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Objective— To investigate the presence of an oestradiol receptor-related protein (P29) in skin and skin oraganelles, and to assess changes in its content during the normal menstrual cycle. Design— An observational study. Setting— King's College School of Medicine and Dentistry, London. Subjects— Twenty-one premenopausal women with regular menstrual cycles undergoing gynaecological surgery. They were allocated to proliferative or secretory phases of the menstrual cycle on the basis of menstrual dating and histological examination of an endometrial sample. Interventions— Small full thickness sections of skin (about 5 mm in depth) taken from the anterior abdominal wall at hysterectomy or laparoscopic sterilization. Main outcome measures— The concentration of the oestradiol receptor-related protein in skin and its organelles was assessed semi-quantitatively, using a monoclonal antibody technique. The intensity of staining was compared between the proliferative and secretory phases of the cycle. Results— The receptor-related protein was consistently observed in epidermis, sebaceous glands, hair follicles and sweat ducts; there was no significant difference in its concentration between the proliferative and secretory phases of the menstrual cycle. The protein was not present in dermis and sweat ducts. Conclusions— Epidermis and some skin organelles contain an oestradiol receptor-related protein and must be considered as oestrogen target tissues. However, the content of this protein does not appear to change significantly during the normal menstrual cycle.

When deprived of steroid in the long term, T-47-D human breast cancer cells lose estrogen sensitivity of cell growth. This loss of response results from an increased basal growth rate in the absence of steroid, not from a loss of estrogen-stimulated growth, and it occurs without any loss of estrogen receptor number or function. Growth factor gene expression and sensitivity have been investigated in this model system in an attempt to unravel the molecular mechanisms underlying the progression to steroid autonomy. The transition was accompanied by a decreased dependence on added serum and by a loss of the stimulatory effects of insulin and basic fibroblast growth factor, but also by an acquired sensitivity to stimulation by transforming growth factor-beta (TGF-beta). An increase in TGF-beta 1 mRNA was detected following loss of steroid sensitivity. There was no increase in epidermal growth factor (EGF) receptor number. These findings are discussed in relation to current knowledge concerning the mechanisms by which estrogens stimulate breast cancer cell proliferation.

Endometria from postmenopausal women taking estrogens exhibit little estradiol dehydrogenase activity and contain appreciable amounts of nuclear estradiol receptor. Ingestion of the synthetic progestin, norethisterone, markedly elevates the dehydrogenase activity and decreases the nuclear estradiol receptor content of the endometria. The quantitative relationship between enzyme activity and receptor binding has been studied in women receiving different daily dosages of norethisterone. Statistically, the best negative correlation is obtained when log receptor is plotted against log dehydrogenase. Thus, a small rise in dehydrogenase activity produces a disproportionate decrease in nuclear receptor. Estradiol dehydrogenase in postmenopausal endometria preferentially metabolizes estradiol into estrone. Separation of endometria into glandular and stromal elements indicates that although the dehydrogenase is present in both fractions it is concentrated in the epithelial glands. The functional significance of changes in receptor and dehydrogenase activity was studied by [3H]-thymidine autoradiography. Induction of the dehydrogenase was accompanied by an inhibition of DNA synthesis in both epithelial and stromal cells and the quantitative interrelationships are complex. The nuclear estradiol receptor content, estradiol dehydrogenase activity and DNA synthesis of the postmenopausal endometria have been compared with premenopausal samples from untreated women.

Two hundred and four patients with progressive locally advanced or metastatic breast cancer not controllable by local therapy alone, and who had had no prior systemic therapy for advanced disease, were treated by primary endocrine therapy according to menopausal status. Premenopausal patients received ovarian irradiation (O) whilst postmenopausal patients received tamoxifen 10 mg b.d. (T). Patients were randomised to receive either no additional treatment or prednisolone 5 mg b.d. (P). In 180 evaluable patients, T + P induced significantly more responses than T alone (26/73 vs 9/72, P less than 0.01) and the addition of P to O in premenopausal patients also induced more responses than O alone (7/16 vs 4/19), but this difference was not significant and accrual of premenopausal patients continues. There was a trend for patients receiving T + P to have a longer survival than those receiving T alone (median 25 vs 16 months). These trends occurred in patients with tumours positive for oestrogen receptors and when receptor status was unknown; patients with receptor-negative tumours had a negligible response to endocrine treatment. P mitigated the occurrence of hypercalcaemia and tumour flare sometimes seen with T alone.

Progression from steroid sensitive to autonomous proliferation can be modelled in several cultured mammary tumour cell lines by long-term withdrawal of steroids. A feature of all the four systems studied thus far is that the basal growth in the absence of steroid increases with duration of steroid withdrawal until it reaches that obtained in the presence of steroid. It cannot be assumed that the increased proliferation in the absence of steroid is modulated by the same pathways as those stimulated by steroids in sensitive cells. Therefore, we feel that mechanisms of progression can best be studied via cell behaviour in the absence of steroid. With both the mouse S115 and human T-47-D systems, changes in sensitivity to several growth factors accompany progression; responses to TGF beta 1 are of particular interest in the T-47-D cells where this growth factor becomes stimulatory in the steroid insensitive state. This is accompanied by upregulation of TGF beta 1 mRNA. This upregulation of TGF beta agrees with the finding that ER - PR - primary human breast tumours contain more TGF beta 1 than do ER + PR + tumours; TGF alpha has the opposite pattern. Furthermore, only 40 and 30 kDa TGF beta species have been detected within cultured cells and primary tumours; TGF alpha exists in a 30 kDa form. The functions of these large forms of TGF alpha and TGF beta are unclear. Our conclusions from these experiments is that the increased proliferation in the absence of steroid accompanying progression may not be mediated by the same pathways as those perturbed by steroids in sensitive cells. Furthermore, TGF beta 1 may have different effects in steroid responsive and unresponsive cells.

