The origin of oestrone sulphate in normal and malignant breast tissues in postmenopausal women.

Conversion of oestrone sulphate to oestrone has been suggested to make a major contribution to the level of oestrone found in breast tissues. In order to examine the ability of breast tissues to take up oestrone sulphate (E1S), 3H E1S or E1-35S was infused into postmenopausal women with advanced breast cancer. For 3 subjects infusion of 3H E1S was repeated after treatment with Danazol, a potential inhibitor of oestrone sulphatase activity. After infusion of 3H E1S significant levels of 3H E1S were detected in normal and malignant breast tissues (tissue: plasma ratios 0.14 +/- 0.13 and 0.24 +/- 0.12 respectively, mean +/- S.D., n = 5). Similar 3H E1S tissue: plasma ratios were detected after infusions of 3H E1 indicating that the 3H E1S detected in breast tissues after infusion of 3H E1S may have originated from the hydrolysis of 3H E1S in tissues other than the breast, with subsequent uptake and sulphation in breast tissues. After infusion of E1-35S no significant levels of radioactivity were detectable in normal or malignant breast tissues. Treatment with Danazol had no significant effect on tissue levels of 3H E1S or on the CRE1S E1 or MCR-E1S. It is concluded that oestrone sulphate, as such, is not taken up by breast tissues and that any contribution that oestrone sulphate makes to the oestrogen content of breast tissues will depend upon prior hydrolysis.     

Modulation of oestrone sulphatase activity in breast cancer cell lines by growth factors

Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E₁) and oestradiol. As the formation of E₁ from its sulphate (E₁S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.     

The role of cytokines and sulphatase inhibitors in regulating oestrogen synthesis in breast tumours

Synthesis of oestrogens within breast tissues makes an important contribution to the high concentrations of oestradiol which are found in breast tumours. The activities of the enzymes involved in oestrogen synthesis, i.e. the aromatase, oestradiol dehydrogenase (E2DH) and oestrone sulphatase (E1-STS), can be stimulated by several growth factors and cytokines. As it is possible that some of these factors may be derived from cells of the immune system (macrophages and lymphocytes), the effects of basic fibroblast growth factor (bFGF) and interleukin-2 (IL-2), which are produced by these cells, on E2DH activity was examined in MCF-7 cells. Treatment of these cells with bFGF resulted in a dose-dependent increase in E2DH reductive activity whereas IL-2 was inactive at the concentration tested. To obtain further evidence that factors produced by macrophages and lymphocytes can modulate the activities of enzymes involved in oestrogen synthesis, conditioned medium was collected from these cells and found to stimulate both E1-STS and E2DH activities. In addition to understanding the control of oestrogen synthesis in breast tumours an inhibitor to block the synthesis of oestrone via the oestrone sulphatase pathway was developed. Oestrone-3-O-sulphamate (EMATE) is a potent, irreversible, inhibitor of E1-STS. A single dose of EMATE (10 mg/kg) inhibited tissue E1-STS activity in rats by more than 95% for up to 7 days, indicating that this compound may have considerable therapeutic potential for the treatment of breast cancer. Evidence is also reviewed that another steroid sulphatase, dehydroepiandrosterone sulphate sulphatase, may have a crucial role in regulating cytokine production and that this may indirectly control tumour oestrogen synthesis.     

The regulation of oestrone sulphate formation in breast cancer cells.

The formation of oestrone sulphate has been examined in MCF-7 (oestrogen receptor positive, ER+) and MDA-MB-231 (ER negative, ER-) breast cancer cells. Using intact cell monolayers and a physiological substrate concentration, progesterone (1 microM) and dexamethasone (1 microM) both increased oestrone sulphate formation in MCF-7 cells. In MDA-MB-231 cells, dexamethasone, but not progesterone, increased conjugate formation. A number of growth factors, cytokines and human serum albumin (HSA), which have previously been found to regulate oestrogen synthesis, were also examined for their ability to regulate oestrone sulphate formation. In MCF-7 cells epidermal growth factor, acidic and basic fibroblast growth factors, insulin-like growth factor-type I and insulin all stimulated oestrone sulphate formation. The cytokines, tumour necrosis factor alpha (TNFalpha) and interleukin-1beta also increased conjugate formation in the ER+ cells, as did HSA. In contrast, in MDA-MB-231 cells TNFalpha was without effect and HSA inhibited oestrone sulphate formation. The ability to modulate oestrone sulphate formation in ER+ cells may be an important mechanism to limit the availability of oestrogen to interact with the ER.     

