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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Abstract |
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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; RTPCR, reverse transcriptionpolymerase chain reaction; SAL, D-saccharic acid 1,4-lactone; SULT, cytosolic sulfotransferase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
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Introduction |
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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-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- and 15
-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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Materials and methods |
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Analysis of OHE2 and MeOE2 metabolites by gas chromatographymass spectrometry (GCMS)
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 GCMS 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, [16,16,172H3]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 (ThermoquestFinnigan, 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 380385, collisionally activate these ions and then monitor the product ions also over a 5 Da range, m/z 300305. 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 (030 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 transcriptionpolymerase chain reaction (RTPCR)
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 I 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 exonexon junction. Each cycle consisted of denaturation at 95°C for 10 s, annealing for 15 s and amplification at 72°C for 3045 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 II
, 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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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 5001000 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, 0.055 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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Results |
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The final mass spectrum shows, in addition to the [M-H-SO3] ion at m/z 301, an ion of m/z 286 (Figure 3A). This ion of m/z 286 presumably arises from homolytic cleavage of the CO 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 3B
, 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 3B
) is indistinguishable from that of the 2-MeOE2-3S standard (Figure 3A
).
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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 II. The analysis of SULT1A1 and SULT1A3 expression in the cell lines is shown in Figure 4C
. Amplification with the primers indicated in Table I
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 4C
, 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 1B
). 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 4C). 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 4C
may represent heteroduplexes (31). The analysis of SULT1A2 mRNA expression is shown in Figure 4D
. 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 I. 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 II
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 4E. The PCR product obtained using the primers designed to amplify the cDNA of SULT1A1 exon IB (Table I
) was expected to be 959 bp and yield fragments of 467 and 492 bp after digestion with StyI (Table II
). 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 4C
). 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 4E) that was consistent with the observed pattern of 2-MeOE2 conjugation (Figure 1B
), 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 I
) 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 2B), 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 381301
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 3A
), 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 5
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Discussion |
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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-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
-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 RTPCR. 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 RTPCR 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 1B). 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 6) 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- and 15
-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 RTPCR 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 1B) 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-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.
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Notes |
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Acknowledgments |
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References |
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