Postmenopausal women receiving conjugated oestrogens 1.25 mg daily continuously were also given dydrogesterone either 5, 10 or 20 mg daily for the first 12 days of each calendar month. Endometrial tissue obtained on the sixth day of combined therapy in the third or subsequent treatment cycle was subjected to histological, ultrastructural and biochemical assessments. Dydrogesterone provoked secretory histological and ultrastructural changes within the endometrium in a dose-dependent manner. A daily dose of 5 mg produced sub-optimal responses but 10 and 20 mg daily produced effects similar to those observed in the secretory phase of the ovulatory cycle. Dydrogesterone 10 mg and 20 mg daily reduced epithelial DNA synthesis and nuclear oestradiol receptor levels to values within the secretory phase range. A dose-response relation was seen in the induction of oestradiol-17 beta and isocitrate dehydrogenase activities; hyperphysiological values were observed with 20 mg of dydrogesterone daily. This study has demonstrated that dydrogesterone exerts potent anti-oestrogenic and progestational effects on the human endometrium which are dose-related. The 10 and 20 mg doses induced responses equal to or greater than those observed in the secretory phase of the ovulatory cycle and both dosages can be recommended for use in combination with exogenous oestrogens in postmenopausal women: and they may also have a role in the management of anovulatory dysfunctional uterine bleeding.

Progression to steroid insensitivity poses a major problem in therapy of breast cancer, but studies of the origin of steroid-insensitive cells have been few and have concentrated in any one system on the loss of response to only one steroid. Since both normal and tumour mammary cells have complex endocrine requirements, we wondered how different steroids might interact during loss of steroid sensitivity. Cloned cells from the androgen-responsive Shionogi 115 mouse mammary carcinoma respond in vitro to both androgens and glucocorticoids in terms of both cellular and molecular parameters but, following prolonged absence of any steroid, these cells become unresponsive. We show here that 2 steroids can interact to prevent the progression to steroid insensitivity since the S115 cells can be protected against any loss of response to either androgen or glucocorticoid with either steroid alone. Androgen protects against loss of glucocorticoid sensitivity and glucocorticoid protects against loss of androgen sensitivity. The clinical implications are discussed.

Endometria from premenopausal women and from postmenopausal women receiving estrogens and progestins have been separated into epithelial and stromal fractions by collagenase digestion. Nuclear and total estradiol receptor content, soluble progesterone receptor and DNA synthesis have been compared in these epithelia and stroma. Epithelial cells from all types of endometria contained similar amounts of nuclear and total estradiol receptor to those found in stroma from comparable endometria. Progesterone receptor content of epithelium was appreciably higher than in stroma. Both epithelium and stroma from secretory phase, premenopausal endometria contained lower amounts of all types of receptor relative to comparable samples from proliferative phase endometria. Epithelial cells from postmenopausal women taking estrogen alone contained more nuclear and total estradiol receptor and progesterone receptor than those exposed to estrogen plus progestin; progestins decreased the stromal estradiol but not progesterone receptor content. In epithelium and stroma from both pre- and postmenopausal endometria, progestin inhibition ofestrogen-induced DNA synthesis was accompanied by a fall in nuclear estradiol receptor. These effects of progestins were accompanied by a marked rise in estradiol dehydrogenase activity in epithelium but not stroma. Thus, there is a need to re-assess the role of estradiol dehydrogenase in regulating estrogen action, at least in the stromal cells. The receptors have been partially characterized. The soluble estradiol receptor from stroma had a higher affinity for estradiol (Kd 0.9 × 10−1 M) than did the epithelial receptor (Kd 3.8 × 10−10 M). Very little specific binding of weaker affinity (Type II sites) was detected in intact endometrium. The soluble progesterone receptors from epithelium and stroma also have similar Kd values (5 × 10−9 M). The nuclear estradiol receptors from epithelium and stroma exhibited similar binding properties and no Type II sites were detected in intact endometrium.

Two hundred and seventy eight patients with advanced breast cancer who had oestrogen receptor (ER) analyses performed on primary or recurrent tumours were studied. Oestrogen receptor (ER) positive (ER ≧ 5 fmole receptor/mg cytosol protein) tumours recurred significantly more commonly in bone and ER negative (ER < 5 fmole receptor/mg cytosol protein) tumours recurred significantly more often in liver and brain. Patients with ER positive tumours had a significantly better survival after relapse. ER analysis of either primary or recurrent tumour gives some indication of the natural history of breast cancer.

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.

Cytosol receptors for estradiol (RE) and progesterone (RP) in breast tumors from women attending one clinic have been analyzed. The analysis involved a single concentration of [3H] ligand and low speed centrifugation of homogenates. Analysis of tumors immediately before the start of first endocrine treatment indicated a poor response rate in patients with tumor RE

The proliferation rate of cultured cells from the mouse mammary carcinoma Shionogi 115 is regulated both by local cell population density and by androgens. Measurement of intracellular levels of cyclic AMP has shown that these levels are constant over a wide range of proliferation rates (mean doubling times varied from 23 hr to more than 200 hr). Addition of dibutyryl cyclic AMP or theophylline to the culture medium resulted in inhibition of growth—even in the presence of androgen. This inhibition of growth and the relationship between cyclic AMP levels and cell proliferation is discussed.

