Role of porcine endometrial estrogen sulfotransferase in progesterone mediated downregulation of estrogen receptor.

Estrogen sulfotransferase (EST) is a progesterone (Pg) induced secretory endometrial enzyme which may effect estrogen receptor levels by esterifying estradiol-17 beta (E2) to an inactive, sulfate form. The effects of this enzyme were studied using specific inhibitors of EST that do not bind to estrogen receptor (ER): 4-nitroestrone 3-methyl ether and 4-fluoroestrone 3-methyl ether. A 1 h pulse with 4 nM E2 caused ERn (i.e. E2-bound, chromatin-bound receptor) to increase 40% in incubations of proliferative gilt endometrium (no EST activity), while the same E2 treatment of secretory endometrium (high EST activity) caused no increase in ERn. ERn accumulation was completely restored in these experiments by preincubating secretory endometrium with 4 microM 4-fluoroestrone 3-methyl ether. Gilt endometrial explants cultured 7 days with 1 nM E2 plus 1 microM Pg (which induced EST activity) possessed half the ERn as explants devoid of EST activity which were cultured in E2 alone. The addition of 10 microM 4-nitroestrone 3-methyl ester to the cultures of secretory endometrium restored ERn to the levels seen in minces cultured with E2 alone. Furthermore, ovariectomized gilts injected daily with 250 micrograms E2 plus 25 mg Pg had much lower ERn (0.06 fmol/micrograms DNA) than gilts injected with E2 only (0.21 fmol/microgram DNA). ERn was restored completely by supplementing the E2 plus Pg injections with 0.5 g 4-nitroestrone 3-methyl ether administered by vaginal suppositories.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine steroid sulfotransferases: porcine endometrial estrogen sulfotransferase.

Porcine endometrial estrogen sulfotransferase has been isolated and its properties examined. This enzyme only appeared in uteri from ovariectomized gilts which had been primed with estrogen and treated with progesterone. The most stable form of the enzyme was obtained via chromatofocusing of the 100,000 g supernatant from secretory endometrium. A molecular weight of 31 KDa was determined for this sulfotransferase by molecular sieve (Sephadex G-200 Superfine) and disk-gel electrophoresis. The active protein displayed a pI of 6.1, pH optimum of 7.6-7.8 and a requirement of 10 mM Mg2+ for maximum transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to estrone (E1). Km of the reaction was 24 +/- 4.7 microM for PAPS and 24 +/- 9.8 nM for E1 as substrate. Porcine endometrial sulfotransferase thus displayed a much greater affinity for E1 than a similar enzyme previously isolated from bovine adrenals. As has been observed of sulfotransferases from other tissues, an endogenous substrate (presumed to be E1) accompanies the enzyme throughout its purification.

Endocrine steroid sulfotransferases: steroid alcohol sulfotransferase from human breast carcinoma cell line MCF-7.

Steroid alcohol sulfotransferase (SAS) has been isolated from the cytosol of a human breast carcinoma cell line, MCF-7. This enzyme from Sephadex G-200 chromatography displayed a mol. wt of 118 KDa. The conditions for optimal enzymic activity of SAS were determined to be 20 min incubations at 45 degrees C in 0.2 M Tris buffer (pH 7.5) containing 0.06 M Mg2+. Chromatofocusing chromatography also yielded a single peak of SAS with a pI of 5.8. Results from the incubations of a series of androstane analogues revealed that SAS required a 3 beta-hydroxyl on a steroid with the trans bridge between the A and B rings. Neither the 3 beta-allylic hydroxyl group nor the A-ring phenolic 3-hydroxyl accepted the sulfate group from 3'-phosphoadenosine-5'-phosphosulfate. D-ring beta-hydroxyl groups were tolerated by the enzyme, however, alpha-hydroxyl groups on the D-ring appeared to interfere with the reaction. Sulfurylation of steroids by SAS was related inversely to the sum of the displacements of the 3-hydroxyl plus that of the 17-hydroxyl groups relative to the plane of symmetry of the dehydroepiandrosterone nucleus. This enzyme was also capable of sulfurylating short chain aliphatic alcohols, although at greatly reduced rates. 3 beta-Chloro-5-androstene-17-one and 2-nitroestradiol. 17 beta proved to be the best inhibitors of SAS.

Induction of porcine uterine estrogen sulfotransferase activity by progesterone.

Studies have been carried out which were designed to examine the hormonal requirement for the appearance of estrogen sulfotransferase activity in porcine uteri. Mature, ovariectomized (OVX) gilts were housed for 3 weeks before being treated with various regimens of estradiol-17 beta (E2) and progesterone (P). Uteri were then removed, minced, incubated for 2 h with [3H] E2 (10(-8) M) and Na2 35SO4 (10(-4) M) and the labeled metabolic products were extracted and analyzed. Endometrial samples were also taken for the determination of E2 and P cytoplasmic and nuclear receptors (R). It was found that 4 daily injections of 250 micrograms of E2 was sufficient to bring plasma E2 concentrations to that representative of a normal estrous cycle (approx. 30 pg/ml) and to induce cytoplasmic PR to high levels (7000--19000 fmol/mg DNA). Estrogen sulfotransferase activity, which was negligible in OVX and E2-treated pigs, increased to near normal secretory levels (4 pmol product/h per 0.4 g tissue) only in pigs primed with E2 and subsequently treated with E2 and P (25--250 mg/day, 3 days). This treatment also brought about the translocation of PR to the nuclear compartment. The steroid alcohol sulfotransferase activity in these tissues decreased upon ovariectomy and remained unaffected by the hormone treatments. Endometria from treated and untreated pigs were cultured for a period up to 7 days. During this time E2 (10(-8) M) induced and/or maintained PR and P (10(-6) M) was shown to stimulate estrogen sulfurylation concomitant with the translocation of PR to the nucleus. These studies have demonstrated that, in OVX pigs and endometrial cultures, P stimulated uterine estrogen sulfotransferase activity to a level normally found in secretory uteri. In order for P to bring about elevated levels of estrogen sulfurylation it was necessary that the endometrium contain adequate concentrations of cytoplasmic PR (which required E2 priming of the system) and the P receptor complex must display nuclear translocation.

Inhibition by 2',3'-dideoxythymidine of retroviral infection of mouse and human cells.

Infection of mouse and human cells with type C murine leukemia/sarcoma viruses was inhibited by the nucleoside analogue 2',3'-dideoxythymidine (ddT). Cell growth and virus synthesis by previously infected cells were not affected by the analogue. Infection of rat cells with murine retroviruses was not inhibited by ddT.