SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells

Barbara C. Spink1, Barbara H. Katz1, Mirza M. Hussain1, Shaokun Pang1,2, Steven P. Connor1, Kenneth M. Aldous1,2, John F. Gierthy1,2 and David C. Spink1,2,3

1 Wadsworth Center, New York State Department of Health, Albany,NY 12201-0509 and
2 Department of Environmental Health and Toxicology, University at Albany, State University of New York, Albany,NY 12201-0509, USA


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 Materials and methods
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In a previous study of nine human breast-derived cell lines, rates of metabolism of 17ß-estradiol (E2) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E2 hydroxylation at the C-2, -4, -6{alpha} and -15{alpha} positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE2) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE2) by the action of catechol O-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude ß-glucuronidase/sulfatase preparation showed that most of the 2-MeOE2 present was in conjugated form, whereas 4-MeOE2, 6{alpha}-OHE2 and 15{alpha}-OHE2 were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE2 conjugates in MCF-7 cell media; the presence of 2-MeOE2-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription–polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE2 in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 (213Arg) and SULT1A1*2 (213His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE2 sulfonation activity. The SULT1A1 allelic variants were also expressed in Sf9 insect cells, from which post-microsomal supernatants were used to determine Km values of 0.90 ± 0.12 and 0.81 ± 0.06 µM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE2 as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE2 sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE2 that have been described recently.

Abbreviations: CYP, cytochrome P450; E1, estrone; E2, 17ß-estradiol; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gas chromatography; G/S, ß-glucuronidase/sulfatase; MeOE1, methoxyestrone; MeOE2, methoxyestradiol; 2-MeOE2-3S, 2-methoxyestradiol-3-sulfate; MS, mass spectrometry; OHE2, hydroxyestradiol; RT–PCR, reverse transcription–polymerase chain reaction; SAL, D-saccharic acid 1,4-lactone; SULT, cytosolic sulfotransferase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.


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Estrogens have long been associated with breast cancer. Based on established risk factors, it is thought that the cumulative exposure of a woman to endogenous and exogenous estrogens is an important determinant in susceptibility to this disease. Stimulation of breast-cell proliferation has been proposed as the main effect of estrogens in carcinogenesis. It has been hypothesized that the more rapidly cells proliferate, the greater the chance that random genetic errors will occur and will be propagated (15). Estrogen metabolism has been studied extensively in the context of breast cancer, not only because various metabolic pathways affect estrogen availability, which would in turn affect gene transcription and epithelial cell proliferation, but also because it results in the formation of metabolites with physiological properties quite distinct from those of the parent hormone.

The potential consequences of alterations in estrogen metabolism on the initiation and promotion of breast cancer are varied and complex, as individual pathways have been described as promoting or inhibiting carcinogenesis. The 4- and 16{alpha}-hydroxylation pathways of cytochrome P450-catalyzed metabolism are thought to give rise to metabolites with carcinogenic potential (68). Conversely, the 2-hydroxylation pathway is thought to be anticarcinogenic. 2-Methoxyestradiol (2-MeOE2), which is formed by the methylation of 2-hydroxyestradiol (2-OHE2), catalyzed by catechol O-methyltransferase (COMT), is thought to be protective against breast cancer, as antiproliferative, antiangiogenic and antitumorigenic properties have been attributed to this metabolite (9,10).

Efforts in this laboratory have been focused on characterizing the metabolism of 17ß-estradiol (E2) within human breast epithelial and tumor cells and determining how this metabolism is affected by exposure to endocrine-disrupting xenobiotics (1117). Our previous studies showed that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or non-ortho-substituted polychlorinated biphenyls had marked effects on E2 metabolism through aromatic hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1), an E2 2-, 6{alpha}- and 15{alpha}-hydroxylase (12), and cytochrome P450 1B1 (CYP1B1), an E2 4-hydroxylase with a lesser activity at C-2 (13,14). The enzymology and regulation of phase I metabolism, resulting in formation of the OHE2 metabolites, and the conversion of the catecholestrogens to the MeOE2 metabolites were focal points of these studies. However, it was apparent that phase II metabolism was also active in MCF-7 cells, as the analytical recovery of E2 and estrogen metabolites, notably 2-MeOE2, from the medium of MCF-7 cultures was dependent on treatment with a crude preparation containing both ß-glucuronidase and aryl sulfatase activities (11,16).

In a recent study, we employed a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines to investigate the roles of CYP1A1 and CYP1B1 in estrogen metabolism (17). These lines differ in tumorigenicity, cellular morphology, estrogen receptor (ER) expression and the relative expression of CYP1A1 and CYP1B1 in response to AhR agonists. In this study, we used this same series of breast-derived cell lines to evaluate the phase II metabolism, or conjugation, of endogenously produced estrogen metabolites.


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Cell culture and treatments
Cultures of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cells were propagated and maintained as described (17). For E2 metabolism studies, cultures at confluence were exposed to 10 nM TCDD or the solvent vehicle, 0.1% (v/v) dimethyl sulfoxide (DMSO) for 72 h followed by 1 µM E2 for 6 h. Media were then recovered and adjusted to pH 5 by addition of 10% acetic acid, and 2 ml aliquots were incubated at 37°C with or without ß-glucuronidase/sulfatase (G/S) (type H-2; Sigma, St Louis, MO) for hydrolysis of the metabolite conjugates for 6 or 18 h as indicated. For inhibition studies, the effects of the presence or absence of 1 mM D-saccharic acid 1,4-lactone (SAL; Sigma), 50 mM Na2SO4 or 100 mM NaCl during the incubation with G/S on the analytical recoveries of 2-MeOE2 were evaluated.

Analysis of OHE2 and MeOE2 metabolites by gas chromatography–mass spectrometry (GC–MS)
Samples of control and G/S-treated cell culture media were subjected to solid-phase extraction and preparation of the trimethysilyl derivatives of the metabolites (11). The metabolite derivatives were analyzed by GC–MS with selected-ion monitoring and quantification by stable-isotope dilution as described (11,16).