On the significance of in situ production of oestrogens in human breast cancer tissue

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

A direct assay for oestrone sulphate and its use to investigate the effect of ampicillin on plasma levels of oestrone sulphate

A direct radioimmunoassay for measuring plasma levels of oestrone sulphate has been developed using 8-anilino-2-naphthalene sulphonic acid to displace oestrone sulphate from plasma binding proteins. Oestrone sulphate was assayed by using an antiserum raised against glucuronide which cross-reacted 100% with oestrone sulphate. The direct assay gave a good analytical recovery of oestrone sulphate and there was a good correlation (r = 0.82, P less than 0.001) for plasma levels of oestrone sulphate measured by the direct assay and a method involving steroid conjugate extraction and enzyme hydrolysis. The mean (+/- S.D.) plasma level of oestrone sulphate in men was 1100 +/- 280 pg/ml. The effect of taking the antibiotic, Ampicillin, on plasma levels of oestrone sulphate was investigated in four men. Plasma levels of oestrone sulphate were significantly reduced after taking Ampicillin for 5 days. Ampicillin may act to lower plasma levels of oestrone sulphate by reducing the growth of bacteria in the gut or by inhibiting oestrogen sulphotransferase activity.     

Metabolism of [3H]oestradiol in vivo by normal breast and tumour tissue in postmenopausal women

Tumour and normal breast tissue was obtained from postmenopausal breast cancer patients following [3H]oestradiol infusion (50 mu Ci over a 12 h period). The fraction of radioactivity present as oestradiol or oestrone was measured and the results expressed both as the ratio of oestradiol-oestrone and as the percentage oestrogen present as oestrone, and the findings compared with in vitro measurements of 17 beta-hydroxysteroid dehydrogenase activity. Concentrations of 5-androstene-3 beta, 17 beta-diol, dehydroepiandrosterone and its sulphate and testosterone were measured and related to oestradiol metabolism. The study demonstrated that tumour tissue is less able to metabolise oestradiol to oestrone than is normal breast tissue and indicated that the ability of the tissue to detoxify oestradiol may be dependent on cofactor availability. The results also supported the possibility that increased tissue concentrations of adrenal androgens inhibit oestradiol and thus increase tissue exposure to oestradiol.     

Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.     

The Development of A-ring Modified Analogues of Oestrone-3-O-sulphamate as Potent Steroid Sulphatase Inhibitors with Reduced Oestrogenicity

Steroid sulphatases regulate the formation of oestrogenic steroids which can support the growth of endocrine-dependent breast tumours. The development of potent steroid sulphatase inhibitors could therefore have considerable therapeutic potential. Several such inhibitors have now been developed of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Unexpectedly, this inhibitor proved to be a potent oestrogen. In an attempt to reduce the oestrogenicity, whilst retaining the potent sulphatase inhibitory properties associated with this type of molecule, a number of A-ring modified derivatives were designed and synthesized. A-ring modified compounds included the 2-methoxy, 2/4-nitro, 2/4-n-propyl and 2/4-allyl EMATE analogues. The ability of these derivatives to inhibit oestrone sulphatase activity was examined using placental microsomes. The allyl-substituted EMATE derivatives were more potent inhibitors than the propyl analogues but were all considerably less potent than EMATE. In contrast, the 2-methoxy and 2/4-nitro analogues were potent sulphatase inhibitors with 4-nitro EMATE being 5 times more active than EMATE. The 4-nitro, 2-methoxy, 4-n-propyl and 4-allyl derivatives were also tested in vivo for their oestrogenicity and ability to inhibit sulphatase activity. While both 4-nitro and 2-methoxy EMATE were potent inhibitors in vivo, 2-methoxy EMATE had no stimulatory effect on uterine growth in ovariectomized rats. The identification of a potent steroid sulphatase inhibitor lacking any oestrogenicity, such as 2-methoxy EMATE, should be of considerable value in evaluating the potential of steroid sulphatase inhibition for breast cancer therapy.     

