History of aromatase: saga of an important biological mediator and therapeutic target.

Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens. Initial studies of its enzymatic activity and function took place in an environment focused on estrogen as a component of the birth control pill. At an early stage, investigators recognized that inhibition of this enzyme could have major practical applications for treatment of hormone-dependent breast cancer, alterations of ovarian and endometrial function, and treatment of benign disorders such as gynecomastia. Two general approaches ultimately led to the development of potent and selective aromatase inhibitors. One targeted the enzyme using analogs of natural steroidal substrates to work out the relationships between structure and function. The other approach initially sought to block adrenal function as a treatment for breast cancer but led to the serendipitous finding that a nonsteroidal P450 steroidogenesis inhibitor, aminoglutethimide, served as a potent but nonselective aromatase inhibitor. Proof of the therapeutic concept of aromatase inhibition involved a variety of studies with aminoglutethimide and the selective steroidal inhibitor, formestane. The requirement for even more potent and selective inhibitors led to intensive molecular studies to identify the structure of aromatase, to development of high-sensitivity estrogen assays, and to "mega" clinical trials of the third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, which are now in clinical use in breast cancer. During these studies, unexpected findings led investigators to appreciate the important role of estrogens in males as well as in females and in multiple organs, particularly the bone and brain. These studies identified the important regulatory properties of aromatase acting in an autocrine, paracrine, intracrine, neurocrine, and juxtacrine fashion and the organ-specific enhancers and promoters controlling its transcription. The saga of these studies of aromatase and the ultimate utilization of inhibitors as highly effective treatments of breast cancer and for use in reproductive disorders serves as the basis for this first Endocrine Reviews history manuscript.

Placental characteristics and reduced risk of maternal breast cancer.

BACKGROUND: Women who have preeclampsia during pregnancy are at reduced risk of subsequent breast cancer. We examined whether other markers of reduced placental size or function, including increased blood pressure during pregnancy, predict a reduction in maternal breast cancer. METHODS: The Child Health and Development Studies is a 40-year follow-up of pregnant women enrolled in the Kaiser Permanente health plan between 1959 and 1967. We identified 3804 white women for whom data were available on placental examinations and other study variables. As of 1997, 146 women had developed invasive breast cancer. Proportional hazards models were used to estimate associations of breast cancer with markers of placental function. All statistical tests were two-sided. RESULTS: A blood pressure increase between the second and third trimesters exhibited a linear relationship with breast cancer rate, with the highest quartile showing a 51% reduction (95% confidence interval [CI] = 20% to 70%) that was not explained by preeclampsia. Smaller placental diameter was independently associated with a reduced breast cancer rate; the association increased with age at first pregnancy (P =.008). Maternal floor infarction of the placenta was associated with a 60% reduction in breast cancer rate (95% CI = 12% to 82%). In combination, placental risk factors were associated with a reduction in the breast cancer rate of as high as 94% (95% CI = 80% to 98%). CONCLUSIONS: Smaller placentas, maternal floor infarction of the placenta, and increasing blood pressure during pregnancy were associated with reduced maternal breast cancer. In the case of smaller placental diameter, the larger reduction observed with older age at first pregnancy suggests a process in which promotion of an existing lesion is blocked. Elucidating the mechanisms for these associations could provide clues to breast cancer prevention and treatment.

Insulin and endometrial cancer.

Elevated insulin levels may explain part of the increased risk of endometrial cancer observed in obese postmenopausal women. Circulating sex hormones and fasting C-peptide levels were measured in sera obtained from 165 postmenopausal endometrial cancer cases accrued between June 1, 1987, and May 15, 1990, from hospitals in Chicago, Illinois; Hershey, Pennsylvania; Irvine and Long Beach, California; Minneapolis, Minnesota; and Winston-Salem, North Carolina, and 180 community and hysterectomy controls. Women with a personal history of diabetes were excluded. Among controls, C-peptide was positively correlated with body mass index (BMI) ((r = 0.44), waist-to-thigh circumference ratio ((r = 0.24), estrone ((r = 0.18), and estradiol ((r = 0.28) (albumin-bound (r = 0.45), and free (r = 0.37)) and negatively correlated with sex hormone-binding globulin (r = -0.48). In age-adjusted analyses, the odds ratios and 95% confidence intervals for tertiles of C-peptide and endometrial cancer were, from lowest to highest: 1.0 (reference), 0.78 (95% confidence interval (CI) 0.43-1.4), and 2.2 (95% CI 1.3-3.7). Further adjustment for BMI substantially attenuated the odds ratios for the highest tertile of C-peptide (odds ratio = 1.2, 95% CI 0.63-2.1), and adjustment for body mass index and other risk factors for endometrial cancer eliminated the association (odds ratio = 1.0, 95% CI 0.55-2.0). In contrast, adjustment for C-peptide had little influence on the magnitude of the positive associations between body mass index (odds ratio for highest vs. lowest tertile, without and with adjustment for C-peptide = 4.1 (95% CI 2.3-7.5) and 3.7 (95% CI 1.9-7.1), respectively) or several steroid hormones and endometrial cancer. These data are not consistent with the hypothesis that the effect of obesity on endometrial cancer risk is mediated through high insulin levels.

Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.

Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated.

Reproducibility studies and interlaboratory concordance for assays of serum hormone levels: estrone, estradiol, estrone sulfate, and progesterone.

We conducted studies to measure sources of assay variability for estrone, estradiol, estrone sulfate, and progesterone for postmenopausal women (n = 5) and for women in the mid-follicular (n = 5) and mid-luteal (n = 5) phases of the menstrual cycle. A single blood sample from each woman was divided into 2.5-ml aliquots and stored at -70 degrees C, and sets of two aliquots were sent at monthly intervals to each of three laboratories (four for progesterone). Each aliquot was analyzed in duplicate. Thus, within each menstrual category, we were able to estimate the components of variance due to variation among women, variation among aliquots, variation among duplicate measurements, and variation among the 4 analysis days. Using the logarithm of assay measurements, we estimated the percentage of variance attributable to variation among women in each menstrual category, 100 rho, is the estimated intraclass correlation. For each assay, 100 rho exceeded 90% for mid-follicular and mid-luteal women. For postmenopausal women, values of 100 rho exceed 84% for estrone in two laboratories. Values of 100 rho were lower for progesterone in postmenopausal women, although a value of 84% was estimated from one laboratory. These studies indicate that estrogen assays over a period of 3 months permit reliable comparisons among women in a given menstrual category. Progesterone measurements are likewise reliable for women in the mid-follicular and mid-luteal phases but somewhat less satisfactory for postmenopausal women. These assessments of variability pertain only to laboratory techniques and do not allow for secular variation in intra-woman hormone levels. Moreover, although these measurements tend to be reliable enough for making comparisons among women, estimates of coefficients of variation for estrogens are about 10% for mid-follicular and mid-luteal phase women and about 11-20% for postmenopausal women. Coefficients of variation for progesterone are about 10% for mid-luteal, 20% for mid-follicular, and 30% for postmenopausal women.

Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction.

The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.

Case-control study of endogenous steroid hormones and endometrial cancer.

BACKGROUND: It has been suggested that identified risk factors for endometrial cancer operate through a single etiologic pathway, i.e., exposure to relatively high levels of unopposed estrogen (estrogen in the absence of progestins). Only a few studies, however, have addressed this issue directly. PURPOSE: We assessed the risk of developing endometrial cancer among both premenopausal and postmenopausal women in relation to the circulating levels of steroid hormones and sex hormone-binding globulin (SHBG). The independent effect of hormones was assessed after adjustment for other known risk factors. METHODS: The data used in the analysis are from a case-control study conducted in five geographic regions in the United States. Incident cases were newly diagnosed during the period from June 1, 1987, through May 15, 1990. The case patients, aged 20-74 years, were matched to control subjects by age, race, and geographic region. The community control subjects were obtained by random-digit-dialing procedures (for subjects 20-64 years old) and from files of the Health Care Financing Administration (for subjects > or = 65 years old). Additional control subjects who were having a hysterectomy performed for benign conditions were obtained from the participating centers. Women reporting use of exogenous estrogens or oral contraceptives within 6 months of interview were excluded, resulting in 68 case patients and 107 control subjects among premenopausal women and 208 case patients and 209 control subjects among postmenopausal women. The hormone analyses were performed on blood samples obtained from case patients or from hysterectomy control subjects before surgery. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by use of an unconditional logistic regression analysis after we controlled for matching variables and potential confounders. All P values were two-sided. RESULTS: High circulating levels of androstenedione were associated with 3.6-fold and 2.8-fold increased risks among premenopausal and postmenopausal women, respectively, after adjustment for other factors (P for trend = .01 and < .001, respectively). Risks related to other hormone fractions varied by menopausal status. Among postmenopausal women, a reduced risk was associated with high SHBG levels and persisted after adjustment was made for obesity and other factors (OR = 0.51; 95% CI = 0.27-0.95). High estrone levels were associated with increased risk (OR = 3.8; 95% CI = 2.2-6.6), although adjustment for other risk factors (particularly body mass index) diminished the effect (OR = 2.2; 95% CI = 1.2-4.4). Albumin-bound estradiol (E2), a marker of the bioavailable fraction, also remained an important risk factor after adjustment was made for other factors (OR = 2.0; 95% CI = 1.0-3.9). In contrast, high concentrations of total, free, and albumin-bound E2 were unrelated to increased risk in premenopausal women. In both premenopausal and postmenopausal groups, risks associated with obesity and fat distribution were not affected by adjustment for hormones. CONCLUSION: High endogenous levels of unopposed estrogen are related to increased risk of endometrial cancer, but their independence from other risk factors is inconsistent with being a common underlying biologic pathway through which all risk factors for endometrial cancer operate. IMPLICATIONS: Further research should focus on alternative endocrinologic mechanisms for risk associated with obesity and body fat distribution and for the biologic relevance of the increased risk associated with androstenedione in both premenopausal and postmenopausal disease.

