. Studies have failed to demonstrate that aromatase has any significant power as an independent prognostic indicator for breast cancer outcome (21, 22). STS messenger RNA (mRNA) expression was an independent predictor of recurrence in one study (21), but not in another (23). However, STS is more commonly detected than aromatase expression, being found in up to 90% of breast tumors (23, 23) compared with 60–70% for aromatase (21, 22). Pasqualini et al. (3) estimate that STS activity is 50–200 times greater than aromatase activity in both premenopausal and postmenopausal breast tumors. However, activity reflects the combination of maximum velocity (Vmax), Km, and substrate availability. The much greater Vmax of sulfatase in breast tumors may be partly offset by the higher affinity interactions between aromatase and its substrate compared with the affinity of estrone sulfate for STS.

Although supportive, these observations only provide an indirect assessment of a possible role for intratumor STS. For example, it is not known whether the levels of STS activity are sufficient to support the growth of estrogen-dependent cells in vivo. We now describe an in vivo model, using ovariectomized nude mice supplemented with an estrogen sulfate and bearing STS-transduced human breast cancer cells, to test this hypothesis directly. Our data demonstrate that stable expression of high levels of STS activity can be obtained, and that this is sufficient to support the growth of estrogen-dependent tumors in mice supplemented with 17ß-estradiol sulfate. Hepatic metabolism is not a major contributor of estrogen sulfate metabolism in these animals. Thus, our data directly support the hypothesis that STS activity can significantly contribute to the high intratumor 17ß-estradiol concentrations seen in the tumors of some premenopausal and postmenopausal breast cancer patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines
The estrogen-responsive MCF-7/2 human breast cancer cell line was obtained from the Lombardi Cancer Center’s Tissue Culture Shared Resource (provided by Dr. Michael Johnson, Lombardi Cancer Center, Washington, DC). MCF-7/2 is a subline of the parental MCF-7 cells derived by single cell cloning of the parental MCF-7 cells. MCF-7/2 cells are reproducibly estrogen responsive. The production of a high incidence of rapidly growing MCF-7/2 tumors in ovariectomized nude mice and proliferation in vitro are estrogen dependent. Thus, MCF-7/2 cells were routinely grown in Improved MEM containing phenol red (Biofluids, Rockville, MD) and supplemented with 5% FBS.

Estrogens are retained for prolonged periods in breast cancer cells in vitro (24). Thus, where experiments required either that cells be grown in the absence of estrogens and/or estrogenic supplementation, we first applied a rigorous stripping regimen to remove endogenous steroids (25). Briefly, cells were extensively washed and then maintained in Improved MEM without phenol red (Biofluids) and supplemented with 5% calf serum stripped of endogenous estrogens by treatment with dextran-coated charcoal and STS (26). Cell culture prepared in this manner contains less than 10 fM 17ß-estradiol (27). Monolayers were washed with the stripped medium three times on the first day, twice on the second day, and once on the third day. All further treatments, e.g. with estrogens or sulfated estrogens, began on the fourth day (25).

Transduction of complementary DNAs (cDNAs) into MCF-7/2 cells
The full-length sulfatase cDNA was obtained from American Type Culture Collection (Manassas, VA). The LXSN retroviral expression vector contains appropriate cloning sites placing the cDNA of interest downstream of the 5'-long terminal repeat, with a constitutively expressed neomycin resistance gene as the selectable marker. A 2.4-kb STS cDNA was ligated into the EcoRI site of the LXSN vector (28) using T4 DNA ligase (Promega Corp.). The ligated product was stably transfected into GP+E86 packaging cell line (29) by the calcium phosphate coprecipitation method (30). Cells were grown to confluence, and the supernatant was collected. A transinfection with the viral supernatant was performed using the PA317ß packaging cell line (31). Cells were selected, and individual colonies were collected and expanded, in Improved MEM containing phenol red and 600 µg/ml G418 and supplemented with 10% FBS (selection medium). Titration of the recombinant retrovirus stock showed that the highest titer was 8 x 105 colony-forming units/ml. This supernatant was tested for the helper virus using a standard helper virus detection assay protocol and was free of the helper virus. MCF-7/2 cells were infected by exposure to the virus, and G418-resistant colonies were isolated. Single cell clones, derived from these resistant colonies, were expanded for further study in Improved MEM containing phenol red and supplemented with 5% FBS. This estrogenic environment precluded the selection for either estrogen- independent or estrogen-supersensitive cells, which require prolonged estrogen deprivation both in vivo (32) and in vitro (32, 33). Clones transduced with retroviral vectors containing the STS genes were designated STS Clone #, e.g. STS Clone 20; those transduced with the vector but lacking the STS gene were designated Vector#, e.g. Vector 3.

Nucleic acid probes
The STS probes for Southern and Northern hybridizations were prepared using 25 ng of a 2.4-kb STS cDNA labeled with [32P]deoxy-ATP (Amersham Pharmacia Biotech, Arlington Heights, IL) by random priming (34). Radiolabeled probes were purified by chromatography on a Quick Spin Column, Sephadex G-50 (Roche Molecular Biochemicals, Indianapolis, IN). To control for RNA loading on Northern hybridization analyses, the blots were probed with a radiolabeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe. For ribonuclease (RNase) protection analyses, a progesterone receptor (PgR) riboprobe was used that produces a 240-bp protected fragment (35). The pS2 riboprobe protects a 300-bp fragment, whereas the 36B4 riboprobe (loading control) (36) produces a 220-bp protected fragment (35).