Steroid sulphatase inhibitors for breast cancer therapy

In contrast to aromatase inhibitors, which are now in clinical use, the development of steroid sulphatase (STS) inhibitors for breast cancer therapy is still at an early stage. STS regulates the formation of oestrone from oestrone sulphate (E1S) but also controls the hydrolysis of dehydroepiandrosterone sulphate (DHEA-S). DHEA can be reduced to 5-androstenediol (Adiol), a steroid with potent oestrogenic properties. The active pharmacophore for potent STS inhibitors has now been identified, i.e. a sulphamate ester group linked to an aryl ring. This has led to the development of a number of STS inhibitors, some of which are due to enter Phase I trials in the near future. Such first generation inhibitors include the tricyclic coumarin-based 667 COUMATE. Aryl sulphamates, such as 667 COUMATE, are taken up by red blood cells (rbc), binding to carbonic anhydrase II (CA II), and transit the liver without undergoing first-pass inactivation. 667 COUMATE is also a potent inhibitor of CA II activity with an IC₅₀ of 17 nM. Second generation STS inhibitors, such as 2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE), in addition to inhibiting STS activity, also inhibit the growth of oestrogen receptor negative (ER⁻) tumours in mice and are anti-angiogenic. As the active pharmacaphores for the inhibition of aromatase and STS are now known it may be possible to develop third generation inhibitors that are capable of inhibiting the activities of both enzymes. Whilst exploring the potential of such a strategy it was discovered that 667 COUMATE possessed weak aromatase inhibitory properties with an IC₅₀ of 300 nM in JEG-3 cells. The identification of potent STS inhibitors will allow the therapeutic potential of this new class of drug to be explored in post-menopausal women with hormone-dependent breast cancer. Second generation inhibitors, such as 2-MeOE2bisMATE, which also inhibit the growth of ER⁻ tumours should be active against a wide range of cancers.     

4- and 6-(p-Sulphamoylphenyl)androstenediones: Studies of aromatase inhibitor-based oestrone sulphatase inhibition

4-(p-Sulphamoylphenyl)androstenedione (3) and 6α-p-sulphamoylphenyl analogues 12-14 were synthesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aromatase with apparent K_i values ranging between 30 and 97 nM. In addition, the aromatase inhibitory activities of 6α-p-hydroxyphenyl compounds 9-11, which may be produced from their respective sulphamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75 nM of the K_i values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of oestrone sulphatase with more than about 200 μM of IC₂₅ values. Although the present findings of the oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment of oestrogen-dependent breast cancer.     

Pharmacokinetics of oestrone-3-O-sulphamate

The sulphatase pathway is thought to be the major route of oestrogen synthesis in breast tumours in postmenopausal women. There is currently considerable interest in developing a potent steroid sulphatase inhibitor to block oestrogen synthesis by this route. One of the most potent inhibitors discovered so far is oestrone-3-O-sulphamate (EMATE) which is active in vivo. In this study we report the preparation of a formulation for the administration of EMATE by the oral route. A method, using high-performance liquid chromatography (HPLC), was also established to measure concentrations of EMATE in rat plasma after its oral or i.v. administration. Using the oral formulation and HPLC assay, EMATE was readily detected in rat plasma after oral administration. Plasma EMATE concentrations were related to the dose of drug administered orally over the 10–40 mg/kg range. To examine the pharmacokinetics of EMATE, the compound (40 mg/kg, single dose) was administered either orally (in the formulation) or i.v. (in propylene glycol) with plasma samples being collected for up to 6 h. After oral administration, EMATE was rapidly absorbed, with the peak plasma concentration being detected at 30 min, after which plasma concentrations rapidly decreased. After i.v. administration a plasma EMATE concentration was detected at 1 h similar to that after oral administration. The clearance of EMATE from plasma followed a bi-phasic curve, showing an initial half-life of 30 min, followed by a slower half-life of 4 h 30 min. Little evidence was obtained for any metabolism of EMATE to oestrone. Rat liver sulphatase activity was almost completely inhibited (>99%) within 30 min of oral or i.v. administration of EMATE.     

Levels of androgen conjugates and oestrone sulphate in patients with breast cysts

Plasma and cyst fluid were obtained from patients with palpable breast cysts and analysed for androgen conjugates and oestrone sulphate content by radioimmunoassay. Concentrations of androgen conjugates in cyst fluids varied from 15.6 to 475.5 mumols/l. These levels were much greater than those in plasma (1.3-5.2 mumols/l) and there was no association between values in cyst aspirates and plasmas obtained from the same individuals. Levels of oestrone sulphate in breast cyst fluids (1.5-744.0 nmol/l) were also generally in excess of those in plasma (2.0-59.9 nmol/l) and again no relationship was evident between concentrations in cyst fluid and the circulation. Neither was there a relationship between levels of androgen conjugate and oestrone sulphate in plasma. In contrast, a highly significant correlation (P less than 0.001) was identified between the androgen conjugate and oestrone sulphate content of cyst fluids. Levels of both androgen conjugates and oestrone sulphate were also significantly different in groups of cysts subdivided according to electrolyte classification, cysts with low Na+:K+ ratios having higher steroid concentrations than those with high Na+:K+ ratios. The biological significance of the relationship between the two conjugates in cyst fluids remains unclear but it is suggested that the accumulation of these steroids involves a common mechanism.     