Specimens of endometrial carcinoma were obtained from 8 women, four of whom had previously been treated with oral medroxyprogesterone acetate 200 mg daily for 7 days. The activities of oestradiol-17β and isocitric dehydrogenases and nuclear oestradiol receptor concentrations were measured in the homogenised tissue and both enzymes were located histochemically. Histochemical evidence of oestradiol dehydrogenase activity was found in all but one of the specimens with biochemical activity. This anomalous specimen was obtained from a woman who had not been treated with MPA and whose endometrium exhibited only low levels of enzyme activity. The histochemical staining caused by isocitric dehydrogenase was intense but bore no relation to the biochemical measurement of enzyme activity in the homogenate. The modified technique for the histochemical demonstration of oestradiol dehydrogenase activity although not quantitative gave results similar to the biochemical methodology. It may therefore be useful as a simple test of the prediction of the sensitivity of endometrial carcinoma to progestogens.

Four hundred and forty-seven women with operable (TNM stage I or II) breast cancer in whom oestrogen receptors (ER), progesterone receptors (PgR) or both receptors had been assayed were studied. Receptor status was independent of axillary nodal status, but infiltrating duct carcinomas that were ER-, PgR- or ER-PgR- were more likely to be anaplastic (P less than 0.001). Four hundred patients with follow-up and uniform treatment were analysed for post-operative disease-free interval (DFI) and survival. No significant difference in DFI existed between patients with ER+ and ER- tumours or PgR+ and PgR- tumours, although there was a trend for longer DFI for ER+ and PgR+. DFI was longer in patients with better-differentiated (grade 1 and 2) tumours than with anaplastic (grade 3) tumours. In patients with ER+ tumours, those with grade 1 and 2 tumours had a longer DFI than those with grade 3 tumours (P less than 0.005). Survival was significantly longer in patients with ER+ tumours compared to those with ER- tumours (P less than 0.001), but there was no such association between tumour PgR and survival. Survival of patients with ER+PgR+ tumours was significantly longer than those with ER-PgR- tumours (P less than 0.025) and, in patients with no evidence of axillary nodal involvement, significantly longer than those with ER+PgR- tumours. Survival in patients with nodal involvement was influenced by histological grade, being longer in those with grade 1 or 2 tumours compared to those with grade 3 tumours. For ER+ tumours, survival was longer in patients with grade 1 or 2 than with grade 3 tumours. These results suggest that steroid receptors significantly affect survival but not DFI. This effect is most closely related to ER content, with relatively little additional information accruing from analysis of PgR. Histological grade influences both DFI and survival, and analysis of both grade and ER content may give a more accurate indication of prognosis in operable breast cancer.

Serum prolactin (HPr) has been measured in 459 patients 1 day before (HPr-1) and in 433 patients 10 days after (HPr-2) treatment. These came from an unselected sequence of 739 patients with operable breast cancer who had been referred to Guy's Hospital over a period of 5 yr. In addition HPr was measured in 100, or more, women at 3, 6 or 12 months after mastectomy. The median levels of either HPr-1 or HPr-2 were higher in pre-menopausal compared with postmenopausal patients (P = 0.03 and 0.06, respectively). Mastectomy was associated with increased serum HPr in both pre- and post-menopausal patients (P less than 0.001 in both cases). Average levels at 3 months, or after, were similar to those found before treatment. Nulliparous women had a higher median amount of HPr-1 than parous which was significant in premenopausal patients (P less than 0.008) whilst HPr-2 levels were not related to parity. Thus the rise in HPr associated with surgery was greater in parous than nulliparous women. Prolactin levels were not related to nodal status or tumour size. However, the amounts of HPR-2 were significantly greater in women with histological grade 3 tumours than those with grade 1 or 2. Standardising for either nodal status, tumour size or histological grade seven situations were found in which HPr-1 or HPr-2 levels were of prognostic significance. Although some of these significant associations could be fortuitous all shared a common feature that the least favourable prognosis was associated with the highest HPr levels.

A simple method is described for isolating epithelium and stroma from pre- and postmenopausal endometria by means of collagenase digestion. This has been used to measure the activities of certain progestin-sensitive enzymes in endometria both from premenopausal women at different stages of the menstrual cycle and from postmenopausal women receiving oral estrogens or estrogens plus progestins. Isocitric dehydrogenase and estradiol dehydrogenase activities are markedly increased by progestins in epithelial but not stromal cells and therefore can be used as indices of epithelial progestin sensitivity. Acid and alkaline phosphatase activities are increased by progestins when measured in intact endometrium but give less clear-cut results after separation into epithelium and stroma.

The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.