Relative weight, weight change, height, and breast cancer risk in Asian-American women.

BACKGROUND: Breast cancer incidence rates have historically been four to seven times higher in the United States than in China or Japan, although the reasons remain elusive. When Chinese, Japanese, or Filipino women migrate to the United States, their breast cancer risk rises over several generations and reaches that for white women in the United States, indicating that modifiable exposures are involved. In a previous report on this case-control study of breast cancer in Asian-American women, designed to take advantage of their diversity in risk and lifestyle, we demonstrated a sixfold gradient in risk by migration history, comparable to the international differences in breast cancer incidence rates. PURPOSE: In this analysis, we have examined the roles of adult height, adiposity, and weight change in breast cancer etiology. METHODS: A population-based, case-control study of breast cancer was conducted among women of Chinese, Japanese, and Filipino ethnicities, aged 20-55 years, living in San Francisco-Oakland (CA), Los Angeles (CA), and Oahu (HI) during the period from April 1, 1983, through June 30, 1987. We successfully interviewed 597 (70%) of 852 eligible case subjects and 966 (75%) of 1287 eligible control subjects from August 1985 through February 1989. Subjects were asked about current height, usual adult weight, and usual weight in each decade of life, excluding the most recent 3 years and any periods of pregnancy. RESULTS: Height, recent adiposity (weight in the current decade of life/height 1.5), and recent weight change (between the current and preceding decades of life) were strong predictors of breast cancer risk after adjustment was made for accepted breast cancer risk factors. Risk doubled (relative risk [RR] = 2.01; 95% confidence interval [CI] = 1.16-3.49) over the 7-inch (17.8-cm) range in height (two-sided P for trend = .003), with comparable effects in both premenopausal and postmenopausal women. Except for reduced risk in the heavy, younger women (weight/height 1.5 > 29 kg/m 1.5 and < 40 years old), risk was positively associated with usual adult adiposity. Trends in risk became more striking as adiposity in each succeeding decade of adult life was considered. Women in their 50s and in the top quintile for their age group had twice the breast cancer risk (RR = 2.13; 95% CI = 1.17-3.87) of women in the bottom quintile (two-sided P for trend = .004). Women in their 50s, above the median adiposity for their age group, and with a recent gain of more than 10 pounds had three times the risk (RR = 3.01; 95% CI = 1.45-6.25) of women below the median adiposity and with no recent weight change. Recent weight loss was consistently associated with reduced risk (RRs of approximately 0.7) relative to no recent weight change. CONCLUSIONS: Adult adiposity, weight change, and height are critical determinants of breast cancer risk. Increased adiposity and weight gain in the decade preceding diagnosis are especially influential, suggesting that excess weight may function as a late stage promoter. IMPLICATIONS: Weight maintenance and/or reduction as an adult, possibly accompanied by specific changes in diet and physical activity, may have a significant and rapid impact on breast cancer risk.

Separation of estrogens by micellar electrokinetic chromatography.

