Effects of oestrogen and the antioestrogens, tamoxifen and LY117018, on four oestrogen-regulated RNAs in the EFM-19 breast cancer cell line

The EFM-19 cell line is a new breast cancer cell line whose proliferation has been reported to be stimulated by oestrogens and inhibited by the antioestrogen tamoxifen. Oestrogen receptor mRNA levels are higher in EFM-19 cells than in other oestrogen-responsive cell lines. The levels of four oestrogen-inducible RNAs [pNR-1, pNR-2, pNR-25 and pNR-100] were measured in EFM-19 cells. Oestradiol treatment increased the levels of the four regulated RNAs between 3-fold (pNR-100) and greater than 100-fold (pNR-2). The induction was half maximal between 1.5 x 10(-11) and 1.5 x 10(-10) M oestradiol. The effects of two antioestrogens, tamoxifen and LY117018, were measured on the expression of the oestrogen-regulated RNAs. Tamoxifen was a partial oestrogen agonist for the induction of the pNR-1 and pNR-25 RNAs but had very little effect on the pNR-2 and pNR-100 RNA levels. The pNR-2 RNA levels were less induced by tamoxifen in EFM-19 cells than in MCF-7 cells. LY117018 did not increase the levels of any RNA. The oestrogen-induced levels of the four RNAs were reduced by both antioestrogens to the RNA levels present in cells treated with the antioestrogens alone. LY117018 was at least 100-fold more potent than tamoxifen as an oestrogen antagonist.     

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.     

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.     

The oestrogen paradox: a hypothesis

Epidemiological and observational studies suggest that oestrogens, when used as hormonal therapy in post-menopausal women, can increase the risk of breast cancer if used long term. However, more recent data suggest that short-term use in sub-groups of post-menopausal women significantly decreases the risk of breast cancer. This beneficial effect is also observed when high-dose oestrogen is administered to post-menopausal women with breast cancer to cause tumour regression, a phenomenon which commonly occurs. We consider these divergent responses to oestrogen to represent a "paradox". Data from our own and other investigative groups suggest a hypothesis to explain this paradox. Deprivation of oestradiol in model systems causes cells to adapt and to undergo apoptosis in response to oestrogen. This occurs through the Fas/Fas ligand death receptor pathway and through alterations in apoptotic mechanisms mediated by mitochondria. This process of programmed cell death may explain the regression of established breast cancer with oestrogen administration and the diminution in the rate of new breast cancer diagnoses recently reported. Our hypothesis is based upon pathological data indicating the presence of a "reservoir" of undiagnosed breast cancer in the population of women who would be starting on oestrogens as menopausal hormonal therapy. The long-term increased risk of breast cancer may then reflect different mechanisms. Oestrogens can cause mutations through enhancement of the rate of cell division and concomitantly the error rate in DNA replication. In addition, oestrogens can be metabolised to directly genotoxic compounds. These carcinogenic processes take much longer, since a number of mutations must accumulate before resulting in breast cancer. These hypotheses regarding oestrogen-induced apoptosis in the short term and carcinogenesis in the long term now require rigorous verification but would serve to explain the "oestrogen paradox".     

Regulation of androgen receptor mRNA and protein level by steroid hormones in human mammary cancer cells.

The regulation of the human androgen receptor (AR) by steroid hormones in human mammary cancer cells was investigated using immunocytochemical and ligand binding assays for its protein and Northern blot analyses for the corresponding mRNA. MFM-223 cells contain high levels of ARs and are growth-inhibited by dihydrotestosterone (DHT). The AR protein is down-regulated to 57% of the control by 10 nM DHT after 24 h, and the corresponding mRNA is also reduced. The nonsteroidal antiandrogen hydroxyflutamide had no effect on the AR level, whereas after incubation with 1 microM cyproterone acetate a slight down-regulation was observed. The AR level was restored completely after release from a 7 day treatment with DHT. However, only 60% of the control level was restored, if the cells wer grown in the presence of DHT for 6 weeks. In androgen-pretreated cells the proliferation rate remained decreased even after the withdrawal of DHT. Concomitantly the distinct growth inhibition was lost. Transfection experiments demonstrated a reduced activity of the residual androgen receptor in these pretreated cells. In addition to the AR, EFM-19 cells also contain significant amounts of estrogen and progesterone receptors. EFM-19 cells are not growth inhibited by physiological concentrations of DHT. Autoregulation of AR was also found in this cell line. Additionally, reduced levels of AR protein and mRNA were found in EFM-19 cells after treatment with the synthetic progestin R5020. The maximum effect of R5020 was observed at the high concentration of 1 microM. Estrogen treatment with 10 nM 17 beta-estradiol for 3 days reduced the AR level only by 25%.     