Monoclonal antibodies have been prepared against a soluble oestradiol receptor (REC) preparation partially purified from human myometrium by oestradiol affinity chromatography. The antibodies were detected by their ability to immunoprecipitate receptor bound [125I] oestradiol. One of the antibodies (D5) has been studied in detail. It will only precipitate REC after activation by salt, heat, low pH or KCNS and will not react with nuclear RE. It will not react with androgen, progesterone or glucocorticoid receptors nor with sex hormone binding globulin; it will only combine with REC from human sources. D5 recognizes a cytoplasmic 29 kdalton protein (p29) that can be separated from both type I and II soluble oestradiol binding proteins. p29 can react with activated REC and is qualitatively and quantitatively related to REC. IRMA and histochemical methods have been developed for quantitating p29 and relating its amount to receptors in human breast tumours. With both methods, highly significant (P less than 0.001) correlations with REC but not RP have been obtained. Both methods indicate that many REC-RP+ tumours contain p29. The histochemical method detects marked cellular heterogeneity in some tumours. The function of p29 is not known. It is an REC-related antigen that may be a previously undetected component of the oestradiol receptor machinery.

Cytoplasmic 8 and 4 S, and nuclear 5 S oestradiol receptors have been detected in oestrogen dependent primary, oestrogen-dependent transplant and oestrogen-independent transplant tumours. There was no correlation between receptor content of the different tumours and their oestrogen responsiveness. The association of oestradiol with the nuclear receptor is acid-labile and oestrogen-specific. Evidence from protamine sulphate, N-ethyl maleimide and KCl studies suggest that the 8 and 4 S receptors are related, and are similar to those found in rat uterine cytoplasm. Normal hamster uterus has maximal number of cytoplasmic binding sites/cell on day 2 of the cycle. Immature or ovariectomised uteri contained 8 S cytoplasmic receptor whilst mature uteri contained both 8 and 4 S receptors. The proportions of these receptors depended on the stage of the oestrus cycle. Nuclei contained 5 S receptor.

Resume Le metabolisme du ( 6,7- 3 H) oestradiol- 17 β a eteetudie, chez des hamsters mâles, dans des tumeurs renales, dont la croissance etait soit dependante, soit independante des oestrogenes. L'etude etait faite 1 2 heure et 4 heures apres l'injection sous-cutanee de ( 6,7- 3 H) oestradiol- 17 β. Apres 1 2 heure, la fraction “steroide libre”, dans les 2 types de tumeurs, ne contenait que de l'oestradiol- 17 β, et, dans les fractions conjuguees, on ne trouavit, a ce moment, que des traces de tritium. Apres 4 heures, la plus grande partie du tritium se trouvait dans les fractions conjuguees. Divers metabolites furent mis en evidence dans le foie et dans le rein, apres 1 2 heure et apres 4 heures. La tumeur primitive, oestrogeno-dependante, captait jusqu'a 10 fois plus de ( 6,7- 3 H) oestradiol- 17 β que la tumeur independante transplantee. Cette constatation ne pouvait pas s'expliquer par le site de la tumeur. Ni l'une ni l'autre des tumeurs ne metabolisaient le ( 6,7- 3 H) oestradiol- 17 β in vitro ; on en conclut que la captation differente, observee in vivo , etait due a une perte ou a modification de la structure du recepteur d'oestrogenes, dans la tumeur independante. Ceci indiquerait une difference fondamentale de la capacite d'utilisation de l'oestradiol- 17 β par la tumeur dependante et par la teumur independante. La tumeur renale RCA 1 , qui ne repond pas aux oestrogenes, captait moins de tritium que la tumeur independante.

SUMMARY The uptake and intracellular localization of [6,7-3H]oestradiol has been studied in liver, kidney and mammary adenocarcinoma from female rats treated with dimethylbenzanthracene (DMBA). The tumour is similar to other oestrogen-responsive tissues in that the major oestrogen* within the tissue is oestradiol which is mainly associated with cell nuclei. The tumour has the ability to retain the oestrogen for at least 4 hr. Virtually no oestrogen is associated with the nuclei from liver and this result is discussed in relation to the intranuclear localization of oestrogen and the mechanism whereby oestrogen can enter the nuclei of some cells. A significant amount of oestradiol is associated with nuclei from kidney.

A comparison has been made of the survival of contemporary patients treated by mastectomy in whom the original diagnosis was made by either Tru-cut biopsy or by excision biopsy. After a minimum of 5 years of follow-up there was no difference in survival between those biopsied by either method. When subdivided by tumour size again no differences emerged. Among those patients in whom the original Tru-cut biopsy was false negative there was no difference in survival compared with those treated by excision biopsy. This suggests that Tru-cut biopsy is a safe method for obtaining a histological diagnosis in operable breast cancer. Furthermore, when the receptor status of the tumours from the two groups was determined, there was no difference in the percentage of oestrogen receptor positive tumours, nor in the mean receptor value. However, there were more progesterone receptor positive tumours among the Tru-cut group, suggesting that a better tumour sample could have been submitted for analysis. This may be yet another advantage for the use of Tru-cut biopsy for the diagnosis of breast cancer.

Summary. Using a monoclonal antibody raised against an oestrogen receptor-related protein, p29, and an indirect immunoperoxidase technique to stain human tissue, the presence of the antigen was investigated in normal, dysplastic and malignant tissue of the uterine cervix. In normal tissue p29 was present throughout the ectocervix during the menstrual cycle and virtually absent from the endocervix. In dysplastic cervical tissue there was a decreasing p29 content with increasing severity of the dysplasia, and very low levels were seen in the carcinomatous tissues.