Capillary electrophoresis of the sex hormone estrogens using different buffer components was investigated. Free zone electrophoresis with 10 mM phosphate buffer (pH 11.5) or 10 mM phosphate buffer with 10-20% methanol was not effective in separating the ten estrogens used in this study. However, nine estrogens were resolved by micellar electrokinetic chromatography using a 10 mM borate buffer (pH 9.2) containing 100 mM sodium cholate. In addition, some estrogens were partially separated using sodium dodecyl sulfate (SDS) micellar buffers; however, the addition of modifiers such as organic solvents or cyclodextrins improved resolutions significantly. Using a 10 mM phosphate buffer (pH 7.0) containing 50 mM SDS and 20% methanol, or a 10 mM borate buffer (pH 9.2) containing 50 mM SDS and 20 mM gamma-cyclodextrin, all ten of the tested estrogens were separated. However, the cyclodextrin-modified buffer allowed faster separation.

Reproducibility of laboratory assays for steroid hormones and sex hormone-binding globulin.

The relationship of serum hormones to cancer risk has recently been pursued in epidemiological studies, but few have reported on the reproducibility of laboratory findings. Prior to conducting a study of endogenous hormones and endometrial cancer, we evaluated the reproducibility of measurements for several hormones (estrone, estradiol, free estradiol, albumin-bound estradiol, and androstenedione) and sex hormone-binding globulin. We obtained a single unit of blood from each of six women and prepared aliquots of serum for repeated testing. Three laboratories analyzed multiple samples on consecutive working days from which estimates of intraassay and interassay measurement variability were obtained. For estrone and estradiol, a log transformation of the data produced distributions which were nearly normal and permitted the use of parametric statistical tests. In general, we found measurements for most hormones varied considerably between assays. Moreover, differences were observed in the absolute values of sex hormone-binding globulin and of the hormones, particularly for estrone and estradiol, from one laboratory to the next. Our findings suggest that variability of current laboratory procedures may hamper efforts to study the association between disease and endogenous hormones in epidemiological studies. In addition, validation of hormone assays is essential in order to assure standardized results and enable comparisons of data across studies.

Squirrel monkey corticosteroid-binding globulin: primary structure and comparison with the human protein.

Squirrel monkey (Saimiri sciureus) corticosteroid-binding globulin (CBG) is the product of a 1.6-kilobase mRNA in the liver. Analyses of two overlapping cDNAs revealed that the squirrel monkey CBG precursor comprises 406 amino acids, the first 22 residues of which exhibit 91% identity with the human CBG leader sequence. The mature form of squirrel monkey CBG, therefore, very likely comprises 384 amino acids and has a polypeptide mol wt of 42,854. Compared to human CBG, the squirrel monkey protein contains an additional residue (threonine) at position 144, and the two proteins exhibit 86% sequence identity if this is taken into account. Squirrel monkey CBG contains five consensus sites for N-glycosylation, four of which are located in analogous positions in human CBG, and has two cysteine residues in the same relative positions as the cysteines in human CBG. Unlike CBG in most other species, squirrel monkey CBG appears to circulate as a dimer, and its affinity for glucocorticoids is remarkably low. We, therefore, expressed cDNAs for human and squirrel monkey CBGs in Chinese hamster ovary (CHO) cells and compared the physico-chemical properties of the products with those of the corresponding serum proteins. Squirrel monkey CBG is produced by CHO cells as a dimer, and its subunit size heterogeneity is similar to that associated with CBG in serum. In addition, the cortisol-binding affinity of squirrel monkey CBG produced by CHO cells is similar to that of the natural protein and is 5- to 8-fold lower than that of natural or recombinant human CBG. Mutants in which a threonine at position 144 was either added to human CBG or subtracted from squirrel monkey CBG were also expressed in CHO cells. This demonstrated that this additional amino acid in the squirrel monkey CBG sequence may actively contribute to its propensity for spontaneous dimerization, but does not account for its relatively low steroid-binding affinity.

Aromatase gene is amplified in MCF-7 human breast cancer cells.

The levels of the aromatase gene and its expression in MCF-7 human breast cancer cells and seven additional cultured cells were investigated. Using normal human foreskin fibroblasts as the control, the aromatase gene appeared to be amplified in MCF-7 cells as shown by Southern and DNA slot blot analyses utilizing human placental aromatase cDNA as the probe. However, the promoter I.1 and the first exon of the aromatase gene were not amplified in MCF-7 cells based on results obtained from DNA slot blot analysis using oligonucleotide probes having sequences derived from those regions of human aromatase gene. Aromatase was expressed at a very low level in this cell line as indicated by Northern blot analysis to measure the level of aromatase mRNA, immunoprecipitation analysis to measure the level of aromatase protein, and aromatase activity measurement. Furthermore, nucleotide sequence analysis of the aromatase cDNA obtained from MCF-7 cells by PCR techniques, revealed no sequence difference from that of the enzyme expressed in placenta. These results lead us to conclude that the expression of aromatase in MCF-7 cells is under the control of an unusual promoter and aromatase gene expression is repressed at the transcriptional level in these cells.

