Pentti K. Siiteri

The serum transport of steroid hormones.

Publisher Summary This chapter discusses the serum transport of steroid hormones. Steroid hormones are extensively bound to plasma proteins including albumin, corticosteroid binding globulin (CBG), and sex hormone binding globulin (SHBG). Because of its high concentration, albumin binding is important in determining the magnitude of the nonprotein bound or free fraction of a steroid in plasma. The generally accepted model of steroid hormone action suggests that free steroid (in equilibrium with circulating binding proteins) diffuses passively through target cell membranes and binds to a soluble intracellular receptor. The steroid-receptor complex apparently moves into the nucleus where it modifies the chromatin transcriptional activity which results in, among other things, altered levels of protein synthesis. CBG has been differentiated from the intracellular glucocorticoid and progesterone receptors by its inability to bind synthetic glucocorticoids and progestins.

Studies of human placental aromatase

Abstract Kinetic analysis of aromatization of androstenedione (Δ4) indicates that 3 mol of oxygen and NADPH are consumed in the formation of estrone (El). During the steady state, the 3d oxidation appears to be the rate limiting step since both 19-hydroxy and 19-oxo-androstenedione accumulate prior to El. Cytochrome P-450 participation in each hydroxylation step is suggested by (1) inhibition by known P-450 inhibitors other than CO, (2) inhibition by an antibody to NADPH-cytochrome c reductase, (3) spectral studies of P-450 binding of substrate and intermediates, and (4) solubilization and partial resolution of enzyme components using digitonin and DEAE cellulose. A survey of steroidal compounds has revealed that 5α-reduced androstenedione is a potent competitive inhibitor which may be of physiologic importance in control of ovarian estrogen synthesis. Finally, many steroidal drugs used in treatment of breast cancer are competitive inhibitors. The non androgenic compound 1-ene-testololactone is effective both in vitro and in vivo suggesting that antitumor activity of steroidal drugs may be due to inhibition of estrogen synthesis rather than direct androgen action.

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.

Associations of maternal and umbilical cord hormone concentrations with maternal, gestational and neonatal factors (United States).

Objective: Risks of some cancers in adults have been associated with several pregnancy factors, including greater maternal age and birth weight. For hormone-related cancers, these effects are hypothesized to be mediated through higher in utero estrogen concentrations. In addition, racial differences in pregnancy hormone levels have been suggested as being responsible for differences in testicular and prostate cancer risk by race. However, data on hormonal levels related to these characteristics of pregnancy are sparse, particularly those from studies of the fetal circulation. Methods: Estrogen and androgen concentrations were measured in maternal and umbilical cord sera from 86 normal, singleton pregnancies. Results: Birth size measures (weight, length and head circumference) were positively correlated with maternal estriol (r = 0.25–0.36) and with cord DHEAS concentrations (r = 0.24–0.41), but not with estrogens in cord sera. Maternal age was inversely correlated with maternal DHEAS, androstenedione and testosterone concentrations (r = −0.30, −0.25 and −0.30, respectively), but uncorrelated with estrogens in either the maternal or cord circulation. Black mothers had higher androstenedione and testosterone concentrations than white mothers, however, there were no racial differences in any of the androgens in cord sera. Cord testosterone concentrations were higher in mothers of male fetuses, while both maternal and cord concentrations of estriol were lower in these pregnancies. Conclusions: These data demonstrate associations between hormone concentrations and pregnancy factors associated with offsprings cancer risk, however, the hormones involved and their patterns of association differ by whether the maternal or fetal circulation was sampled. Hormone concentrations in the fetal circulation in this study are not consistent with the hypothesis that greater estrogen concentrations in high birth weight babies mediate the positive association with breast cancer risk observed in epidemiologic studies, or with the hypothesis that higher testosterone exposure in the in utero environment of black males explains their higher subsequent prostate cancer risk.

