Carcinogenesis, Vol. 21, No. 11, 1947-1957,
November 2000
© 2000 Oxford University Press
Cancer Biology |
SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells
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
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
and -15 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-OHE2 and 15-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.
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