This report demonstrates that androgens as well as glucocorticoids can regulate MMTV RNA production in the short term. In S115 mouse mammary tumour cells, MMTV RNA accumulation is regulated within hours by androgen, at a time before any increase in DNA synthesis can be detected, thus providing a marker of an early postreceptor molecular event in steroid action on these cells. Androgen acts via its own receptor and not by cross-binding to the glucocorticoid receptor. The effects are at transcription and not just on stabilisation of RNA because they are blocked by actinomycin D. However, the androgen action shows some partial dependence on simultaneous protein synthesis since cycloheximide is inhibitory. The androgen regulation of MMTV RNA is compared and contrasted with that by glucocorticoids in these cells.

The relevance of steroid-receptor content of mammary tumour cells to the growth promoting effect of steroids is discussed by reference to the effects of androgens and oestrogens on growth of mouse mammary tumour cells (S115 cells) in cell culture. Proliferation of these cells is increased by physiological levels of androgen and evidence is presented that the response is related to the presence of androgen receptors in S115 cells. The S115 cells also contain an oestradiol receptor that is unrelated to proliferative responses. Thus one cannot automatically equate receptor content to proliferation rate.

At high (μM) concentrations and in the absence of protein, [ H ]-progesterone, [ 3 H]-oestradiol and [ 3 H]-diethylstilboestrol form aggregates which elute in the void volume of Sephadex G-25 and G-75 columns. This could be a potential source of error in calculating the amount of receptor actually present in the eluate from an affinity column.

The properties of a monoclonal antibody (D5) that can immunoprecipitate human oestradiol receptor (ER) under some but not all conditions are described. The antibody recognises a 29-kDa serine phosphoprotein that is qualitatively and quantitatively related to ER but not other steroid receptors or binding proteins. p29 will not complex with untreated cytosol ER but, after ammonium sulphate, KCl, heat or phosphatase treatments, interaction occurs that can be detected by immunoprecipitation with D5; molybdate and GTP inhibit complex formation. In human endometrium, p29 is increased by oestrogen and decreased by progestins. IRMA and histochemical assays for p29 have been developed and applied to a large series of human breast tumours. Most, but not all ER+ tumours are p29+, whilst ER-tumours are rarely p29+ unless they are also PR+. p29 predicts for clinical response to hormone therapy. ER+ p29+ tumours have a higher response rate than the ER+ p29-tumours. We do not know if p29 is a previously undetected component of the oestradiol receptor machinery or whether it is a product of oestrogen action.

A major problem in treatment of cancers arising in steroid-sensitive cells is their inevitable progression to a steroid-insensitive state; current therapies are based on the assumption that hormone insensitivity is associated with loss of receptor. We demonstrate for the first time that breast tumor cells can progress to steroid insensitivity in spite of functional steroid receptors. Transfection of the steroid-inducible LTR-C3 gene into unresponsive S115 mouse mammary tumor cells results in full inducibility of that gene with both androgen and glucocorticoid. Thus, although all known endogenous inducible parameters are lost, the steroid sensitivity of a transfected exogenous gene demonstrates that the machinery for steroid responsiveness is still fully functional. Furthermore, these transfected genes retain steroid sensitivity only while steroid is present; on prolonged withdrawal of steroid, they lose responsiveness, implying an epigenetic mechanism is involved.

Soluble oestrogen (R E ) and progesterone (R P ) receptors have been measured in primary breast tumours from English and Japanese women. The only difference between the two groups of patients was that the Japanese had a higher proportion ( 31% ) of R E negative tumours than the English group ( 19% ).

Sera raised in sheep immunised with human myometrial estradiol receptor purified by affinity chromatography on an estradiol-hemisuccinate matrix have been shown to contain antibodies to cytoplasmic estradiol receptor (Re). Ultracentrifugation studies in sucrose gradients indicated that RE-antisera crossreacted with [3H]-estradiol-labelled cytoplasmic RE components from human uterus, breast tumors and MCF-7 cells, rat uterus and pituitary gland and calf uterus. RE-antibodies did not react with either human or rat progesterone receptors, dihydrotestosterone receptors from human breast tumours or rat ventral prostate, sex steroid binding globulin from human plasma or rat α-foetoprotein. No crossreaction could be detected between the antisera and calcium stabilized (4.6S) or trypsinized (4S) forms of rat uterine RE or with the 5S nuclear re component from rat uterus. As the antisera cross-reacts with eytoplasmic rat uterine and pituitary 4S RE in high salt sucrose gradients, absence of immunocomplex formation with proteolytically digested rat uterine re may indicate loss of antigenic determinants from these subunits.

Androgen responsive and unresponsive Shionogi 115 mouse mammary carcinoma cells have been examined for anchorage-independent growth and tumorigenicity in nude mice. The two cell types exhibit transformed and normal growth characteristics respectively, but both give rise to tumours in nude mice. No correlation between tumorigenicity and transformed characteristics including anchorage-independent growth could be demonstrated.

Monoclonal antibody D5, raised against cytosolic human estrogen receptor (ER) reacts with p29, a receptor-associated cytoplasmic serine phosphoprotein which does not bind steroid, While p29 selectively binds GTP and to a lesser extent ATP, in vitro GTP binding does not result in p29 phosphorylation. Under ER activating conditions, p29 associates with cytosolic ER; GTP, ATP and sodium molybdate block formation of immunoprecipitable p29-ER complexes. Nucleotide binding data suggest a role for p29 in the estrogen response machinery, possibly at the level of phosphate or nucleotide metabolism.