Effects of the antioestrogen, ICI 164,384, on oestrogen induced RNAs in MCF-7 cells

The effects of ICI 164,384 on the expression of six oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-13, pNR-17, pNR-25 and pNR-100) and the 46 kDa secreted protein were measured in MCF-7 cells. In marked contrast to tamoxifen, an antioestrogen commonly used in the treatment of breast cancer, ICI 164,384 administered alone had little or no effect on the RNAs or protein. ICI 164,384 completely inhibited the induction of the RNAs and 46 kDa protein by oestradiol. Although ICI 164,384 has an affinity for the human oestrogen receptor only slightly less than that of oestradiol, half maximal inhibition of oestradiol action was attained with between a 50 and 150-fold molar excess of ICI 164,384. The pNR-1 RNA is induced by tamoxifen but this induction was abolished by ICI 164,384. Thus, ICI 164,384 acts as a potent antioestrogen for the regulation of the expression of specific oestrogen-responsive genes in human breast cancer cells.     

The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor (TNF), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

A novel binding site for a synthetic progestagen in breast cancer cells

A novel, high-affinity saturable binding site for the synthetic 19-nor testosterone progestagen, 17 alpha-ethinyl-13 beta-ethyl 17 beta-hydroxy-4,15-oestradiene-3-one (gestodene) has been detected using a sensitive affinity chromatography technique. This binding site is present in a range of malignant breast-derived cells lines, regardless of the presence of oestrogen and progesterone receptors, but is absent from endometrial carcinoma cells that contain both oestrogen and progesterone receptors. Competition studies show that this binding is not attributable to the receptors for the progestagens, androgens, glucocorticoids or mineralocorticoids. Cytosolic gestodene binding is refractory to competition with oestradiol but nuclear gestodene binding is completely abolished by oestradiol. The binding of oestradiol to the oestrogen receptor is reduced 40-50% by competition with gestodene. Non-dissociating polyacrylamide gel electrophoresis and size-exclusion high performance liquid chromatography reveal that this binding activity is associated with a protein of mean molecular mass 47 +/- 9 kDa. Ligand binding studies with a range of other cell lines indicates that this binding site appears to be specific to breast cancer cells. These data show the presence of a partly oestrogen competable novel binding protein in breast cancer cells which does not appear to be due to any of the conventional steroid receptors.     

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 relationship between factors affecting endogenous oestradiol levels in postmenopausal women and breast cancer

Breast cancer accounts for 1 in 4 of all female cancers worldwide; approaching 13,000 women dying per year in the UK alone. Seventy five per cent of all diagnosed breast cancers are oestrogen receptor (ER) positive. Ovarian synthesis of oestrogens ceases at menopause and as breast cancer is more prevalent in postmenopausal women the non-ovarian sources of oestrogen are important in disease progression. There is now considerable evidence that associates increased breast cancer risk with prolonged exposure to oestrogens hence greater attention is now being given to determining whether the measurement of plasma oestrogen may assist in identifying chemoprevention target groups. Studies suggest that in most postmenopausal patients the intra-tumoural concentrations of oestrogens are up to 20-fold higher than those present in the plasma however, while the extent of biosynthesis of oestrogens within breast tissue is a major determinant of local exposure, plasma levels are a useful indicator of overall metabolism in peripheral tissues. As such it is important to understand factors that influence these measurements. This review summarises the impact of lifestyle such as body mass index, together with the role of genetic polymorphisms placed within the context of designing future epidemiological studies and breast cancer risk algorithms.     

Molecular epidemiology of breast cancer: genetic variation in steroid hormone metabolism

The age-specific incidence rate of breast cancer in women rises until menopause, levels off and then rises again at a much lower rate indicating a possible hormonal influence on the disease risk. A large amount of evidence has implicated hormones and other compounds with oestrogen activity in the pathogenesis of certain endocrine cancers, particularly breast cancer. Widely dispersed hormone-like chemicals, capable of disrupting the endocrine system and interfering with proliferation, have been described. Compounds such as dioxins, some polychlorinated biphenyls and the plastic ingredient bisphenol-A have been shown to interfere with human reproduction and hormonal regulation. The levels of these foreign compounds as well as the levels of endogenous oestradiol may influence the risk of breast cancer. Endogenous oestradiol is synthesised in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast cancer tissue, express all the necessary enzymes for this synthesis: CYP17, CYP11a, CYP19, hydroxysteroid hydrogenase, steroid sulphatase as well as enzymes further hydroxylating oestradiol such as CYP1A1, CYP3A4, CYP1B1. Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.     