Sex steroids and the immune system-I. Sex difference in autoimmune disease in NZB/NZW hybrid mice

Abstract Hybrid NZB/NZW (B/W) mice develop an autoimmune disease that has many of the characteristics of human systemic lupus erythematosis including marked sex differences. Studies were done to investigate the role of gonadal steroids in modulating expression of disease. Estrogens accelerate and androgens slow the time course of appearance of antibodies to nucleic acids and death when administered by implants. Castration of females does not influence survival whereas castrated males have earlier development of antibodies and accelerated mortality similar to intact females. In general, administration of estradiol or progesterone accelerates the appearance of IgG antibodies to both DNA and Poly A whereas dihydrotestosterone or testosterone retarded antibody formation. Surprisingly, coadministration of estradiol and dihydrotestosterone increased the mortality rate in both sexes. Studies with estradiol demonstrated receptor-like binding in cytosol and nuclear extracts prepared from the thymus of several strains of mice whereas DHT binding could not be found. Administration of estradiol elevated total receptor binding in the thymus of male B/W mice. While complex mechanisms with other systems are involved, it appears that the sex steroids can influence antibody formation by interaction with the thymus gland.

Differential actions of progesterone and cortisol on lymphocyte and monocyte interaction during lymphocyte activation — relevance to immunosuppression in pregnancy

Progesterone and glucocorticoids share important anti-inflammatory and immunosuppressive properties. Both hormones have potent anti-proliferative effects in MLR, mitogen activation and cytotoxic T-cell generation. We investigated the cellular target of this in vitro anti-proliferative activity by comparing the effects of progesterone and cortisol on lymphocyte-monocyte interaction in concanavalin (Con A) induced human T-cell activation. Three different in vitro systems for assessing monocyte dependent T-cell activation by Con A were used: (1) limiting concentration of monocyte, (2) preincubation of isolated populations of monocytes and T cells with steroids and (3) role of steroid on action of Interleukin-1 (IL-1) activity. Monocytes separated from human peripheral blood leukocytes by flotation gradients and adherence to plastic were cultured at concentrations of 0.5-10% with constant numbers of isolated autologous T cells. Inhibition of Con A activation in cortisol (0.1-10 micrograms/ml) treated cultures was inversely proportional to percent monocytes, whereas in progesterone (2.0-20 micrograms/ml) treated cultures, inhibition was independent of monocyte concentration. Separated monocytes preincubated with progesterone and cultured with fresh T cells supported normal (108 +/- 7% control) levels of activation, but progesterone treated T cells and fresh monocytes responded at about 60% control levels. Similar experiments with cortisol (1 or 10 micrograms/ml) revealed significantly reduced responses when either cell population was preincubated with steroid. IL-1 induced by LPS stimulation of monocytes was blocked in its ability to stimulate Con A induced T cell proliferation with either steroid present during the assay of IL-1. These data provide additional support for local immunosuppression by steroids in the placenta during pregnancy. They suggest that progesterone selectively blocks T cell activation by a direct effect on T cells, whereas cortisol interferes with both monocytes and T cells.

Suppression of murine allogeneic cell interactions by sex hormones.

Investigations have been carried out on the action of several steroid hormones on lymphocyte functions in inbred strains of mice. The recognitive, proliferative and effector phases of allogeneic cell interactions in vitro were assessed using a mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). In MLR containing Balb/c (responder) and C57bl/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of progesterone, cortisol or estradiol. In CML, progesterone and estradiol (1-5 microgram/ml) blocked in vitro generation of cytotoxic lymphocytes, while cultures with cortisol were partially inhibited. None of these hormones suppressed the cytotoxic activity of previously sensitized effector cells generated in vitro. Cultures containing testosterone expressed both normal DNA synthesis in MLR and cytotoxic activity in the CML test. These findings suggest a selective pattern of immunosuppression by sex hormones which may be important in preventing graft rejection or graft-versus-host interactions which may arise as a consequence of fetal engraftment during pregnancy.

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

SummaryRecent 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.

A re-examination of the interaction of estradiol with target cell receptors

Abstract Two intranuclear estradiol receptor complexes can be distinguished by sucrose density gradient ultracentrifugation of the immature rat uterus following either in vivo or in vitro administration of radiolabelled estradiol. Time course studies demonstrate that the earliest detectable complex sediments as a 4 S form whereas a 5 S form accumulates during prolonged exposure to hormone (> 10 min). Pulse chase experiments suggest that the 5 S complex arises from the lighter 4 S complex in a precursor-product manner. The properties of both the intranuclear 4 S and 5 S forms are shown to be those of authentic estrogen receptors. These observations are not in agreement with the generally held view that receptor 4 S to 5 S transformation occurs in the cytoplasm and is a necessary prerequisite to nuclear translocation.

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.