Analysis of 2-methoxyestradiol-3-sulfate (2-MeOE2-3S) by electrospray ion-trap MS
For the analysis of 2-MeOE2-3S, 10 µl containing 3 pmol of the internal standard, [16,16,17–2H3]2-MeOE2-3S, in electrospray buffer (1:1, CH3CN:H2O containing 1 mM ammonium acetate) was added either to media from cells exposed to 2-MeOE2 or to enzyme incubation mixtures with 2-MeOE2 as substrate. These samples were extracted twice with an equal volume of hexane to remove 2-MeOE2, and then twice with an equal volume of n-butanol for recovery of 2-MeOE2-3S. The n-butanol was evaporated under nitrogen and the residue was dissolved in 50 µl of electrospray buffer for analysis on an LCQ ion-trap mass spectrometer (Thermoquest–Finnigan, San Jose, CA) equipped with an electrospray ion source. The system was operated in the negative-ion mode at an electrospray potential of 4 kV. Samples were introduced without chromatographic separation by injection via a 5 µl loop into electrospray buffer flowing at 100 µl/min delivered by a model 510 HPLC pump (Waters, Milford, MA). Nitrogen was used as both the sheath and auxiliary gases. The capillary was maintained at 220°C and the capillary and tube offset potentials were both –35 V.

The electrospray ion-trap system was programmed to perform three stages of MS (MS3) or to monitor specific MS/MS transitions by using a selected-reaction monitoring technique. For analysis of 2-MeOE2-3S in the MS3 mode, the [M-H] ion of 2-MeOE2-3S of m/z 381 was isolated and collisionally activated by application of end-cap voltage (18,19), and the resultant [M-H-SO3] ion of m/z 301 was in turn isolated and collisionally activated; the final mass spectrum was then acquired by scanning from m/z 80 to 400. For quantitative determinations of 2-MeOE2-3S, the selected-reaction monitoring technique was used for the MS/MS transition resulting from the loss of SO3 from the [M-H] ion. In order to monitor this transition for the 2-MeOE2-3S analyte, m/z 381-> m/z 301, and simultaneously the corresponding transition for the [2H3]2-MeOE2-3S internal standard, m/z 384-> m/z 304, it was necessary to initially isolate ions over a 5 Da range, m/z 380–385, collisionally activate these ions and then monitor the product ions also over a 5 Da range, m/z 300–305. To determine the amount of 2-MeOE2-3S in a sample, the ratio of the peak area of the response at m/z 301 relative to that at m/z 304 was determined from selected-reaction monitoring analysis, and these values were compared with an 11-point calibration curve prepared by analyzing a series of standards containing a fixed amount of the [2H3]2-MeOE2-3S internal standard (3 pmol) and a variable amount of 2-MeOE2-3S (0–30 pmol).

The 2-MeOE2-3S standard was prepared by a facile synthesis with 2-methoxyestrone (2-MeOE1; Steraloids, Newport, RI) as the starting material. Sulfonation of 2-MeOE1 at the C-3 hydroxyl was achieved by reaction with the pyridine sulfur trioxide complex (20), which was obtained from Aldrich (Milwaukee, WI); reduction of the C-17 keto group with sodium borohydride (Aldrich) gave 2-MeOE2-3S. The deuterium-labeled analog of 2-MeOE2-3S was prepared using standard labeling techniques (11,21). Deuterium incorporation at C-16 of 2-MeOE1 was achieved by exchange in the presence of 2HCl in 2H2O (Aldrich); after the sulfonation reaction, reduction with sodium borodeuteride (Aldrich) yielded [16,16,17-2H3]2-MeOE2-3S.

Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA was isolated as described (22) from each cell line and 5 µg samples were reverse transcribed in a total volume of 20 µl using 200 units of Superscript II (Life Technologies, Grand Island, NY) followed by treatment with RNase H as recommended by the manufacturer. A 0.2 µl aliquot of the reverse-transcription reaction product was amplified by PCR in a total reaction volume of 44 µl containing core reagents from Perkin Elmer (Foster City, CA), TaqStart antibody (Clontech, Palo Alto, CA), Taq extender (Stratagene, La Jolla, CA) and 1.8 mM MgCl2. The primers for amplification of the cDNAs of five cytosolic sulfotransferases (SULTs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in Table IGo together with the appropriate annealing temperatures and PCR cycle numbers. Primers were designed using the GCG Wisconsin Sequence Analysis Package (Madison, WI) and were designed such that each amplified product spanned an exon–exon junction. Each cycle consisted of denaturation at 95°C for 10 s, annealing for 15 s and amplification at 72°C for 30–45 s followed by a final elongation at 72°C for 5 min. For analysis of the cDNAs of SULT1A1, SULT1A2 and SULT1A3 (for nomenclature of the SULT superfamily, see references 23 and 24), 12 µl of the PCR product mixture was digested with the restriction enzymes listed in Table IIGo, resolved on a 2% agarose gel and stained with 0.75 µg/ml ethidium bromide for visualization by UV-activated fluorescence.


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Table I. Primers used for the amplification of SULT and GAPDH cDNA sequences
 

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Table II. Restriction endonuclease digestion of SULT cDNA sequences
 
Cloning of SULT1A1 from MCF-7 cells
The entire coding sequence of SULT1A1 was amplified from cDNA prepared from MCF-7 cells using the high-fidelity Vent DNA polymerase (New England Biolabs, Beverly, MA). Because of the high degree of homology among the coding sequences of SULT1A1, SULT1A2 and SULT1A3, a reverse primer with an added BglII site (Table IGo) specific for SULT1A1 was designed in order to avoid amplifying artifactual chimeric cDNAs (25). A portion of the highly conserved start sequence, CAGGAACATG, was provided by the PCR Blunt vector (CAGG); the other portion, containing the Kozak sequence (26), was provided by the foward primer (AACATGG). After denaturation for 1 min at 95°C, 1 µl of cDNA in a reaction volume of 100 µl was amplified for 27 cycles, each consisting of denaturation at 97°C for 15 s, annealing at 70°C for 15 s and elongation at 72°C for 3 min, followed by a final elongation for 10 min at 72°C. After electrophoresis through 2% agarose, the band corresponding to 962 bp was excised, extracted and cloned into the PCR Blunt plasmid (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Plasmid DNA isolated from the clones was screened by restriction endonuclease digestion with EcoRI and BglII; positive clones were verified by sequencing the entire SULT1A1 coding region.