Postmenopausal women all receiving treatment by oral estrogen replacement therapy (ERT) for climacteric symptoms (either Progynova Harmongen or Premarin) gave endometrial samples to determine levels of estrone estradiol estradiol dehydrogenase and nuclear estradiol receptor and to compare differences in these levels among women taking estrogen-only formulations and those taking estrogen plus norethisterone at time of curettage. Data from samples obtained when Premarin was administered alone showed a significant excess of estradiol over estrone in nucleus whereas the converse applied in plasma. Therefore estradiol seems clearly the predominant intranuclear estrogen and the nucleus apparently has the ability to take up estradiol selectively as compared to estrone; this selective process may also apply to both the specifically bound and free estrogen fraction of cytosol (data scant). When coupled with norethisterone the nuclear content of estradiol but not estrone was decreased. So whereas there was a 4-fold excess of estradiol over estrone in endometria from women taking estrogen-only preparations this ratio diminished to near-unity when norethisterone was included in the regimen. The diminished nuclear estradiol content in endometrium treated with progestin correlated with the lower nuclear estradiol receptor content and increased estradiol dehydrogenase activity. Again in combined preparation-treated women there was an excess of estrone over estradiol in the plasma. Therefore it seems that in postmenopausal women estradiol is the most active intranuclear estrogenic form and that concurrent progestin use can diminish the nuclear estradiol content presumably through a mechanism involving estradiol dehydrogenase. A brief experiment to characterize nuclear estradiol receptor in estradiol-primed endometrial samples showed according to labeled estradiol binding at both 4 and 30 degrees centigrade that the nuclear receptor behaved as if it were uncomplexed with estradiol. The same behavior was observed with highly purified nuclei.

The efficacy of commercially available progestin preparations were investigated with a view toward determining the optimum type, dose, duration, and route of administration required to protect the endometrium. Biochemical indices of estrogen and progestin action in endometria from postmenopausal women receiving various hormone therapies were monitored. The premenopausal samples obtained during the proliferative and secretory phases of the cycle can be compared with physiologically normal activities. Estrogen effects were monitored by nuclear estradiol receptor (REN) and soluble progesterone receptor (RP) content and DNA synthesis by autoradiography after [3-H]-thymidine labelling. Progestin action was assayed by inhibition of estrogen-induced REN and DNA synthesis by induction of isocritic and estradiol dehydrogenases and by morphological criteria. Postmenopausal patients were attending the menopause clinics at King's College Hospital or the Chelsea Hospital for Women in London for symptoms associated with the climacteric. Premenopausal samples were obtained from women attending the above hospitals as well as St. Thomas Hospital in London. There are no differences in REN or estradiol receptor content (RET) between epithelium and stroma for any of the groups. Progestins, regardless of whether they are derived from exogenous (postmenopausal) or endogenous (premenopausal sources, decrease REN and RET in both fractions. Progestins also decreased DNA synthesis in both cell types and this suppression correlates with the fall in REN. The RP content of epithelium is greater than stroma, but the 2 enzymes are markedly stimulated by progestins in epithelium but not stroma. The lower RP content of the stromal fraction could be because of cellular heterogeneity, differential loss of receptor during processing, or to genuine differences between epithelium and stroma. Estrogen induced DNA synthesis is inhibited by progestins in both epithelium ans stroma but the induction of some enzymes is dissimilar in the 2 cell populations. Marked increases in activity of isocitric and estradiol dehydrogenases take place in epithelium but not stroma under the influence of progestins. Enzymes such as acid and alkaline phosphatase do not exhibit this uneven cellular distribution. For the clinical studies on progestin effects, it was decided to analyze DNA synthesis, REN, and estradiol and isocritric dehydrogenase activities. All estrogenic medications in clinical use in the U.K. produce levels of REN and RP that are at least equivalent to those found in the proliferative phase of the premenopausal endometrium. No differences in REN, RP, or DNA synthesis were observed between the 0.625 and 1.25 mg doses of Premarin so it appears that even the low dose of estrogen is maximally stimulating the endometrium. The study results strongly indicate that the addition of a progestin to estrogen medication is efficacious in preventing continued cell multiplication of both epithelium and stroma. They also show that unnecessarily high doses of progestin are in current use.

156 patients with advanced breast cancer of known estrogen receptor (ER) and progesterone receptor (PgR) status treated by endocrine therapy were studied. Regarding values for ER and PgR greater than or equal to 5 fmole/mg cytosol protein as positive, patients were divided into 4 phenotypic subgroups: ER+PgR+ (43%), ER+PgR- (26%), ER-PgR+ (8%), and ER-PgR- (23%). In patients with tumor phenotype ER+PgR+, responses were seen in 20/30 (67%) assessable initial treatments when receptor assays were performed on tumor recurrence or on primary tumor immediately before endocrine therapy, and in only 11/32 (34%) assessable initial treatments when receptor analysis was performed on primary tumor and there was intervening local therapy before endocrine therapy was started for tumor recurrence (P less than 0.05). Responses to first endocrine therapy for each tumor phenotype were ER+PgR+ 50%, ER+PgR- 27%, ER-PgR+ 27%, and ER-PgR- 6%. Four of 16 (25%) patients with ER+PgR+ tumors responded to subsequent secondary endocrine therapy, but such responses were not observed in 20 patients with other tumor phenotypes. Duration of response was similar for each phenotype, but patients with ER-PgR- tumors had a significantly shorter survival from time of initial endocrine treatment than patients of any other phenotype. These results suggest that repeat steroid receptor assays on accessible tumor immediately before endocrine therapy may result in improved predictability.