Southern and Northern hybridizations and RNase protection studies
Genomic DNA was isolated from cultured cells by the DNAzol method (Life Technologies, Inc., Gaithersburg, MD). Total RNA was obtained using the TRIzol reagent method (Life Technologies, Inc.). Southern hybridizations were performed using standard techniques (34). Northern hybridizations and RNase protection studies were performed as previously described (30, 37). RNA loading controls were GAPDH (Northern hybridizations) and 36B4 (RNase protection analyses). Where appropriate, phosphorimage analyses were performed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Isolation of subcellular fractions
Cellular homogenates of transduced clones, vector, and wild-type MCF-7/2 cells were prepared by the method of MacIndoe et al. (38). Cells were harvested by scraping and centrifuged at 2000 x g for 5 min, the cell pellets were allowed to swell on ice for 10 min in ice-cold 1.5 mM MgCl2, and the cells were disrupted with a Dounce homogenizer. An equal volume of 0.04 M Tris-HCl buffer, pH 6.5, was added, and the homogenate was centrifuged at 200,000 x g for 30 min. Microsomal fractions (pellet) were washed once in 1 ml 0.02 M Tris-HCl buffer, pH 6.5, and further spun at 2000 x g for 5 min. These pellets were resuspended in Tris-HCl buffer, sonicated, and stored at -20 C until used.

STS biochemical assay
We used a modified method of MacIndoe et al. (38) to measure activity in cells growing either in vitro or in vivo. For in vivo tissues, tumors were obtained at necropsy, immediately frozen, and stored at -80 C until used. Samples were diluted to the appropriate protein concentration, i.e. each protein sample contained 0.05–0.15 mg protein/100 µl, and 100 µl were added to reaction buffer (200 µl 0.02 M Tris-HCl, pH 6.5). Next, 100 µl Tris-HCl buffer, pH 6.5, containing 0.4 nM [3H]estrone sulfate (SA, 49 Ci/mmol; NEN Life Science Products, Boston, MA), diluted with a 100-fold excess of unlabeled estrone sulfate (Sigma, St. Louis, MO), were added to each tube to obtain the required final molarity. After incubation for 1 h, the reaction was terminated by placing the samples on ice for 10 min. To measure unconjugated radiolabeled metabolites, 100 µl of each sample were mixed with 5 ml of a highly nonpolar scintillation fluid (666 ml dioxane, 330 ml xylene, 80 g naphthalene, and 5 g 2,5-diphenyloxazole), the samples were vortexed, and radioactivity was measured by scintillation spectrometry. Subsequently, 5 ml dH2O were added, each sample was vortexed, and scintillation spectrometry was repeated to assess radioactivity incorporated into the polar metabolites.

Cell cycle analyses
Cell monolayers were grown at 3 x 106 cells/T-75 cm2 flasks and stripped of endogenous steroids for 3 days with extensive washing, as described above. After the 3-day stripping procedure, cells were treated with estrogen with or without antiestrogen (17ß-estradiol 3-sulfate, 1 nM; ICI 182,780, 100 nM; tamoxifen 1 µM) in Improved MEM without phenol red-free medium and supplemented with 5% stripped calf serum. Cell cycle distribution was measured 48 h later. Briefly, 106 cells were suspended in a citrate buffer (250 mM sucrose, 40 mM trisodium-citrate, and 5% dimethylsulfoxide), and cell cycle analysis was performed using standard techniques (39) in the Lombardi Cancer Center Flow Cytometry Resource with a FACStar flow cytometer (Becton Dickinson and Co., Palo Alto, CA).

Anchorage-independent growth
Anchorage-independent colony formation was performed as previously described (26). Briefly, cells were stripped of endogenous steroids (25), 4 x 106 cells were suspended in 0.5 ml Improved MEM without phenol red-free medium and supplemented with 5% stripped calf serum, then added to a mixture containing 1.5 ml 1.2% agar (Difco, Detroit, MI) solution and 0.5 ml of treatment/vehicle solution. The suspension was poured onto a layer of solidified agar and incubated for 14 days at 37 C in a humidified 5% CO2/95% air atmosphere. Colonies of 50 cells or more (>=60 µm in diameter) were counted using an Omicron 3600 image analysis system (Artek, Farmingdale, NY). Five replicates were made for each sample.

In vivo tumor growth
Ovariectomized nude mice were used as a model because these animals have serum estrogen levels similar to those observed in postmenopausal women (40, 41). Although many breast tumors can convert estrone to 17ß-estradiol, we were concerned that the level of 17ß- hydroxysteroid dehydrogenase type 1 activity in the STS Clone 20 and Vector 3 cells might be low. Consequently, we used 17ß-estradiol sulfate rather than estrone sulfate for the in vivo studies. This approach should ensure that any failure to support tumorigenicity could be essentially attributed to an inability of STS activity to generate biologically relevant intratumor 17ß-estradiol concentrations. To control for the possible contribution of hepatic STS activities, mice received STS Clone 20 cells on one flank and control Vector 3 cells on the opposite flank.

Cells growing in vitro were used for the xenograft inocula. Briefly, subconfluent monolayers (80%) were removed by gentle scraping, the cell suspensions were spun for 5 min at 1000 x g, and the pellets were resuspended in growth medium. Cell viability was estimated by trypan blue dye exclusion, and 2 x 106 viable cells were sc inoculated into the right and left flanks of 6- to 8-week-old, specific pathogen-free, ovariectomized, athymic, NCr-nu/nu nude mice (Taconic Farms, Germantown, NY). Estrogens were administered as sc implants of 60-day release pellets (Innovative Research of America, Sarasota, FL). The 17ß-estradiol sulfate pellets were custom made by Innovative Research of America using 17ß-estradiol 3-sulfate (Sigma). Mice received STS Clone 20 cells in one flank and Vector 3 cells in the opposite flank. Body weights were obtained on each group of animals twice weekly. The response to 17ß-estradiol sulfate was determined by measuring tumor incidence, a standard end point for many in vivo studies (42, 43). Tumor incidence was defined as the proportion of proliferating tumors, i.e. those tumors that consistently increased in size throughout the study. To facilitate this determination, tumor size was recorded weekly.