Stable transfection of SULT1A1 in MDA-MB-231 cells
SULT1A1 cDNA recovered from the PCR Blunt plasmid was subcloned into the HindIII/NotI sites of the expression vector, pcDNA 3.1(+) (Invitrogen, Carlsbad, CA) and used to transfect MDA-MB-231 cells. Cells were cultured in 6-well plates until they reached 80% confluence, after which 12 µl of Lipofectamine (Life Technologies) in 100 µl of serum-free medium and 2 µg of the vector in 100 µl of serum-free medium were combined, incubated at room temperature for 30 min, diluted to 1 ml with 800 µl serum-free medium and applied to each well of cells that had been previously rinsed with serum-free medium. After 6 h, the medium was replaced with DC5 medium (27) without phenol red. Seventy-two hours after transfection, cells were subcultured at 1:16 in DC5 medium containing 800 µg/ml Geneticin (Life Technologies). The cells were allowed to grow for ~3 weeks, after which colonies containing 500–1000 cells were isolated using cloning cylinders. These clones were maintained thereafter in medium containing 600 µg/ml Geneticin. After sufficient growth, cells were seeded into 6-well plates, grown to confluence and exposed to medium containing 2-MeOE2 for assay of 2-MeOE2-3S formation as described above.

Expression of SULT1A1 in Sf9 cells
The SULT1A1 cDNA recovered from the PCR Blunt plasmid was subcloned into the EcoRI/BglII sites of the baculovirus transfer vector, pVL1393, and co-transfected with 0.5 µg BaculoGold DNA (Pharmingen, San Diego, CA) into Sf9 cells according to the manufacturer's protocol. For expression, Sf9 cells were plated at a density of 2 x 105 cells/cm2 in TNM-FH medium containing 10% fetal bovine serum and amplified recombinant baculovirus was applied at a multiplicity of infection of 10 p.f.u./cell. After 24 h, medium was replaced and cells were cultured for an additional 48 h before harvesting.

Subcellular fractionation and assay of SULT1A1 activity
Post-microsomal supernatant was prepared from Sf9 cells expressing SULT1A1 or from confluent cultures of untreated MCF-7 cells. Sf9 cells were washed twice with phosphate-buffered saline, harvested and lysed in 50 mM potassium phosphate buffer, pH 7.0, with 10% glycerol and 100 µg/ml phenylmethylsulfonyl fluoride (Sigma) with three 6 s bursts with a Polytron homogenizer (Brinkmann, Westbury, NY), followed by three 35 s bursts with a series 4710 sonicator (Cole Palmer, Vernon Hills, IL). The lysate was centrifuged at 10 000 x g for 20 min. The supernatant was recovered and centrifuged at 100 000 x g for 1 h and the post-microsomal supernatant was recovered and stored at –80°C prior to determination of SULT activity. The same protocol was followed for preparation of the post-microsomal supernatant from MCF-7 cells, except that the cells were lysed by sonication only. Incubation mixtures for determination of SULT1A1 activity contained 5 or 45 µg of post-microsomal supernatant from Sf9 or MCF-7 cells, respectively, 0.05–5 µM 2-MeOE2, 10 µM 3'-phosphoadenosine 5'-phosphosulfate (PAPS; Sigma) and 10 mM potassium phosphate, pH 7.0, in a final volume of 300 µl. After incubation for 10 min (Sf9) or 15 min (MCF-7 cell supernatant) at 37°C, [2H3]-2-MeOE2-3S was added as an internal standard and samples were rapidly extracted for analysis of 2-MeOE2-3S by electrospray ion-trap MS with selected-reaction monitoring as described above.


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Conjugation of E2 metabolites in a series of human breast-derived cell lines
We previously reported the effects of pretreatment with TCDD on the metabolism of E2 in a series of human breast-derived cell lines (17). Figure 1Go shows these previous data but illustrates the effects of treatment with G/S on metabolite recovery by inclusion of the metabolite determinations without treating the media with G/S. Exposure of MCF-7 cultures to TCDD, which induces expression of CYP1A1 and CYP1B1, greatly enhanced the rate of metabolism of E2 to 2-MeOE2, 4-MeOE2, 6{alpha}-OHE2 and 15{alpha}-OHE2. On comparing the levels of these metabolites recovered from the media of TCDD-treated MCF-7 cells with and without treatment with G/S (Figure 1AGo), it was apparent that 96% of the 2-MeOE2 produced was in a conjugated form that was hydrolyzed by treatment with G/S, whereas only 27% of the 4-MeOE2 was conjugated, and 6{alpha}-OHE2 and 15{alpha}-OHE2 were not conjugated. Conjugation of 2-MeOE2 was also evident in MCF-7 cultures that had not been exposed to TCDD. In these control cultures, the medium contained 0.21 nM 2-MeOE2 without treatment with G/S and 2.6 nM 2-MeOE2 after treatment with G/S, indicating that 92% of the 2-MeOE2 was in conjugated form. In the control cultures, the medium contained 1.6 nM 4-MeOE2 without treatment with G/S and 2.0 nM 2-MeOE2 after treatment with G/S, indicating that 19% of the 4-MeOE2 was in conjugated form. The effect of treatment with G/S on the analytical recovery of 2-MeOE2 in a series of nine human-derived breast-cell lines pretreated with TCDD (17) is shown in Figure 1BGo. Extensive conjugation of 2-MeOE2 was evident in BT-20 cells and the ER{alpha}-positive cell lines, T-47D, ZR-75–1 and MCF-7.