Structure and function of the hepatic form of 11 beta-hydroxysteroid dehydrogenase in the squirrel monkey, an animal model of glucocorticoid resistance.

Both cortisol and aldosterone bind to and activate the mineralocorticoid receptor. Cortisol concentrations are generally 100- to 200-fold higher than aldosterone concentrations, yet mineralocorticoids clearly exert effects different from glucocorticoids. One hypothesis is that 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts cortisol to biologically inactive cortisone, protects the mineralocorticoid receptor from cortisol. The circulating concentrations of cortisol in the squirrel monkey are 20- to 50-fold higher than human cortisol concentrations, yet this animal has no evidence of glucocorticoid or mineralocorticoid excess. We used this experiment of nature to test the hypotheses that the known (hepatic) form of 11 beta-HSD protects renal mineralocorticoid receptors from the action of cortisol and that it modulates glucocorticoid concentrations in target tissues. Using a long oligonucleotide based on the rat sequence, we cloned the squirrel monkey 11 beta-HSD complementary DNA and gene. The encoded monkey amino acid sequence is 75% and 91% identical to the corresponding rat and human sequences, respectively. The tissue abundance of the messenger RNA for the monkey enzyme was similar to or less than that seen for the rat and human enzymes. Both the monkey and human 11 beta-HSD complementary DNAs were cloned into an expression vector and used to transfect cultures of Chinese hamster ovary cells. Both vectors were transcribed and translated into equivalent amounts of 11 beta-HSD enzyme. The monkey enzyme was slightly more efficient than the human enzyme in converting [3H]cortisol to cortisone, and estimates of the Michaelis-Menten constant and maximum velocity of both enzymes are similar. These data indicate that the abundance and activity of the hepatic form of 11 beta-HSD are insufficient to inactivate the very high concentrations of cortisol in the squirrel monkey, suggesting that this form of 11 beta-HSD does not defend the mineralocorticoid receptor or protect tissues from high cortisol concentrations. Rather, this enzyme appears to favor conversion of cortisone to cortisol, thus maximizing tissue concentrations of cortisol to overcome glucocorticoid resistance associated with a 50% reduction in glucococorticoid receptors.

A mechanism for virilization of female spotted hyenas in utero.

Female spotted hyenas exhibit male-like genitalia and dominance over males. Hyena ovarian tissues incubated in vitro produced large quantities of the steroid hormone precursor androstenedione. The activity of aromatase, which converts androstenedione to estrogen, was one-twentieth as great in hyena versus human placental homogenates. In comparison, the activity of 17 beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, was equal in the two homogenates. The limited aromatase activity may allow the hyena placenta to convert high circulating concentrations of androstenedione to testosterone, which results in virilization of the fetal external genitalia and possibly destruction of fetal ovarian follicles. Androstenedione production by residual ovarian stromal cells during reproductive life accounts for the epigenetic transmission of virilization in female spotted hyenas.

Hormonal correlates of 'masculinization' in female spotted hyaenas (Crocuta crocuta). 2. Maternal and fetal steroids.

Concentrations of androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone), oestrogen and progesterone were measured in relation to pregnancy in the spotted hyaena (Crocuta crocuta). The gestation period was estimated to be about 110 days. There was a marked progressive rise in all the steroids starting in the first third of gestation. Chromatographic separation of plasma showed that much of the oestrogen is not oestradiol (only 12% of total measured) and that a significant fraction of the 'testosterone' may be dihydrotestosterone. In the final third of pregnancy, concentrations of androgen (especially testosterone plus dihydrotestosterone) in the female circulation reached the maximal values of adult males; the percentage of dihydrotestosterone relative to total testosterone plus dihydrotestosterone was higher in females (44 +/- 3.9%, n = 20) than in males (29.5 +/- 3.5%, n = 17). Plasma androstenedione was also significantly higher in females, but the increment was less than for oestrogen, testosterone and progesterone, and the temporal pattern was less clear. Samples from the maternal uterine and ovarian circulation showed that androstenedione is largely of ovarian origin and metabolized by the placenta, while testosterone, progesterone and oestrogen are primarily of placental or uterine origin. Fetal samples were taken from two mixed-sex sets of twins and one male singleton. Gradients across the placenta measured in the fetal circulation confirmed that the placenta metabolizes androstenedione and is a source of testosterone for the female fetus; there were no consistent differences in androgens between male and female fetuses. It is suggested that the conspicuous masculinization of the female spotted hyaena, especially evident in the external genitalia at birth, is a result, at least in part, of high placental production of testosterone or dihydrotestosterone derived from the metabolism of high maternal androstenedione.