Statistical analyses
Lineweaver-Burke transformations were fitted by simple least square linear regression, and the 99% confidence interval for each fit was estimated. These analyses were performed using the algorithms in SigmaPlot version 5.0 (Jandel Scientific, Carlsbad, CA). Statistical tests were performed using SigmaStat version 2.0 (Jandel Scientific). {chi}2 analyses were performed to compare tumor incidence (proportions) among treated and control groups. ANOVA was used to compare multiple groups. Where only two groups were compared, Student’s t test was applied. Values are represented as the mean ± SE unless otherwise indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Southern and Northern analyses of STS in transduced and control cells
Effective incorporation of the STS cDNA into the estrogen-responsive MCF-7/2 cells was demonstrated by Southern analysis. Expression of the appropriate size band from the XbaI/BbsI restriction digest of 5.2 kb was detected only in the STS-transduced cells (Fig. 1AGo). Placenta produces bands of 9.6, 3.0, 2.6, and 1.6 kb. MCF-7/2, Vector 3, and STS-transduced cells produced bands of 3.0, 2.6, and 1.6 kb, representing the endogenous STS gene.



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  		Figure 1. STS nucleic acid analyses. A, Southern blot analysis of genomic DNA isolated from transduced STS cells and wild-type MCF-7/2 cells. B, Northern blot analysis of total RNA isolated from STS clones, MCF-7/2 cells, and human placenta. RNA loading was assessed by comparing expression of the GAPDH mRNA.

 
Data from Northern hybridizations confirmed the expression of increased levels of the appropriately sized RNA transcript. Clones 1, 3, 12, and 20 synthesized an RNA transcript of 4.8 kb. No expression was seen in Clone 2 (Fig. 1BGo). A 5.2-kb band representing the placental RNA was observed (lane 2), consistent with the report by Yen et al. (44). However, these transcripts were not detected in placenta by others using similar Northern hybridization analyses (45, 46). STS mRNA expression in STS Clone 3 was approximately equivalent to that in placenta, with expression in Clone 12 and Clone 20 being approximately 2-fold higher than that in placenta. It is difficult to provide precise estimates relative to other controls because the MCF7/2 and Vector 3 values are consistently undetectable. As described by others (45, 46), Northern hybridizations do not detect the endogenous STS transcripts in MCF-7 cells that should be similar to our MCF-7/2 and Vector 3 cells. These transcripts are readily detected by RT-PCR (45).

STS enzyme activity in control and transduced cells
STS enzyme activity was measured in cellular homogenates, cytosol, and microsomal fractions. As expected, activity was detected in both the homogenate and cytosol (not shown); the highest activity was found in the microsomal fractions. Microsomal fractions were used in all subsequent studies. STS activity displayed linear enzyme kinetics up to 0.50 mg protein/ml at 37 C for 1 h. Nonlinear kinetics were observed above this concentration (not shown). A level of 0.50 mg protein/ml was used in all other reactions.

Sulfatase activity was assessed from 30 min to 4 h. At 1 h, placenta produces 26.2 ± 0.27 pmol estrone /mg protein·h, MCF-7/2 produces 13.0 ± 0.42 pmol estrone/mg protein·h, Vector 3 produces 12.0 ± 0.26 pmol estrone/mg protein·h, and STS Clone 20 produces 64.2 ± 0.14 pmol estrone/mg protein·h. After 1 h, STS Clone 20 cells hydrolyze 87% of the [3H]estrone sulfate, and STS Clone 12 cells hydrolyze 70% of the estrone sulfate. STS Clone 3 cells had the lowest percentage of enzyme activity, and this was comparable to that seen in MCF-7/2 (36%) and Vector 3 (42%) cells. The higher enzyme activity in the transfectants was less than might be predicted by the Northern hybridizations. However, it is not unusual to detect levels of mRNA expression among transfected cells that do not fully reflect the levels of an active protein.

As sufficient sulfatase activity could be detected with an incubation of 1 h in each of the cell lines, all sulfatase assays were performed using a 1-h incubation unless otherwise indicated. The optimum pH was estimated by incubating the microsomal fraction of STS clones, placental, vector, and MCF-7/2 cells for 1 h with [3H]estrone sulfate in Tris-HCl buffer over a pH range of 5.0–8.0. The optimum pH for all the samples in all experiments was 6.5–7.5 in Tris-HCl buffer. Subsequent samples were assayed at a pH of 6.5. These data are consistent with the most frequently reported optimum pH of 6.5 for the sulfatase assay of MCF-7 cells (38, 47).

Consistent with the data from the Southern and Northern analyses, STS Clone 20 and STS Clone 12 cells express significantly elevated levels of STS activity, relative to MCF-7/2 and Vector 3 cells, as evidenced by their higher Vmax estimates (Table 1Go). The estimated STS Km values in the transduced cells (Table 1Go) are variable, but broadly comparable to both controls (MCF-7/2, Vector 3, and placenta) and previously published data (38, 48). Detection of endogenous STS activity in MCF-7/2 and Vector 3 cells is consistent with previous reports of this activity in other MCF-7 cells (49).


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  		Table 1. Catalytic properties of steroid sulfatase transduced cells

 
These data clearly demonstrate that the STS mRNA expressed is translated into functional protein in at least two of the clones studied (STS Clone 12 and STS Clone 20). This is supported by the comparability of the estimated Km values with previously published studies (38, 48) and the increased Vmax for estrone sulfate metabolism in the transduced cells. The STS activities detected in STS Clone 12 (0.61 nmol/min·mg) and STS Clone 20 (0.72 nmol/min·mg) cells are broadly comparable with the levels seen in breast tumors expressing high levels of STS activity (8, 23, 50). For example, a range of 0–0.40 nmol/min·mg1 was reported in 93 of 104 human breast cancers (23).