The effect of three antiandrogens, flutamide, hydroxyflutamide and cyproterone acetate on the growth of an androgen responsive mouse mammary tumour cell line has been investigated. These effects have been correlated with binding of the compounds to the androgen receptor. The growth inhibitory activities of hydroxyflutamide and cyproterone acetate are clearly related to their ability to inhibit androgen binding to cytoplasmic and nuclear receptors. Flutamide, however, inhibits cell growth in both the presence and absence of testosterone but does not inhibit DHT binding to the receptor.

Progression from a steroid sensitive to insensitive state is characteristic of breast tumors, but little is known about the molecular mechanisms involved. Changes in steroid receptor can be associated with the progression. This paper reviews the cell culture data pertaining to loss of response and concludes that loss of receptor is a consequence rather than a cause of insensitivity. This view is based on evidence that loss of all response parameters occurs despite the presence of fully functional receptors as determined by transfection experiments. The postreceptor defect appears to be at the level of the hormone response element of the responsive genes arid may involve DNA methylation. The implications of the model for human breast cancer biology are discussed.

Of 54 patients with advanced breast cancer, 21/37 (57%) with oestrogen receptor-positive (ER+) tumours and 11/17 (65%) with oestrogen receptor-negative (ER-) tumours responded to cytotoxic chemotherapy. These data and a survey of the literature indicate that oestrogen receptor status is not a determinant of response to chemotherapy. However, ER+ tumours seem to grow more slowly than ER- ones and the response to chemotherapy tends to be longer in ER+ patients.

Most of the information we have about the regulation of breast tumor growth relates to hormones, especially steroid hormone effects. Such actions are diverse and complex involving direct effects on the tumor cells and indirect effects via other endocrine and non-endocrine organs. Because of this complexity, it is extremely difficult to interpret in vivo data at the required level of cellular and molecular organization so cell culture systems have to be extensively used. Despite the general dogma that ‘cancer cells have escaped from growth control’, establishment of cultures from primary breast tumors has been difficult, more difficult in fact than with normal mammary cells [1]. Furthermore, the establishment of meaningful hormonal regulation of such cultures has been elusive. This has led many workers, ourselves included, to use established cell lines to answer the question of how do steroids affect mammary tumor cell growth? Cell lines clearly have the advantage of relative cellular homogeneity, reproducibility from one experiment to another, ease of experimental manipulation and an approximately defined medium in which growth can be studied. The word ‘approximately’ is used because, although serum free conditions have been used for looking at short to medium-term effects of steroids [2–4], serum is present in the majority of such experiments and is always used for long-term maintenance of such cultures.

The role of steroids in promoting cell proliferation is well established1–3 but the molecular mechanisms are not clear. The S115 mouse mammary tumour cell line provides a model system for molecular studies in vitro in that it exhibits in tissue culture both a positive proliferative response to androgens and a change from a transformed phenotype in the presence of androgen to a normal phenotype when androgen is removed. We have considered here the possible involvement of mouse mammary tumour virus (MMTV) in these processes. We have demonstrated the presence in S115 cells of MMTV-related sequences which are transcribed into RNA only in the long-term presence of androgen. Prolonged culture in the absence of androgen, which results in loss of proliferative response to androgen, is accompanied by loss of MMTV-related RNA and increased methylation of MMTV-related sequences.

SUMMARY The radioactive material in rat pituitary, liver and uterus 1 hr. after the s.c. injection of either 0·73 or 7·3 μg. [6,7-3H]oestradiol has been investigated. Oestrone and oestradiol were the only compounds detected in pituitary and uterus whereas at least six compounds were present in liver. Cell fractionation studies have been carried out on anterior pituitary and liver from female rats which had received an injection of either 0·73 or 7·3 μg. [6,7-3H]oestradiol in vivo. Most of the tritium in the anterior pituitary was associated with the nuclear fraction whereas most of the tritium in the liver was in the soluble fraction. The uptake of [6,7-3H]oestradiol by the anterior pituitary reached a maximum ½-1 hr. after injection of the steroid and remained at this level for the next 3 hr. The intracellular distribution of this steroid at different times after injection suggests that the steroid passes into the cytoplasm and then into the nucleus.

SUMMARY The uptake of [6,7-3H]oestradiol in vivo and in vitro by cell fractions from regions of rat brain and the anterior pituitary gland has been studied. Cytoplasmic and nuclear receptors were detected in anterior and posterior hypothalamus but not in brain cortex. After labelling in vivo, tissues took up [6,7-3H]oestradiol in the following order of magnitude: anterior pituitary > anterior hypothalamus > posterior hypothalamus > cortex. With the exception of the cortex, all extracts from mature tissues had a higher uptake/mg protein than did extracts from immature animals. In the in-vitro system, oestradiol-17β competed with [6,7-3H]oestradiol-17β in the hypothalamus whereas progesterone, testosterone and oestradiol-17α did not. In the pituitary, oestradiol-17α and 17β competed for binding sites. A single injection of testosterone propionate on the second day of life affected [6,7-3H]oestradiol binding in later life. By 28 days of age, the androgenized animals had a lower nuclear and higher cytoplasmic uptake of [6,7-3H]oestradiol in anterior hypothalamus. This effect was not seen in the posterior hypothalamus or cortex. Binding was decreased in all fractions from the pituitary. In mature animals (60 days old), binding fell in both nuclear and cytoplasmic fractions from anterior hypothalamus and pituitary. The nuclei from posterior hypothalamus also took up less [6,7-3H]oestradiol after androgenization. Androgenization affected specific binding in uteri at both 28 and 60 days of age.