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Fig. 1. Effect of treatment with G/S on the recovery of E2 metabolites from the media of human breast-derived cell lines in culture. (A) Confluent cultures of MCF-7 cells were treated with medium containing 10 nM TCDD or 0.1% (v/v) DMSO as control for 72 h followed by exposure to medium containing 1 µM E2 for 6 h. Aliquots (2 ml) of these media were incubated for 18 h at 37°C with or without G/S containing 5000 U ß-glucuronidase and 190 U aryl sulfatase and analyzed for OHE2 and MeOE2 metabolites. Rates of formation of 2-MeOE2 (open bars), 4-MeOE2 (black bars), 15{alpha}-OHE2 (vertically hatched bars), and 6{alpha}-OHE2 (horizontally hatched bars) were as indicated. (B) The formation of 2-MeOE2 in a series of nine breast-derived cell lines pretreated with TCDD was determined as in (A) and the media were treated with (open bars) and without (black bars) G/S before metabolite analysis. Metabolic rates are expressed relative to total cellular protein content and are the mean ± SD of four determinations. *Metabolite not detected (<0.5 pmol/h/mg).

 
Inhibition of G/S activities by SAL and Na2SO4
The conjugate of 2-MeOE2 formed in MCF-7 cells was hydrolyzed by treatment with a crude G/S preparation; however, it was not known which component, the ß-glucuronidase or the aryl sulfatase, catalyzed the hydrolysis. To investigate whether MCF-7 cells metabolize 2-MeOE2 to a sulfate or glucuronide metabolite, confluent cultures of MCF-7 cells were exposed to 3 nM 2-MeOE2 for 4 h. Media containing the 2-MeOE2 conjugate were treated for 6 h with G/S in the presence or absence of SAL, a glucuronidase inhibitor (28) or Na2SO4, a sulfatase inhibitor (29), before determination of unconjugated 2-MeOE2. Figure 2AGo shows that, at the end of the 4 h period, the majority of the 2-MeOE2 was present in MCF-7 cultures as a conjugated form that was hydrolyzed by treatment with G/S; unconjugated 2-MeOE2 in the medium was 0.55 nM. When the media were incubated with G/S in the presence of SAL, no effect on conjugate hydrolysis by G/S was observed. However, when the media were treated with G/S in the presence of 50 mM Na2SO4, conjugate hydrolysis catalyzed by the G/S preparation was inhibited. NaCl at 100 mM did not inhibit the hydrolysis of 2-MeOE2-3S catalyzed by G/S (data not shown). Identical experiments with untreated cultures of MDA-MB-231 cells showed no effects of G/S, SAL or Na2SO4 (Figure 2BGo), indicating a lack of 2-MeOE2-conjugating activity.



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Fig. 2. Inhibition of conjugate hydrolysis in MCF-7 and MDA-MB-231 cell media by SAL and Na2SO4. Media from confluent cultures of MCF-7 cells (A) or MDA-MB-231 cells (B) exposed to 3 nM 2-MeOE2 for 4 h were treated without or with G/S containing 3300 U ß-glucuronidase and 125 U aryl sulfatase only or in the presence of 1 mM SAL or 50 mM Na2SO4 for 6 h at 37°C. Unconjugated 2-MeOE2 was analyzed as described in Materials and methods. Data are the mean ± SE of four determinations.

 
Analysis of 2-MeOE2-3S by electrospray ion-trap MS
Since the results of the inhibitor studies suggested the formation of a sulfate conjugate of 2-MeOE2 in MCF-7 cells, we investigated analytical methods for the analysis of the putative sulfate conjugate of 2-MeOE2. The sulfate conjugates of E2 and estrone (E1) have been analyzed with high sensitivity by electrospray ionization tandem MS in the negative-ion mode (30). We investigated the use of similar techniques for the analysis of putative sulfate conjugates of 2-MeOE2. Experiments with synthetic 2-MeOE2-3S were undertaken to determine the fragmentation of 2-MeOE2-3S and to develop methods for the analysis of this metabolite. A scan function of MS3 was devised for the analysis of 2-MeOE2-3S, which started with the isolation of the molecular anion, [M-H], at m/z 381. This was followed by excitation and collisional activation, causing the loss of SO3 (80 Da) from the [M-H] ion, producing an ion with m/z 301. This loss of SO3 from the [M-H] ion occurs with estrogen 3-sulfates but not with estrogen 17ß-sulfates (30), probably because the negative charge is stabilized as a phenolate ion on fragmentation of the estrogen 3-sulfates. The m/z 301 ion was then isolated and collisionally activated and the final mass spectrum was recorded by scanning from m/z 80 to 400.

The final mass spectrum shows, in addition to the [M-H-SO3] ion at m/z 301, an ion of m/z 286 (Figure 3AGo). This ion of m/z 286 presumably arises from homolytic cleavage of the C–O bond of the methoxy group, resulting in a loss of a methyl radical (15 Da) from the [M-H-SO3] ion. An advantage of the MSn experiments afforded by the ion-trap mass spectrometer is that due to the high specificity of the fragmentation pathways of individual compounds, these experiments can be performed with impure samples. In Figure 3BGo, analysis of the n-butanol fraction from extraction of MCF-7 cell culture medium by using the same MS3 sequence, m/z 381->301->scan, is shown. The MCF-7 culture was exposed to 10 nM TCDD for 72 h followed by 1 µM E2 for 6 h; the final mass spectrum for analysis of this extract of culture medium (Figure 3BGo) is indistinguishable from that of the 2-MeOE2-3S standard (Figure 3AGo).



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Fig. 3. Analysis of 2-MeOE2-3S by electrospray ion trap MS. (A) One hundred picomoles of 2-MeOE2-3S standard in 5 µl of electrospray buffer was introduced into the mass spectometer by loop injection. The MS3 sequence, m/z 381->301->scan 80–400, was performed, producing the final mass spectrum as shown. The inset structure shows the proposed cleavages of 2-MeOE2-3S resulting in the losses of SO3 (80 Da) and CH3 (15 Da). (B) The n-butanol fraction from extraction of culture medium from TCDD-treated MCF-7 cells, which initially contained 1 µM E2, was analyzed by using the same MS3 scan function.