Effects of sulfated estrogens on cell cycle distribution
Cell cycle analysis of STS Clone 20, MCF-7/2, and Vector 3 cells treated with 17ß-estradiol sulfate in the presence or absence of either ICI 182,780 (100 nM) or tamoxifen (1 µM) was determined by flow cytometry (Table 2Go). Consistent with the reported effects of estrogens on cell cycle distribution (51), 1 nM 17ß-estradiol sulfate increased the proportion of cells in the proliferative fraction (S+G2/M), with a consequent reduction in G0/G1. The greatest change was evident in the STS Clone 20 cells, reflecting their higher levels of STS activity. To confirm that these are estrogenic effects, we determined the ability of antiestrogens to block the changes in cell cycle distribution induced by 17ß-estradiol sulfate. Treatment of STS Clone 20 cells with either ICI 182,780 or tamoxifen increased the proportion of cells in Go/G1 while reducing the proportion in S+G2/M. These data clearly indicate that STS-transduced cells retain responsiveness to antiestrogens.


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  		Table 2. Effect of 17ß-estradiol sulfate on cell cycle distribution in vitro

 
Estrogenic effects of estrogen sulfates in vitro
The effects of sulfated estrogens and 17ß-estradiol on anchorage-independent colony-forming ability are shown in Fig. 2Go. At the physiologically relevant concentration of 1 nM, the sulfated estrogens act as potent mitogens for colony formation in both STS Clone 20 and MCF-7/2 cells. As expected, these data are broadly comparable to the changes in cell cycle profiles seen in Table 2Go. These mitogenic effects appear dose dependent, as 5-nM treatments are generally more effective than 1-nM treatments and are consistently higher in STS Clone 20 cells compared with either the MCF-7/2 or Vector 3 cells. These data reflect the higher level of STS expression in STS Clone 20 relative to endogenous STS activity. 17ß-Estradiol is equally effective in all three cell lines.



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  		Figure 2. Induction of anchorage-independent colony formation by estrogens and estrogen sulfates. Data represent the mean ± SE of each of five replicates in two or more experiments. *, P < 0.05; **, P < 0.01 (compared with control).

 
Expression of the estrogen-regulated genes PgR (Fig. 3AGo) and pS2 (Fig. 3BGo) were evaluated in the presence of 1 nM estrone sulfate, 17ß-estradiol sulfate, and 17ß-estradiol. Estrone sulfate does not stimulate the expression of PgR in any of the cell lines (not shown). Both 17ß-estradiol sulfate and 17ß-estradiol increase PgR mRNA expression in STS Clone 20, but only 17ß-estradiol is effective in MCF-7/2 and Vector 3 cells (Fig. 3AGo). In contrast, pS2 expression is induced by estrone sulfate in all three cells (Fig. 3BGo). The expression pattern seen for the apparently less estrogen-responsive PgR gene is consistent with higher STS activity in STS Clone 20 cells. Santner et al. (49) suggest that both PgR and pS2 are relatively insensitive to stimulation, requiring at least 1 µM estrone sulfate for induction of expression. Although we used only a single concentration (1 nM), this was clearly sufficient to induce pS2 in all cells (estrone sulfate) but to induce PgR expression only in STS clones (17ß-estradiol sulfate). Cells that acquire an estrogen-independent phenotype also up-regulate pS2 mRNA, but not PgR expression, suggesting that pS2 induction is more sensitive to changes in estrogenicity than PgR (37).



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  		Figure 3. Effects of estrogens and estrogen sulfates on transcription of the PgR (A) and pS2 mRNAs (B). In the RNase protection assays, the PgR probe produces a protected fragment of 250 bp, the pS2 probe protects a 300-bp fragment, and the 36B4 probe a generates a protected fragment of 220 bp.

 
Effect of intratumor STS activity on tumorigenicity
To address the possible in vivo relevance of estrogens released by intratumor estrogen sulfatase activity, we evaluated the ability of 17ß-estradiol sulfate to support the establishment of estrogen-dependent tumors in ovariectomized athymic nude mice (Table 3Go). When evaluating endocrine therapies in these models, it is common to first establish tumors with 17ß-estradiol, and then administer the endocrine manipulation, e.g. removal of the estrogenic stimulus by administration of an estrogen antagonist. Our experimental design is more rigorous and appropriate for the assessment of the effects of estrogen sulfates because it requires the hormone to promote the survival and establishment of a relatively small number of cells. In this regard, our approach is similar to that used to determine the ability of aromatase overexpression to support tumor growth in vivo. However, we did not implant cells embedded in Matrigel, an artificial basement membrane, which is required for the maintenance of MCF-7 cells transfected with the aromatase gene (52).


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  		Table 3. Effect of 17ß-estradiol sulfate on tumor incidence in ovariectomized athymic nude mice

 
The growth rate of nonproliferating tumors could not be measured. We observed marked variation in tumor growth within each group, which would limit our ability to statistically detect modest differences in tumor growth rate. However, we were more interested in whether proliferating tumors could be established than in the rate at which any tumors might grow once established. From a clinical perspective, the presence of a proliferating, estrogen-dependent tumor is more relevant than the rate at which an established tumor proliferates. Thus, we compared the incidence of proliferating tumors in mice bearing vector control vs. transfectant (STS Clone 20) xenografts. The rate of growth is a useful prognostic indicator is some cases, but is not particularly informative in predicting response to endocrine therapies. Although toxicity was not anticipated, body weight measurements were obtained on each group of animals during the study. No significant difference in body weights was seen among the groups.