Summary The work of our group on 3H-estradiol-binding is reviewed with respect to the following points. 1. What is the nuclear acceptor molecule for the nuclear receptor? Evidence is presented that it might be DNA. There is a tissue specificity about the ability of both nuclei and chromatin to accept uterine nuclear receptor. This specificity does not reside in the DNA and it is suggested that histones may play a part in the acceptor mechanism both by retaining the receptor on the DNA and by making different regions of the DNA available in different types of chromatin. The non-histone proteins would also be important from the latter aspect. 2. Is the estradiol-binding mechanism influenced by endogeneous hormones by routes other than competition for receptor sites? The answer appears to be yes and the endogeneous hormones act at more than one locus. Both qualitative and quantitative changes in uterine binding occur after ovariectomy and hypophysectomy but not adrenalectomy. The qualitative effect of ovariectomy can be reversed by estradiol but not progesterone. Ovariectomy also decreases the ability of both uterine nuclei and chromatin to accept 3H-estradiol receptor. 3. Are there estradiol receptors in several uterine cell types? Yes, cytoplasmic receptors have been found in both epithelium and stroma. The stromal receptors are more labile than the epithelial ones. 4. What are the properties of estradiol-receptors in hormone responsive and unresponsive, DMBA-induced rat mammary tumours and estrogen induced hamster kidney tumours? In most respects the responsive tumours bind estradiol by a mechanism similar to that in uterus. Evidence is presented that, in the mammary tumour, nuclear receptor-acceptor interaction may be weaker than in other estrogen sensitive cells. The relatively unresponsive transplanted kidney tumour has many cytoplasmic receptors as other estrogen sensitive cells. 5. Hypothalamic binding of estrogen. Specific receptors exist in both the anterior and posterior portions of the hypothalamus. These can be influenced by neonatal administration of testosterone propionate. Some evidence is presented that the hypothalamus can influence uterine estradiol binding. 6. References are quoted for specific estradiol binding in anterior pituitary gland, vagina, human endometrium, fallopian tube and endometrial carcinoma and rat kidney. 7. Preliminary data is presented to suggest that different types of protein migrate from cytoplasm to nucleus in different types of cell.

Sex hormones play a decisive role in the determination of hypothalamic function. In the neonatal rat, the “basic” condition appears to be the female type, in which the hypothalamus, at about the time of puberty, exerts a cycling effect on the sex hormone-dependent cells; this results in the estrous cycle (1,2,3). In male rats this potential cyclic activity of the hypothalamus is irreversibly switched off at, or near to birth by a process that seems to require androgen (4,5). The role of sex steroid-binding proteins in these processes has not been fully delineated and this paper will present data that is pertinent to this topic. An experimental tool that has been much used in the study of hypothalamic development is the neonatally androgenized rat. Injection of certain androgens, notably testosterone propionate, into neonatal female rats produces, in adult life, a state of persistent vaginal estrous, polyfollicular ovaries, cystic glandular hyperplasia of the uterus, sterility and male-type behavior (4,5). Pfeiffer (6), who first discovered this syndrome, thought that it was due to effects on the anterior hypophysis, but it is now known that the main effect is on the anterior hypothalamicpreoptic region of the brain, with the primary effect being on the latter region (5,6). In the adult female rat this region controls the cyclic release of gonadotrophins via the hypothalamus and anterior hypophysis.

The in vivo incorporation of [ 3 H] thymidine into rat mammary gland has been studied under different conditions. Incorporation into DNA was higher in glands obtained at oestrus than at prooestrus and decreased after ovariectomy. The effect of ovariectomy was prevented by oestradiol injection. The incorporation of [ 3 H] thymidine was greater in the terminal buds of the mammary gland than in the central area of the gland. DMBA markedly inhibited incorporation into DNA of glands obtained at both oestrus and prooestrus. It also inhibited the incorporation into uterine DNA. The non-carcinogenic hydrocarbon, benzanthracene, also inhibited DNA synthesis in mammary gland. It is concluded that the DMBA inhibition of DNA synthesis is not directly related to the carcinogenic action of this hydrocarbon.

Intracellular oestrogen metabolism has been investigated in endometrial tissue from postmenopausal women receiving oestrogen therapy either alone or in combination with a progestogen. During oestrogen therapy alone, there was a 3.2 fold predominance of oestradiol over oestrone within the endometrial cell nucleus and the mean nuclear oestrogen receptor content was 1.40 pmol/mg DNA. The addition of norethisterone decreased the nuclear oestradiol/oestrone ratio to 1.4:1 by lowering the oestradiol mass. A concurrent reduction in the mean level of the nuclear oestrogen receptor to 0.58 pmol/mg DNA indicated a decrease in oestrogenic stimulation. The activity of oestradiol-17 beta dehydrogenase was significantly increased. There was a plasma excess of oestrone over oestradiol during oestrogen therapy alone and oestrone/oestradiol ratio was not significantly altered following norethisterone administration. The ability of the endometrium to incorporate oestradiol selectively into the nucleus is discussed in relation to the risk of endometrial hyperplasia with unopposed oestrogen therapy. The profound biochemical changes induced by norethisterone help elucidate mechanisms whereby progestogens lower oestrogenic potency and thereby protect the endometrium against excessive stimulation.