 
Analysis of SULT mRNA expression by RT–PCR
Having established that MCF-7 cells convert 2-MeOE2 to 2-MeOE2-3S, we next initiated studies to ascertain which member(s) of the SULT family catalyzed this sulfonation. To determine which members of the SULT gene family were expressed in the breast-cell lines under investigation, we analyzed the mRNA expression patterns of several of the more important members of the SULT family by using RT–PCR. Analysis of expression of five SULT genes and the GAPDH gene in nine breast-cell lines and in human liver is shown in Figure 4Go. In order to detect differences among the expression levels of the mRNAs, 25 cycles of PCR were used for all amplifications of SULT cDNAs, whereas 21 cycles were used for amplification of the GAPDH cDNA, as the mRNA encoding GAPDH is very abundant. Predicted product sizes after amplification of SULT and GAPDH cDNAs are presented in Table IGo. Representative PCR product from amplification with each of the primer sets was sequenced to verify the identity of the amplified DNA.



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Fig. 4. PCR amplification of sulfotransferase and GAPDH cDNAs from nine breast cell lines. RT–PCR was performed as described in Materials and methods with primers as indicated in Table IGo. Expected product sizes and/or restriction endonuclease fragment sizes are listed in Tables I and IIGoGo. Sample loadings were: lane 1, 100 bp DNA ladder; lane 2, no cDNA control; and lanes 3–12, cDNA from 184A1 (lane 3), MCF-10A (lane 4), T-47D (lane 5), ZR-75-1 (lane 6), MCF-7 (lane 7), BT-20 (lane 8), MDA-MB-231 (lane 9), MDA-MB-157 (lane 10), MDA-MB-436 (lane 11) and human liver (lane 12). The expression of SULT2A1 (A), SULT1E1 (B), SULT1A3 and SULT1A1 (C), SULT1A2 (D), exon 1B of SULT1A1 (E) and GAPDH (F) was analyzed; the arrows denote the expected sizes of the PCR products or expected fragments after restriction endonuclease digestion with StyI (C and E) or HindIII (D).

 
Primers specific for the amplification of SULT2A1 cDNA (Table IGo) were used in the analysis shown in Figure 4AGo. No detectable amplification of the SULT2A1 sequence was observed with cDNA from any of the cell lines. Only with cDNA of human liver was the expected 277 bp PCR product of SULT2A1 cDNA obtained (Figure 4AGo, lane 12), whereas the GAPDH cDNA sequence was readily amplified from cDNA of each of the cell lines and of human liver (Figure 4GoF). Primers specific for the amplification of SULT1E1 cDNA were used in the analysis shown in Figure 4BGo. Amplification of cDNA of the 184A1 and MCF-10A lines of immortalized breast epithelial cells showed the expected 464 bp product for the SULT1E1 cDNA (Figure 4BGo, lanes 3 and 4), as did amplification of human liver cDNA (Figure 4BGo, lane 1 and 2), but cDNA of none of the other cell lines gave rise to appreciable SULT1E1 PCR product.

Due to the very high homology among the SULT1A1, SULT1A2 and SULT1A3 nucleotide sequences, it was not feasible to design PCR primers to amplify the SULT1A1, SULT1A2 and SULT1A3 cDNAs individually. As an alternative approach, we designed a set of primers that would amplify the three cDNAs simultaneously. We then used restriction endonuclease digestion of the PCR products with StyI and HindIII to distinguish among the three SULT cDNAs. Predicted sizes of the fragments produced by digestion of the SULT1A1, SULT1A2 and SULT1A3 cDNAs with StyI and HindIII are indicated in Table IIGo. The analysis of SULT1A1 and SULT1A3 expression in the cell lines is shown in Figure 4CGo. Amplification with the primers indicated in Table IGo should produce 767 bp cDNA products of SULT1A1, SULT1A2 and SULT1A3. Treatment with StyI should cleave the SULT1A1 PCR product into fragments of 437 and 330 bp. The 437 and 330 bp fragments were observed for analysis of human liver cDNA (Figure 4CGo, lane 12) and at varying levels on analysis of all of the cell lines except for MDA-MB-231 (lane 9) and MDA-MB-436 (lane 11). This pattern of expression of SULT1A1 mRNA correlated well with the observed conjugation of 2-MeOE2 in these cell lines, as neither MDA-MB-231 nor MDA-MB-436 cells showed conjugation of 2-MeOE2 (Figure 1BGo). The same expression pattern of SULT1A1 mRNA was observed in cultures pretreated with 10 nM TCDD for 72 h as in the DMSO-treated control cultures (results not shown).

In a similar way, treatment with StyI is expected to cleave the SULT1A3 PCR product into fragments of 656 and 111bp. The 656 bp band representing the cleavage fragment of the SULT1A3 cDNA was observed from cDNA of each of the cell lines and, at a very low level, with cDNA from human liver (Figure 4CGo). These results indicate that expression of SULT1A3 mRNA did not correlate with the observed pattern of 2-MeOE2 conjugation in the cell lines, as SULT1A3 expression was observed in MDA-MB-231 and MDA-MB-436 cells, while neither cell line conjugated endogenously produced 2-MeOE2. The undigested 767 bp band observed in several lanes of Figure 4CGo may represent heteroduplexes (31). The analysis of SULT1A2 mRNA expression is shown in Figure 4DGo. Treatment with HindIII should cleave the SULT1A2 PCR product into fragments of 468 and 299 bp. These fragments were not observed on analysis of cDNA of human liver or of any of the cell lines, indicating that the SULT1A2 mRNA was not expressed at an appreciable level.

Amplification of 5'-noncoding variants of SULT1A1 cDNA
Results of previous molecular studies indicate that there are several forms of SULT1A1 mRNA that differ in the 5' untranslated region, resulting from alternative promoter and splice sites of the RNA. To determine which of these variants were expressed in the cell lines under investigation, specific primers were designed to amplify the 5'-untranslated regions of SULT1A1 cDNA, exons IB, IA (32), and the intron region between exons IA and II, referred to as exon IIA (33) or intron IA (34). The sequences of these primers are presented in Table IGo. Representative PCR products were sequenced to verify the identity of the amplified DNA. Twenty-five cycles of PCR were used for amplification such that only the more abundant cDNAs would be detected. It was necessary to digest with the restriction enzymes listed in Table IIGo to identify the SULT1A1 PCR product, since it is possible to amplify SULT1A2 and SULT1A3 cDNAs from homologous 5'-untranslated regions that occur in these cDNAs (32).