Mice receiving both STS Clone 20 cells and 17ß-estradiol sulfate exhibited the highest incidence of proliferating tumors, more than 3-fold higher than that in their nonsupplemented controls (Table 3Go; P < 0.001). Although 17ß-estradiol sulfate also supported the growth of some Vector 3 cells, tumor incidence was not significantly increased compared with the incidence in nonsupplemented mice (Table 3Go; P = 0.37). These data are consistent with the increased STS activity (~4-fold higher compared with Vector 3 tumors; P < 0.001) in the STS Clone 20 cells and indicate continued expression of high levels of the active enzyme in vivo. Tumorigenicity in the absence of 17ß-estradiol sulfate supplementation was equivalent in both STS Clone 20 and Vector 3 cells (P = 0.84). Similarly, the tumorigenicity of STS Clone 20 and Vector 3 cells was equivalent in the presence of 17ß-estradiol (P = 1.00). Thus, the differences in tumor incidence are not due to altered basal tumorigenicity between STS cells and controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To address the possible functional relevance of the STS gene further, we have overexpressed the STS cDNA in estrogen-dependent human breast cancer cells. Previous studies were limited to those characterizing endogenous levels from breast cancer tissues or various breast cancer cell lines. The STS-transduced clones we have generated now permit studies to address the potential importance of this activity in the production of biologically relevant concentrations of intratumor estrogens. These transfectants clearly exhibit differential responses to estrogen sulfates in vitro and in vivo, consistent with their elevated STS expression. For example, equimolar concentrations of estrogen sulfates are more effective in STS-transduced cells relative to controls in vitro. This is evident for estrogenic effects on PgR mRNA expression, cell cycle distribution, and anchorage-independent growth. Modest activity in controls is consistent with the low level of endogenous STS in the MCF-7/2 and Vector 3 cells and other MCF-7 populations (49).

Consistent with the estrogenic effects on cell cycle distribution and anchorage-independent growth, 17ß-estradiol sulfate is a potent mitogen in vivo. In ovariectomized mice supplemented with 17ß-estradiol sulfate, STS Clone 20 tumors arise with a higher incidence compared with that in untreated controls inoculated into the opposite flanks of the same mice. As control tumors are not supported by 17ß-estradiol sulfate, any endogenous STS activity in these cells is not sufficient to support full tumorigenesis.

Hydrolysis of estrone sulfate can stimulate the growth of N-nitroso-N-methylurea-induced mammary adenocarcinomas in castrated rats. This observation provides only circumstantial evidence, because a direct requirement for sulfatase activity in the in situ synthesis of estrogens was not demonstrated (53). For example, hepatic sulfatases could release 17ß-estradiol into the blood. Supplementation with 17ß-estradiol sulfate is not sufficient to support an increase in the tumorigenicity of Vector 3 cells. If hepatic sulfatases released biologically relevant concentrations of 17ß-estradiol, the incidence of Vector 3 tumors would have been increased. However, the small increase in Vector 3 tumor incidence, from 22% to 33%, is not statistically significant (P = 0.37). In marked contrast, the incidence of STS Clone 20 tumors, which arise in the opposite flanks of the same mice, is significantly increased (P < 0.001). These data, which provide a more direct assessment of the likely importance of intratumor STS in supporting tumor growth, clearly indicate that serum-derived estrogen sulfates can support estrogen-dependent tumorigenicity. Furthermore, hepatic STS activity seems biologically less important than intratumor STS.

Extrapolation of these data to human breast cancer requires a degree of caution. There may be differences in the pharmacokinetics of estrogen sulfates between mice and humans. For example, mice have different metabolic rates that require consideration in pharmacological/toxicological studies (54). MCF-7 cells probably reflect only one of several possible endocrine-responsive breast cancer phenotypes. Nonetheless, the consistent in vitro and in vivo responses exhibited by the STS-transfected cells, relative to their appropriate controls, strongly imply a likely role for this enzyme and serum estrogen sulfates in the biology of breast cancer. The in vivo data demonstrate that relevant levels of STS expression can support tumorigenesis and demonstrate the utility of this model to evaluate the role of this enzyme and estrogen sulfates further.

The in vitro and in vivo estrogenic effects of estrogen sulfates we observed are most likely a consequence of the liberation of free estrogens, as only free estrogens can bind and activate estrogen receptors (16). Treatment of STS Clone 20 cells with either ICI 182,780 or tamoxifen induces Go/G1 arrest, as is widely reported for MCF-7 cells (51). Antiestrogens primarily function by competing for estrogen activation of estrogen receptors (55). Thus, the mitogenic effects of the estrogen sulfates are primarily mediated though activation of estrogen receptors. The ultimate effector for estrone sulfate treatment is probably free intracellular 17ß-estradiol, as MCF-7 cells have detectable 17ß-hydroxysteroid dehydrogenase activity and can convert some estrone to 17ß-estradiol (56).

Our data provide limited evidence that exogenous estrogens may have different biological activity than estrogens released from estrogen sulfates within cells. Indeed, the estrogen sulfates apparently exhibit greater biological activity than equimolar concentrations of 17ß-estradiol, at least for some experimental end points. For example, 17ß-estradiol sulfate is more mitogenic than 17ß-estradiol in vitro, producing a greater increase in anchorage-independent colony formation in the STS Clone 20 cells. These differences are not consistently seen in the Vector 3 and MCF-7/2 cells. Although tumor volumes recorded at the end of a study contain only limited information (42), the volume of STS Clone 20 tumors in the 17ß-estradiol sulfate-treated animals (mean tumor volume, 138 mm3; n = 36) is greater than that of tumors arising in animals supplemented with 17ß-estradiol (mean tumor volume, 51 mm3; n = 6). The number of observations is limited, but the trend reflects our observations in anchorage-independent colony formation assays.

These in vitro and in vivo observations are surprising and clearly require further study. Why the sulfated estradiol may be more effective than the free hormone is unclear. If all estrogen sulfate was converted to 17ß-estradiol, it might be expected that the estrogen sulfates and 17ß-estradiol should be equieffective at equimolar concentrations. However, the increased activity is seen only in the STS cells. As the ability to increase free intracellular estrogen levels will reflect the relative activities of both the STS (increase unconjugated estrogen production) and steroid sulfotransferase enzymes (reduce unconjugated estrogen production), a high proportion of intracellular estrogen may be present as free 17ß-estradiol in STS Clone 20 cells.