Molecular epidemiology of sporadic breast cancer. The role of polymorphic genes involved in oestrogen biosynthesis and metabolism

The major known risk factors for female breast cancer are associated with prolonged exposure to increased levels of oestrogen. The predominant theory relates to effects of oestrogen on cell growth. Enhanced cell proliferation, induced either by endogenous or exogenous oestrogens, increases the number of cell divisions and thereby the possibility for mutation. However, current evidence also supports a role for oxidative metabolites, in particular catechol oestrogens, in the initiation of breast cancer. As observed in drug and chemical metabolism, there is considerable interindividual variability (polymorphism) in the conjugation pathways of both oestrogen and catechol oestrogens. These person-to-person differences, which are attributed to polymorphisms in the genes encoding for the respective enzymes, might define subpopulations of women with higher lifetime exposure to hormone-dependent growth promotion, or to cellular damage from particular oestrogens and/or oestrogen metabolites. Such variation could explain a portion of the cancer susceptibility associated with reproductive effects and hormone exposure. In this paper the potential role of polymorphic genes encoding for enzymes involved in oestrogen biosynthesis (CYP17, CYP19, and 17beta-HSD) and conversion of the oestrogen metabolites and their by-products (COMT, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, GSTT1 and MnSOD) in modulating individual susceptibility to breast cancer are reviewed. Although some of these low-penetrance genes appeared as good candidates for risk factors in the etiology of sporadic breast cancer, better designed and considerably larger studies than the majority of the studies conducted so far are evidently needed before any firm conclusions can be drawn.     

Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors

Since sex steroid hormones and growth factors are known to modulate the proliferation of breast tumors, we have studied the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, which contain low levels of ER, and in two sublines of MCF-7 cells which contain high ER levels. The mRNA levels were measured by Northern blot analysis using lambda OR8, a cDNA probe for ER, and protein levels were measured by hormone binding or Western blot analysis. Treatment of T47D cells with estradiol (E2) caused a 2.5-fold increase in ER mRNA (6.6 kilobases) levels after 48 h. The progestin R5020 evoked a marked decrease in ER mRNA and protein levels to 20% of control values, while the antiprogestin RU38,486 caused no change in ER. In MCF-7 cells, the effect of E2 on ER levels was dependent on the prior growth history of the cells. In cells grown in low estrogen [5% charcoal-dextran-treated calf serum with phenol red for 8 yr (MCF-7-K2)], which are still E2 responsive, treatment with E2, the antiestrogen LY117018, or both produced little change in ER mRNA or protein; in contrast, ER mRNA and protein were reduced by E2 to 40% and 50% of control levels, respectively, in MCF-7 cells (denoted MCF-7-K1) which had been maintained routinely in medium containing 5% calf serum. This decrease in ER mRNA was dose dependent; 10(-11) E2 reduced levels to 60%, and 10(-10) M E2 evoked the maximal drop to 40% of the control level in 2 days. LY117018 alone did not alter ER mRNA levels in these cells, but it completely prevented the down-regulation of ER by E2. Administration of progestin, but not antiprogestin, along with E2 partially prevented the decrease in ER evoked by E2. Addition of epidermal growth factor or insulin-like growth factor-I to MCF-7-K1 cells, which increased cell proliferation, had no detectable effect on ER levels. Treatment with transforming growth factor-beta, which decreased cell proliferation, reduced ER by about 20%.(ABSTRACT TRUNCATED AT 400 WORDS)     

17beta-oestradiol up-regulates longevity-related, antioxidant enzyme expression via the ERK1 and ERK2[MAPK]/NFkappaB cascade

Females live longer than males. Oestrogens protect females against aging by up-regulating the expression of antioxidant, longevity-related genes such as glutathione peroxidase (GPx) and Mn-superoxide dismutase (Mn-SOD). The mechanism through which oestrogens up-regulate those enzymes remains unidentified, but may have implications for gender differences in lifespan. We show that physiological concentrations of oestradiol act through oestrogen receptors to reduce peroxide levels in MCF-7 cells (a mammary gland tumour cell line). Oestradiol increases MAP kinase (MAPK) activation as indicated by ERK1 and ERK2 phosphorylation in MCF-7 cells, which in turn activates the nuclear factor kappa B (NFkappaB) signalling pathways as indicated by an increase in the p50 subunit of NFkappaB in nuclear extracts. Blockade of MAPK and NFkappaB signalling reduces the antioxidant effect of oestradiol. Finally, we show that activation of MAPK and NFkappaB by oestrogens drives the expression of the antioxidant enzymes Mn-SOD and GPx. We conclude that oestradiol sequentially activates MAPK and NFkappaB following receptor activation to up-regulate the expression of antioxidant enzymes, providing a cogent explanation for the antioxidant properties of oestrogen and its effects on longevity-related genes.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

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.     

Regulation and role of the insulin-like growth factor I system in rat luteal cells.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor beta mRNA contents were determined by semiquantitative RT-PCR. The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor beta. Furthermore, whereas IGF-I upregulated oestrogen receptor beta expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor beta. Luteal cells with increased oestrogen receptor beta can respond fully to oestradiol, leading to cell hypertrophy.     

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.