Results from the analysis of expression of the SULT1A1 exon IB transcript are shown in Figure 4EGo. The PCR product obtained using the primers designed to amplify the cDNA of SULT1A1 exon IB (Table IGo) was expected to be 959 bp and yield fragments of 467 and 492 bp after digestion with StyI (Table IIGo). The unresolved doublet of 467 and 492 bp fragments was observed on analysis of cDNA of human liver and cDNAs of each of the cell lines except for MDA-MB-231 and MDA-MB-436. The expression of SULT1A1 exon IB mRNA thus followed a very similar pattern to that seen when the primers that amplify the coding sequence of SULT1A1 cDNA were used (Figure 4CGo). Very little or no PCR product was observed when the primers specific for exon IA and exon IIA cDNA transcripts were used (data not shown). Only with high PCR-cycle numbers did we observe cDNA derived from these 5'-non-coding variant mRNAs. With 37 cycles of PCR, we observed a low level of expression for the exon IA splice variant of the 747 bp and 946 bp SULT1A1 PCR products in ZR-75-1 and T-47D cells. In human liver, the 747 bp variants of the SULT1A1 and SULT1A2 cDNAs were also observed. With 33 cycles of PCR, expression of the exon IIA variant transcript of SULT1A1 was evident in human liver and all the cell lines except for MDA-MB-157, MDA-MB-231 and MDA-MB-436. Expression of the exon IIA variant transcript of SULT1A3 was observed in all of the cell lines examined with the exception of BT-20; it was not found to be expressed in human liver (results not shown).

Cloning of SULT1A1 cDNA from MCF-7 cells
SULT1A1 mRNA, and specifically the exon IB mRNA variant, showed a pattern of expression in the breast-cell lines (Figure 4EGo) that was consistent with the observed pattern of 2-MeOE2 conjugation (Figure 1BGo), suggesting that SULT1A1 may catalyze sulfonation of 2-MeOE2. To investigate this hypothesis, we initiated efforts to clone a SULT1A1 cDNA and express the enzyme for activity determinations. SULT1A1 DNA was obtained by performing PCR with cDNA prepared from MCF-7 cells using a primer set (Table IGo) that spanned the entire coding sequence of SULT1A1 and utilizing a SULT1A1-specific reverse primer. This DNA was cloned into the PCR Blunt plasmid. Sequence analysis of resultant clones confirmed the presence of only SULT1A1 cDNA and revealed the presence of two SULT1A1 sequences that differed at nucleotide positions (numbered from ATG) 153, 162, 600 and 638. The sequences of one series of clones showed the allelic variants 153T, 162A, 600G and 638G (identical to GenBank accession number X78283), and the other series of clones showed the allelic variants 153C, 162G, 600C and 638A (identical to GenBank accession number L19955). Only the nucleotide substitution of A for G at position 638 results in an amino acid substitution, which is histidine for arginine at codon 213. The more common 213Arg variant is referred to as SULT1A1*1, whereas the 213His variant is referred to as SULT1A1*2. We were thus able to clone cDNAs encoding two polymorphic forms of SULT1A1 from MCF-7 cells.

Stable transfection of SULT1A1 cDNAs in MDA-MB-231 cells
To determine whether the SULT1A1 forms catalyze the conversion of 2-MeOE2 to 2-MeOE2-3S, we performed heterologous cDNA-directed expression of SULT1A1*1 and SULT1A1*2. Cultures of MDA-MB-231 cells showed no detectable conjugation of 2-MeOE2 (Figures 1B and 2BGoGo), so this cell line was appropriate for these transfection experiments.

SULT1A1 DNA was cloned into the expression vector, pcDNA 3.1(+) and used to stably transfect MDA-MB-231 cells. Confluent cultures of several clones were exposed to 2-MeOE2 for 1 h and the media were then extracted for analysis of 2-MeOE2-3S by electrospray ion-trap MS. The mass spectrum obtained for the sequence m/z 381->301->scan from the analysis of the medium from a SULT1A1*2-expressing, MDA-MB-231-derived clone after exposure to 2-MeOE2 was indistinguishable from the analogous spectrum recorded for the 2-MeOE2-3S standard (Figure 3AGo), as only peaks of m/z 301 and 286 were observed, and the intensity of the m/z 301 peak was 85% that of the m/z 286 peak (results not shown). The selected-reaction monitoring technique was used to quantify 2-MeOE2-3S formation in cultures of MDA-MB-231-derived clones stably expressing the allelic variants of SULT1A1. Several clones were obtained for both SULT1A1*1 and SULT1A1*2 that were able to convert 2-MeOE2 added to the medium to 2-MeOE2-3S, whereas stable transfection in MDA-MB-231 cells with the vector alone produced only clones that were unable to produce detectable 2-MeOE2-3S. The rates of 2-MeOE2-3S formation of three representative clones were as indicated in Figure 5Go.



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Fig. 5. Stable transfection of SULT1A1 cDNAs in MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with cDNA encoding the SULT1A1*1 (black bars) or SULT1A1*2 (gray bars), cloned into pcDNA3.1(+) or with vector alone (open bars). Transfectants in 6-well plates were exposed to 2 ml of medium containing 100 or 500 nM 2-MeOE2 for 1 h. Media were then collected, internal standard was added and 0.5 ml portions were extracted as described in Materials and methods. The resultant n-butanol fractions were analyzed for 2-MeOE2-3S formation by electrospray ion-trap MS with selected-reaction monitoring. Product formation is expressed relative to total cellular protein content. Data are the mean ± SE of three determinations. *Metabolite not detected (<5 pmol/h/mg).