Biological activity will reflect the concentrations of available intracellular estrogens able to interact with their receptors. Availability will be determined by binding to other extracellular and intracellular proteins (5, 57, 58), sequestration in cellular membranes (59), and distribution within extranuclear compartments. Free estrogen molecules are highly lipophilic, with some partitioning into cellular membranes and affecting membrane function (59, 60) while others eventually reach their nuclear receptors and activate gene transcription (55). In marked contrast, sulfated estrogen molecules are less lipophilic and require removal of the sulfate moiety (8), perhaps within specific plasma membrane domains rich in the STS enzyme, before they can reach the nucleus in substantial numbers. Thus, the cellular pharmacologies of exogenous sulfated and free estrogens may produce significantly different subcellular distributions of those estrogen molecules capable of eventually activating their receptors. This also could contribute to the different activities of free and sulfated estrogens in the STS cells. Studies to further address this hypothesis are currently in progress.

The high levels of intratumor 17ß-estradiol seen in breast cancers are probably multifocal in origin. These levels reflect a combination of uptake of 17ß-estradiol, estrone, and their sulfated metabolites from blood (4, 5) and the metabolism of circulating adrenal androgen precursors in neoplastic epithelium (61) and adjacent adipocytes (62). The predominant pathway may vary among tumors. Nonetheless, our data clearly establish the feasibility of uptake of sulfated estrogens from blood and their conversion to biologically active estrogens within breast tumors. Furthermore, the intratumor release of estrogens from their sulfates appears more important than the production of free estrogens by hepatic activation or other peripheral metabolism.

Our data also suggest that tumors and normal tissues that concurrently express both the aromatase and STS enzymes may be the most efficient at maintaining a highly estrogenic environment. The utility of aromatase inhibitors is well established. Inhibitors of STS have been generated (63), but their clinical utility is unclear, as aromatase inhibitors reduce the available substrate concentrations for STS. Differences in tissue distributions of aromatase and STS could produce a tissue-specific advantage for some STS inhibitors. Perhaps a combination of aromatase and STS inhibitors would produce a greater inhibition of intratumor estrogen concentrations by blocking the activation of any remaining sulfated estrogens. STS Clone 20 cells provide a new model to begin to address several of these issues in more detail.


   Acknowledgments
 
The authors thank Dr. Fabio Leonessa (Lombardi Cancer Center) for assistance with the growth assays, Mr. Robert Williams (Department of Pathology, Howard University) for the human placental tissue, and Ms. Kerrie Bouker for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by awards from the American Institute for Cancer Research (90BW65) and the USPHS (F31-CA-63923 to M.R.J., R01-CA/AG-58022, and USAMRMC BC980629 to R.C.). Several aspects of this work were supported by Shared Resources funded through the Lombardi Cancer Center’s Cancer Center Support Grant (USPHS P30-CA-51008): Animal Research Resource (to R.C.), Macromolecular Synthesis and Sequencing (to T.C.S.), Tissue Culture (to Dr. Michael Johnson), and Flow Activated Cell Sorting (to Dr. Owen Blair). Back