Androgen receptor expression and 5 alpha-reductase activity along the proximal-distal axis of the rat prostatic duct.

The rat prostate consists of a series of branched ducts that eminate from the urethra. Heterogeneity of rat prostatic growth, secretory activity, and cell turnover has been observed along the proximal-distal axis of the branched ductal network. In addition, there are regional differences in androgen sensitivity along the ducts, with the distal ductal tips being highly androgen dependent and the proximal regions being relatively androgen independent. To determine the underlying mechanisms that may regulate these regional differences in androgen responsiveness, androgen receptor (AR) levels and 5 alpha-reductase activity were examined along the proximal-distal axis of microdissected ventral prostatic ducts from 15-, 30-, and 100-day-old rats. As in the murine prostate, DNA synthetic activity was concentrated in the distal tip region of the 15- and 30-day ducts. Immunocytochemistry and autoradiography with [3H] dihydrotestosterone were used to examine AR expression and functional ability to bind ligand, respectively. The results revealed no discernable differences in AR levels or binding activity in any cell type along the ductal length in prepubertal, pubertal, or adult rats. In addition, 5 alpha-reductase activity was the same in the distal and proximal ductal regions. We conclude that regional heterogeneity in prostatic growth and function is not a result of differences in levels of AR and 5 alpha-reductase. Rather, other region-specific structural, intracellular, or paracrine factors may be responsible for the differences in androgen responsiveness along the prostatic duct.

Progestin regulation of insulin and insulin-like growth factor I receptors in cultured human breast cancer cells.

Recent studies indicate that the insulin receptor (IR) content is higher in breast cancer cells than in normal mammary epithelial cells. This observation has been made both in tissue specimens from patients with breast cancer, and in various human cultured breast cancer cell lines. Investigations have now been undertaken to understand the role of progestins in the regulation of the IR and the closely related insulin like growth factors-I receptor (IGF-I-R). Pretreatment of T-47D cultured human breast cancer cell lines with progestins induced a time and dose dependent increase in IR content. This increase was due primarily to an effect of progestins to increase IR mRNA levels. Other steroid hormones including glucocortocoids, estrogen, and testosterone were without effect. In contrast to their up-regulation of the IR, progestins down-regulated the IGF-I-R at the level of mRNA. An analysis of the processes involved revealed that progestins increased the biosynthesis of a ligand for IGF-I receptor, IGF-II. IGF-II in turn down-regulated the IGF-I-R. Thus these studies indicate that progestins have important effects on both the IR and the IGF-I-R. The effects of progestins on these and other growth factor receptors, therefore, may have an important role in the biology of breast cancers.

Regulation of 11 beta-hydroxysteroid dehydrogenase activity in human skin fibroblasts: enzymatic modulation of glucocorticoid action.

The regulation of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) was studied in cultured human skin fibroblasts. 11-Oxo-reductase activity was 5- to 10-fold higher than 11 beta-dehydrogenase activity. Cells treated with 100 nM dexamethasone (Dex) showed a 3-fold increase in the maximum velocity of both activities without a change in the Km values. Dex induction of 11 beta HSD was half-maximal at 48 h and was blocked by glucocorticoid receptor antagonists. Nonglucocorticoid steroids were ineffective. Removal of serum from the culture medium increased maximum velocity values up to 6-fold. Treatment of cells grown in the absence of serum with 8-bromo-cAMP, phorbol esters, or insulin decreased both 11 beta HSD activities. The effects of Dex treatment and serum removal were additive and were blocked by cycloheximide and actinomycin-D. In all experiments both 11 beta HSD activities were modulated in parallel. Both cortisone (200 nM) and cortisol increased the aromatase activity of fibroblasts in the presence of serum. Prior induction of 11 beta HSD by serum removal increased the potency of cortisone from 10-15% to 50% that of cortisol. We conclude that 1) in human fibroblasts 11 beta HSD appears to be a single protein that is under multifactorial regulation; 2) 11 beta HSD may increase or decrease cortisol availability to glucocorticoid receptors; and 3) plasma cortisone levels may be important in assessing glucocorticoid status.