 
Kinetic analysis of SULT1A1*1 and SULT1A1*2 expressed in Sf9 cells
To evaluate the kinetics of SULT1A1*1 and SULT1A1*2 forms with 2-MeOE2 as substrate, soluble preparations of the enzymes were necessary. These were readily obtained by expressing the two enzyme forms in Sf9 cells and preparing post-microsomal supernatant fractions. Sulfotransferase activity in these preparations and similar ones from MCF-7 cells were assayed with 2-MeOE2 as substrate and determination of the 2-MeOE2-3S product by electrospray ion-trap MS with selected-reaction monitoring. Rates of product formation were linear for 14 min for enzyme expressed in Sf9 cells (r2 > 0.994) and 30 min for MCF-7 post-microsomal supernatant (r2 = 0.997) under our assay conditions. Post-microsomal supernatant from Sf9 cells infected with the control vector, pVL1392XylE (Pharmingen, San Diego, CA) showed no activity; however, both polymorphic forms of expressed SULT1A1 showed robust activity with 2-MeOE2 as substrate. The activities of SULT1A1*1 and SULT1A1*2 as a function of varying 2-MeOE2 concentration are shown in Figure 6Go. The values of Km and Vmax derived from these data for expressed SULT1A1*1 and SULT1A1*2 are also presented. Similar determinations with post-microsomal supernatant from MCF-7 cells produced values of 0.88 ± 0.22 mM and 0.070 ± 0.007 nmol/min/mg protein for Km and Vmax, respectively.



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Fig. 6. Effect of varying 2-MeOE2 concentration on the activities of SULT1A1 forms expressed in Sf9 cells. The cDNAs of the two polymorphic forms, SULT1A1*1 and SULT1A1*2, were cloned into pVL1393 and the enzyme forms were expressed in Sf9 cells. Post-microsomal supernatants were prepared and assayed for SULT activity with 2-MeOE2 as substrate and metabolite determination by electrospray ion-trap MS with selected-reaction monitoring. Shown are the rates of sulfation catalyzed by SULT1A1*1 (A) and SULT1A1*2 (B) in the presence of 10 µM PAPS and 0.05, 0.1, 0.2, 0.3, 0.6, 1.0, 2.0 or 5.0 µM 2-MeOE2. Data are the mean ± SE of three determinations and were fitted to the Michaelis–Menton equation to produce the values of Km and Vmax and associated standard errors.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cell lines used in these studies were chosen to represent a wide spectrum of breast-cell types, ranging from normal epithelial cells to highly invasive tumor cells with metastatic potential. In our previous study (17) we found that these cell types varied widely in the expression and inducibility of the E2 hydroxylases, CYP1A1 and CYP1B1. In the lines derived from normal breast epithelial cells and ER{alpha}-positive tumor cells, induction of CYP1A1 was favored over that of CYP1B1, and E2 metabolism was primarily through the 2-hydroxylation pathway, resulting in predominantly 2-MeOE2 formation. In contrast, in the ER{alpha}-negative tumor lines, MDA-MB-231, MDA-MB-157 and MDA-MB-436, induction of CYP1B1 was favored over that of CYP1A1, and rates of formation of 4-MeOE2 exceeded those of 2-MeOE2.

In the present study we observed another aspect of estrogen metabolism that varies among these cell lines, which was the proportion of the 2-MeOE2 produced that was present in conjugated form. In the ER{alpha}-positive lines, T-47D, ZR-75-1 and MCF-7, in which CYP1A1 was highly induced, high rates of 2-MeOE2 formation were observed and >90% of the 2-MeOE2 produced was present in conjugated form. This is in contrast to what was observed with the ER{alpha}-negative cell lines, MDA-MB-157, MDA-MB-231 and MDA-MB-436. In MDA-MB-157 only about half of the 2-MeOE2 was conjugated, and in MDA-MB-231 and MDA-MB-436 cells no conjugation of 2-MeOE2 was observed. Inhibition studies of the G/S-catalyzed hydrolysis of 2-MeOE2 conjugates in MCF-7 media suggested that this conjugation was mainly sulfation rather than glucuronidation, and expression of 2-MeOE2 sulfonation activity in MCF-7 cells was confirmed in assays employing electrospray ion-trap MS.

To investigate which of the SULT enzymes could potentially be responsible for the observed conjugation of 2-MeOE2, we examined the expression of five of the major SULTs at the mRNA level in the nine breast-cell lines by RT–PCR. We did not observe significant expression of SULT2A1 or SULT1E1 mRNAs in any of the tumor-derived breast cell lines, which agrees well with other reports of SULT enzyme activities (3537). SULT2A1 mRNA was expressed in human liver, which agrees with previous reports of its enzyme activity (36,38). A high level of expression of SULT1E1 mRNA was observed in the 184A1 immortalized breast epithelial cell line. We did not detect expression of SULT1A2 mRNA by RT–PCR in the breast-cell lines or in human liver, even when PCR was performed at high cycle numbers. SULT1A1 mRNA was found to be the major transcript in liver compared with SULT1A2 and SULT1A3 mRNAs, which agrees with a previous report (39). SULT1A3 mRNA was expressed in all breast cells lines examined, which is consistent with a previous study of enzyme activities in several of the same cell lines (35).

Only the expression of SULT1A1 mRNA showed a close correlation with the the pattern we observed for extensive conjugation of the 2-MeOE2 formed (Figure 1BGo). Both the level of conjugation of 2-MeOE2 and the expression of SULT1A1 mRNA were particularly high in BT-20, T-47D, ZR-75-1 and MCF-7 cells, and neither conjugation of 2-MeOE2 nor SULT1A1 mRNA expression was observed in MDA-MB-231 or MDA-MB-436 cells. Additionally, we found that, of the different 5'-untranslated forms of SULT1A1 mRNA (32), the variant containing exon IB was expressed at higher levels than those containing exons IA and IIA. Studies in human liver indicate that transcription initiation occurs most often 5' to exon IB (34). In comparing the 5'-flanking sequences upstream from exons IB and II in a chloramphenicol acetyl transferase expression vector transfected in human embryonal kidney cells, Bernier et al. (33) found that exon IB had the higher promoter activity. The significance of these variant 5'-untranslated forms in tissue-specific expression is, at present, unknown.