Received October 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. van Landeghem AA, Poortman J, Nabuurs M, Thijssen JH 1985 Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue. Cancer Res 45:2900–2906[Abstract/Free Full Text]
  2. McNeill JM, Reed MJ, Beranek PA, Bonney RC, Ghilchik MW, Robinson DJ, James VHT 1986 A comparison of the in vivo uptake and metabolism of 3H-oestrone and 3H-oestradiol by normal breast and breast tumor tissues in post-menopausal women. Int J Cancer 38:193–196[Medline]
  3. Pasqualini JR, Chetrite G, Blacker C, Feinstein MC, Delalonde L, Talbi M, Maloche C 1996 Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. J Clin Endocrinol Metab 81:1460–1464[Abstract]
  4. Miller WR 1997 Uptake and synthesis of steroid hormones by the breast. Endocr Related Cancer 4:307–311[Abstract/Free Full Text]
  5. Masamura S, Santner SJ, Gimotty P, Georger J, Santen RJ 1997 Mechanism for maintenance of high breast tumor estradiol concentrations in the absence of ovarian function: role of very high affinity tissue uptake. Breast Cancer Res Treat 42:215–226[CrossRef][Medline]
  6. Poutanen M, Miettinen M, Vihko R 1993 Differential estrogen substrate specificities for transiently expressed human placental 17ß-hydroxysteroid dehydrogenase and an endogenous enzyme expressed in cultured COS-m6 cells. Endocrinology 133:2639–2644[Abstract/Free Full Text]
  7. Falany JL, Falany CN 1996 Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines. Cancer Res 56:1551–1555[Abstract/Free Full Text]
  8. Pasqualini JR, Schatz B, Varin C, Nguyen BL 1992 Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer. J Steroid Biochem Mol Biol 41:323–329[CrossRef][Medline]
  9. Adams JB, Pewnim T, Chandra DP, Archibald L, Foo MS 1979 A correlation between estrogen sulfotransferase levels and estrogen receptor status in human primary breast carcinoma. Cancer Res 39:5124–5126[Abstract/Free Full Text]
  10. Pasqualini JR, Chetrite G, Nestour EL 1996 Control and expression of oestrone sulphatase activities in human breast cancer. J Endocrinol 150:S99–S105
  11. Santner SJ, Feil PD, Santen RJ 1984 In situ estrogen production via the estrone sulfate pathway in breast tumors: relative importance versus the aromatase pathway. J Clin Endocrinol Metab 59:29–33[Abstract/Free Full Text]
  12. Dowsett M 1997 Future use of aromatase inhibitors in breast cancer. J Steroid Biochem Mol Biol 61:261–266[CrossRef][Medline]
  13. Miller WR, Hawkins RA, Forrest AP 1982 Significance of aromatase activity in human breast cancer. Cancer Res 42:3365s–3368s
  14. Fishman J, Nisselbaum JS, Menendez-Botet C, Schwartz MK 1977 Estrone and estradiol content in human breast tumors: relationship to estradiol receptors. J Steroid Biochem 8:893–896[CrossRef][Medline]
  15. Vermeulen A, Deslypere JP, Paridaens R, LeClercq G, Roy F, Heuson JC 1986 Aromatase, 17ß-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normal glandular breast tissue in postmenopausal women. Eur J Cancer 22:515–525
  16. Clarke R, Hilakivi-Clarke LA, Cho E, James MR, Leonessa F 1996 Estrogens, phytoestrogens and breast cancer. Adv Exp Biol Med 401:63–86[Medline]
  17. Cocconi G 1994 First generation aromatase inhibitors–aminoglutethimide and testololactone. Breast Cancer Res Treat 30:57–80[CrossRef][Medline]
  18. Lonning PE, Dowsett M, Powles TJ 1989 Treatment of breast cancer with aromatase inhibitors - current status and future prospects. Br J Cancer 60:5–8[Medline]
  19. Lonning PE, Johannesson DC, Thoresen T 1989 Alterations in the production and clearance rate of estrone and estrone sulfate in breast cancer patients treated with aminoglutethimide. Br J Cancer 60:107–111[Medline]
  20. Lonning PE, Dowsett M, Powles TJ 1990 Postmenopausal estrogen biosynthesis and metabolism: alterations caused by aromatase inhibitors used for the treatment of breast cancer. J Steroid Biochem 35:355–366[CrossRef][Medline]
  21. Evans TR, Rowlands MG, Silva MC, Law M, Coombes RC 1993 Prognostic significance of aromatase and estrone sulfatase enzymes in human breast cancer. J Steroid Biochem Mol Biol 44:583–587[CrossRef][Medline]
  22. Lipton A, Santen RJ, Santner SJ, Harvey HA, Sanders SI, Matthews YL 1992 Prognostic value of breast cancer aromatase. Cancer 70:1951–1955[CrossRef][Medline]
  23. Evans TR, Rowlands MG, Law M, Coombes RC 1994 Intratumoral oestrone sulphatase activity as a prognostic marker in human breast carcinoma. Br J Cancer 69:555–561[Medline]
  24. Strobl JS, Lippman ME 1979 Prolonged retention of estradiol by human breast cancer cells in tissue culture. Cancer Res 39:3319–3327[Abstract/Free Full Text]
  25. Skaar TC, Prasad SC, Sharaeh S, Lippman ME, Brunner N, Clarke R 1998 Two- dimensional gel electrophoresis analyses identify nucleophosmin as an estrogen regulated protein associated with acquired estrogen-independence in human breast cancer cells. J Steroid Biochem Mol Biol 67:391–402[CrossRef][Medline]
  26. Clarke R, Brünner N, Thompson EW, Glanz P, Katz D, Dickson RB, Lippman ME 1989 The inter-relationships between ovarian-independent growth, antiestrogen resistance and invasiveness in the malignant progression of human breast cancer. J Endocrinol 122:331–340[Abstract/Free Full Text]
  27. Darbre P, Yates J, Curtis S, King RJB 1983 Effect of estradiol on human breast cancer cells in culture. Cancer Res 43:349–354[Abstract/Free Full Text]
  28. Miller AD, Rosman GJ 1989 Improved retroviral vectors for gene transfer and expression. BioTechniques 7:980–982[Medline]
  29. Markowitz DG, Goff SP, Bank A 1988 Safe and efficient ecotropic and amphotropic packaging lines for use in gene transfer experiments. Trans Assoc Am Physicians 101:212–218[Medline]
  30. Clarke R, Brünner N, Katz D, Glanz P, Dickson RB, Lippman ME, Kern F 1989 The effects of a constitutive production of TGF-{alpha} on the growth of MCF-7 human breast cancer cells in vitro and in vivo. Mol Endocrinol 3:372–380[Abstract/Free Full Text]
  31. Miller AD, Buttimore C 1986 Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 6:2895–2902[Abstract/Free Full Text]
  32. Clarke R, Brünner N, Katzenellenbogen BS, Thompson EW, Norman MJ, Koppi C, Paik S, Lippman ME, Dickson RB 1989 Progression from hormone dependent to hormone independent growth in MCF-7 human breast cancer cells. Proc Natl Acad Sci USA 86:3649–3653[Abstract/Free Full Text]