Simulation of pregnancy levels of plasma oestrone sulphate by infusion in the non-pregnant mare: A preliminary study

Oestrone sulphate was infused intravenously in saline solution into two, non-pregnant pony mares in repeated trials to simulate levels of the oestrogen found in plasma after midpregnancy in mares. Rates of infusion of oestrone sulphate ranged from 32 to 231 mg of oestrone equivalent per hour. Blood samples were taken from the opposite jugular vein for measurement of oestrone sulphate by radioimmunoassay. Plasma levels rose rapidly from the start of each infusion of the oestrogen and remained elevated over the hour-long periods, with concentrations as oestrone ranging from about 175 to 700 ng/ml. During each subsequent infusion with saline alone the oestrogen levels in plasma fell within 1 h to about 25% of the preceding values. Results were similar for each animal and simulation of plasma levels of oestrone sulphate in pregnancy was achieved.     

Inhibition of steroid sulphatase activity by steroidal methylthiophosphonates: Potential therapeutic agents in breast cancer

The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E₁ S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the K_m and V_max of E₁S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding K_i values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Steroid sequestration and tightly bound oestrogen-protein complexes in human breast tumours and a breast cancer cell line

Homogenates of breast tumours taken at surgery were prepared in phosphate-buffered medium in the presence or absence of the protease inhibitors N-alpha-p-tosyl-L-arginine methyl ester (TAME, 10 mM) or soy bean trypsin inhibitor (STI, 1 mg/ml). Aliquots (0.25 ml) were incubated in 5 ml medium, with the addition of excess trypsin (2 mg/ml) to experimental flasks. Oestrogen was measured by means of a radioreceptor assay (RRA) based on rat or human uterine cytosolic oestradiol receptor. In oestrogen receptor positive (ER +ve) tumour homogenates, TAME decreased while STI increased ethyl acetate extractable oestrogen in these preparations. The addition of trypsin enhanced yields of oestrogen in the TAME, but not in the STI or control (no inhibitor) preparations. None of these treatments affected RRA detectable oestrogen in homogenates of ER -ve tumours. Suspensions of ZR-75-1 cells, prepared in Krebs Ringer bicarbonate (KRBG) incubated with trypsin also gave greatly enhanced yields of extractable oestrogen. Fractionation of oestrogens from both tumour homogenates and from the cell line showed coincidence of RRA detectable steroid with oestradiol and oestrone, and, particularly in trypsin flasks, very non-polar components were also found. In the cell-line extracts, HPLC fractionation combined with specific radioimmunoassays confirmed the presence of both oestradiol and oestrone. The major extracted component was oestrone. The data suggest the existence within breast tumour tissue of sequestered pools of steroid requiring proteolytic action for their release. One possibility, consistent with reports in the literature, is that the steroids may themselves be directly conjugated to protein. Their presence in ER +ve but not ER -ve tumours strongly suggests some relationship to the development of hormone-sensitive disease. Alternatively, the phenomenon may be associated with the rigid compartmentalization of the paracrine function of the tissue.     

Hormonal and physical changes associated with bovine conceptus development.

Conceptus (placental membranes, fetal fluids and fetus) development was characterized between Days 27 and 111 of gestation. Progestagens, oestrone, oestradiol, oestrone sulphate and prostaglandins (PG) F were measured in maternal plasma and allantoic and amniotic fluids. Protein concentrations are described for fetal fluids. The early increase in placental membrane weight from 1.12 g (27 days) to 58.45 g (50 days) was associated with oestrogen production presumably of conceptus origin. Oestrogens increased significantly in allantoic and amniotic fluids throughout the period studied with oestrone being the primary free oestrogen, rising from 2 pg/ml (Day 33) to 144 ng/ml by 111 days in allantoic fluid. Changes in plasma oestrogens of the maternal circulation were not detected until after Day 70 at which time oestrone concentration was greater than that of oestradiol. Fetal fluid concentrations of progestagens, oestrone sulphate and PGF were not related to maternal plasma levels and a sequestration of these hormones by the allantois is postulated.     

Regulation of steroid sulphatase expression and activity in breast cancer

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.