Upon cloning the SULT1A1 cDNA from MCF-7 cells, we found that these cells expressed two previously characterized allelic variants, SULT1A1*1 and SULT1A1*2. Both variants, when expressed in either MDA-MB-231 cells or Sf9 cells, were able to catalyze the sulfonation of 2-MeOE2 at the C-3 hydroxyl. This is the first report of a cytosolic SULT catalyzing the sulfonation of 2-MeOE2. The values of Km (Figure 6Go) for SULT1A1*1 or SULT1A1*2 with 2-MeOE2 as a substrate were similar to the respective values of Km determined when p-nitrophenol was used as the substrate (4042). SULT1A1*2, which is expressed in platelets, has lower thermal stability and lower activity for p-nitrophenol conjugation than SULT1A1*1 (43). The allele frequencies of SULT1A1*1 and SULT1A1*2, respectively, are ~83.2% and 16.8% in Japanese (42), 73.1% and 26.9% in Africans (44) and 68% and 32% in Caucasians (40,44). There is some evidence that the SULT1A1*1 allozyme may be associated with protection against cell or tissue damage during aging, as it has been reported that there is an increasing incidence of SULT1A1*1 homozygosity and decreasing incidence of SULT1A1*2 homozygosity with increasing age (44).

The observed activities of SULT1A1*1 and SULT1A1*2 with 2-MeOE2 as substrate and the ability of breast-cell lines expressing SULT1A1 to conjugate nanomolar levels of 2-MeOE2 suggest that 2-MeOE2 may be a physiological substrate of SULT1A1. SULT1A1 appears to show specificity for 2-MeOE2 as a substrate, as 4-MeOE2 was conjugated to a far lesser extent, and conjugation of 6{alpha}- and 15{alpha}-OHE2 was not observed. Concentrations of unconjugated circulating 2-methoxyestrogens range from 0.15 nM to 0.23 nM in the menstrual cycle (45), and reach 30 nM during pregnancy (45,46). The levels of circulating 2-methoxyestrogens in conjugated form are 6- and 1.5-fold higher than those of the unconjugated metabolites in ovulating and pregnant women, respectively (45). The major circulating 2-methoxyestrogen is 2-MeOE1, although 2-MeOE1 and 2-MeOE2 may be interconverted by the action of 17ß-hydroxysteroid dehydrogenase (9). 2-MeOE1 might also be expected to be a substrate for SULT1A1; this possibility is currently under investigation.

Although the activities of SULT forms with 2-MeOE2 as substrate have not been previously determined, the activities of a number of enzymes of the SULT gene family with several estrogens and other steroid hormones as substrates have been evaluated (47). SULT2A1, which is also referred to as dehydroepiandrosterone sulfotransferase, catalyzes the conjugation of E2 and E1, but only with micromolar levels of substrate. SULT1A3, which is also referred to as the monoamine sulfonating form of phenol sulfotransferase, does not catalyze the sulfonation of estrogens (38). Although both SULT1A1 (48), the phenol-sulfating form of phenol sulfotransferase, and SULT1E1 (49), or estrogen sulfotransferase, catalyze the conjugation of estrogens, SULT1E1 appears to be the most efficient in catalyzing sulfation of E2 and E1 present at physiological, nanomolar concentrations. In MCF-7 breast tumor cells, which do not express significant levels of SULT1E1, heterologous cDNA-directed expression of SULT1E1 suppresses E2-stimulated cell growth and gene expression, presumably by converting E2 to E2-3-sulfate, which does not bind to or activate the ER (37,50). While SULT1E1 is active with E2 and E1 as substrates, it may not act on estrogen metabolites such as 2-MeOE2. Analysis by RT–PCR showed a high level of SULT1E1 mRNA in 184A1 cells. If SULT1E1 is expressed in 184A1 cells, its activity did not result in significant conjugation of 2-MeOE2 (Figure 1BGo) under these conditions where 2-MeOE2 was present in TCDD-treated cultures at 22 nM.

Recent studies indicate that 2-MeOE2 is a unique, protective E2 metabolite (reviewed in reference 9) that inhibits the proliferation of many human cancer cell lines, with breast cancer cells showing the greatest sensitivity to nanomolar concentrations of this metabolite (51,52). 2-MeOE2 also inhibits angiogenesis in vivo and in vitro and at higher concentrations exhibits cytotoxicity (10,53). One biochemical mechanism of 2-MeOE2 that has been identified is that, at micromolar concentrations, 2-MeOE2 inhibits tubulin polymerization by interacting at the colchicine-binding site (54). Cell-free experiments in which tubulin polymerization is inhibited indicate a direct effect of 2-MeOE2 rather than a putative conjugate; the formation of a sulfate conjugate with diminished activity may attenuate this activity. However, this interaction with tubulin may not fully account for the antitumor properties of 2-MeOE2 that have been observed. It is conceivable that other effects of 2-MeOE2 may be mediated by its conversion to 2-MeOE2-3S, as sulfonation of some drugs, carcinogens and endogenous steroids produces more active compounds (23,55). The formation of 2-MeOE2-3S catalyzed by SULTs and the possible hydrolysis of 2-MeOE2-3S catalyzed by sulfatases are metabolic reactions that should be considered in investigations of the physiological effects of 2-MeOE2.

In breast cells in which E2 is converted to 2-MeOE2 through enhanced metabolism elicited by AhR agonists, cell proliferation may be decreased in two ways: by removal of E2, which enhances proliferation in ER{alpha}-positive cells, and by production of 2-MeOE2, a non-estrogenic metabolite that inhibits cell proliferation. SULT1A1 may have a specific role in modulating the antiproliferative effects of 2-MeOE2 in these cell lines by catalyzing the sulfonation of 2-MeOE2. Expression of SULT1A1 may determine which cell types are more or less susceptible to the antiproliferative, antiangiogenic and cytotoxic effects of endogenously produced or exogenously administered 2-MeOE2.


    Notes
 
3 To whom correspondence should be addressed E-mail: spink{at}wadsworth.org Back


    Acknowledgments
 
The authors gratefully acknowledge use of the Wadsworth Center's Biochemistry and Molecular Genetics Core Facilities. This research was supported by NIH grants CA81243, ES04913 and ES03561.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 31, 2000; revised July 26, 2000; accepted August 11, 2000.