  33. Masamura S, Santner SJ, Heitjan DF, Santen RJ 1995 Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. J Clin Endocrinol Metab 80:2918–2925[Abstract/Free Full Text]
  34. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
  35. Brunner N, Boysen B, Jirus S, Skaar T, Holst-Hansen C, Lippman J, Frandsen T, Spang-Thomsen M, Fuqua SAW, Clarke R 1997 MCF7/LCC9: An antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182, 780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen. Cancer Res 57:3486–3493[Abstract/Free Full Text]
  36. LaBorda J 1991 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal protein PO. Nucleic Acids Res 19:3998[Free Full Text]
  37. Brünner N, Boulay V, Fojo A, Freter C, Lippman ME, Clarke R 1993 Acquisition of hormone-independent growth in MCF-7 cells is accompanied by increased expression of estrogen-regulated genes but without detectable DNA amplifications. Cancer Res 53:283–290[Abstract/Free Full Text]
  38. MacIndoe JH, Woods G, Jeffries L, Hinkhouse M 1988 The hydrolysis of estrone sulfate and dehydroepiandrostenedione sulfate by MCF-7 human breast cancer cells. Endocrinology 123:1281–1287[Abstract/Free Full Text]
  39. Vindelov LL, Christensen IJ, Nissen NI 1983 A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3:323–327[CrossRef][Medline]
  40. Seibert K, Shafie SM, Triche TJ, Whang-Peng JJ, O’Brien SJ, Toney JH, Huff KK, Lippman ME 1983 Clonal variation of MCF-7 breast cancer cells in vitro and in athymic nude mice. Cancer Res 43:2223–2239[Abstract/Free Full Text]
  41. Brünner N, Svenstrup B, Spang-Thompsen M, Bennet P, Nielsen A, Nielsen JJ 1986 Serum steroid levels in intact and endocrine ablated BALB nude mice and their intact litter mates. J Steroid Biochem 25:429–432[CrossRef][Medline]
  42. Clarke R 1997 Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models. Breast Cancer Res Treat 46:255–278[CrossRef][Medline]
  43. Clarke R 1996 Human breast cancer cell line xenografts as models of breast cancer: the immunobiologies of recipient mice and the characteristics of several tumorigenic cell lines. Breast Cancer Res Treat 39:69–86[CrossRef][Medline]
  44. Yen PH, Allen E, Marsh B, Mohandas T, Wang N, Taggart RT, Shapiro LT 1987 Cloning and expression of steroid sulfatase cDNA and the frequent occurrence of deletions in STS deficiency: implications for X-Y interchange. Cell 49:443–454[CrossRef][Medline]
  45. Evans TR, Rowlands MG, Luqmani YA, Chander SK, Coombes RC 1993 Detection of breast cancer-associated estrone sulfatase in breast cancer biopsies and cell lines using polymerase chain reaction. J Steroid Biochem Mol Biol 46:195–201[CrossRef][Medline]
  46. Pasqualini JR, Maloche C, Maroni M, Cherite G 1994 Effect of the progestagen promegesterone (R-5020) on mRNA of the estrone sulfatase in the MCF-7 human mammary cancer cells. Anticancer Res 14:1589–1594[Medline]
  47. Rabe T, Kiesel L, Radke G, Runnebaum B 1984 New assay for steroid sulfatase (EC 3.1.6.2) and its application for studies of human placental and skin sulfatase. J Steroid Biochem 20:677–682[CrossRef][Medline]
  48. Pasqualini JR, Gelly C, Lecerf F 1986 Estrogen sulfates: biological and ultrastructural responses and metabolism in MCF-7 human breast cancer cells. Breast Cancer Res Treat 8:233–240[CrossRef][Medline]
  49. Santner SJ, Ohlsson-Wilhelm B, Santen RJ 1993 Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures. Int J Cancer 54:119–124[Medline]
  50. Pasqualini JR, Nguyen B-L 1991 Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs independent human breast cancer cell lines. Breast Cancer Res Treat 18:93–98[CrossRef][Medline]
  51. Taylor IW, Hodson PJ, Green MD, Sutherland RL 1983 Effects of tamoxifen on cell cycle progression of synchronous MCF-7 human mammary carcinoma cells. Cancer Res 43:4007–4010[Abstract/Free Full Text]
  52. Lu Q, Liu Y, Long BJ, Grigoryev D, Gimbel M, Brodie A 1999 The effect of combining aromatase inhibitors with antiestrogens on tumor growth in a nude mouse model for breast cancer. Breast Cancer Res Treat 57:183–192[CrossRef][Medline]
  53. Santner SJ, Levin MC, Santen RJ 1990 Estrone sulfate stimulates growth of nitrosomethylurea-induced breast carcinoma in vivo in the rat. Int J Cancer 46:73–78[Medline]
  54. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE 1966 Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 50:219–244[Medline]
  55. Clarke R, Brünner N 1996 Acquired estrogen independence and antiestrogen resistance in breast cancer: estrogen receptor-driven phenotypes? Trends Endocrinol Metab 7:25–35
  56. Cherite G, Paris J, Botella J, Pasqualini JR 1999 Effect of nomegestrol acetate on estrone-sulfatase and 17ß-hydroxysteroid dehydrogenase activities in breast cancer cells. J Steroid Biochem Mol Biol 58:525–531
  57. Takahashi N, Breitman TR 1989 Covalent binding of 17ß-estradiol and retinoic acid to proteins in the human breast cancer cell line MCF-7. In Vitro Cell Dev Biol 25:1199–1200[Medline]
  58. Anderson E, Vinson GP, Puddefoot JR, Raven PW, Gilmore OJA 1986 Steroid sequestration and tightly bound oestrogen-protein complexes in human breast tumors and a breast cancer cell line. J Steroid Biochem 24:489–495[CrossRef][Medline]
  59. Clarke R, van den Berg HW, Murphy RF 1990 Tamoxifen and 17ß-estradiol reduce the membrane fluidity of human breast cancer cells. J Natl Cancer Inst 82:1702–1705[Abstract/Free Full Text]
  60. Improta-Brears T, Whorton AR, Codazzi F, York JD, Meyer T, McDonnell DP 1999 Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc Natl Acad Sci USA 96:4686–4691[Abstract/Free Full Text]
  61. Brodie AMH, Lu Q, Nakamura J 1997 Aromatase in the normal breast and breast cancer. J Steroid Biochem Mol Biol 61:281–286[CrossRef][Medline]
  62. Bulun SE, Simpson ER 1994 Breast cancer and expression of aromatase in breast adipose tissue. Trends Endocrinol Metab 5:113–120
  63. Duncan L, Purohit A, Howarth NM, Potter BV, Reed MJ 1993 Inhibition of estrone sulfatase activity by estrone-3-methylthiophosphonate: a potential therapeutic agent in breast cancer. Cancer Res 53:298–303[Abstract/Free Full Text]



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