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1.
We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E 2 15 plus oestrogen (E 2) potentiated the specific activity of the brain type creatine kinase (CK) response to E 2 in the rat tissues or skeletal cells. The enhancing CK response of anti E 2 15 plus E 2 was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E 2 15 blocked the stimulatory CK response to E 2. Two other high affinity anti-E 2 anti-bodies, clones 8D 9 and 11B 6, had no effect in augmenting the response of CK to E 2 in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E 2 15 plus E 2 since the intact anti-E 2 in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E 2 15 abolished the CK response to E 2 in rat tissues and not to anti-E 2 15 plus E 2 whereas tamoxifen completely blocked the CK response to anti E 2 plus E 2. Anti E 2 15 may therefore serve as a specific carrier in delivering E 2 to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E 2 effects in vivo and in vitro. 相似文献
2.
The interconversion of estrone (E 1) and 17β-estradiol (E 2), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using 14C-labeled E 1, E 2, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E 2 as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E 2→E 1, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology. 相似文献
3.
Estradiol-17β (E 2) is a mitogen in vivo for the proliferation of granulosa cells in the rat ovary. E 2 is synthesized by the preovulatory follicle through a series of gonadotrophin-dependent events: LH stimulates thecal cells to synthesize androgens (androstenedione and testosterone) which are substrates for FSH-induced aromatization to estrogens in granulosa cells. More recently, we have found that transforming growth factor-β (TGF-β) stimulates DNA synthesis in rat granulosa cells in vitro and this effect is augmented by FSH. Since E 2 is a mitogen in vivo and TGF-β is the only known growth factor to stimulate proliferation in vitro, the possible link between the actions of E 2 and TGF-β were examined. E 2 stimulated the secretion of a TGF-β-like factor by rat granulosa cells in culture, and with time DNA synthesis was stimulated. The mitogenic action of E 2 was enhanced in the presence of FSH, and attenuated by a neutralizing antibody to TGF-β. The latter observations have identified TGF-β as the “missing-link” in the mitogenic actions of E 2 on rat granulosa cells. In addition to the growth-promoting actions of TGF-β plus FSH, TGF-β enhanced FSH-induced aromatase activity. Consequently, FSH plus TGF-β stimulates both the proliferation and aromatization capacity of rat granulosa cells. We propose that interactions between FSH, E 2 and TGF-β lead to the exponential increase in serum E 2 levels that occurs during the follicular phase of the cycle. Similarly, FSH stimulates the aromatization of exogenous androgens to estrogen by Sertoli cells isolated from immature rat testes, and there is a correlation between FSH-induced aromatization and mitotic activity. We have shown that FSH plus TGF-β stimulates DNA synthesis in Sertoli cells. Since E 2 increases the secretion of TGF-β by Sertoli cells, interactions between FSH, E 2 and TGF-β may provide the mitogenic stimulus for Sertoli cells during the prepubertal period. In summary, our findings suggest that the estrogen-induced growth of rat granulosa cells is mediated through the production of TGF-β, which acts as an autocrine regulator of proliferation. We also propose that the growth-promoting actions of FSH on Sertoli cells may depend upon a cascade series of events involving estrogens and TGF-β. 相似文献
4.
The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E 1 S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E 1 S to E 2 very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E 1 to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E 2) in hormone-dependent cells, but oxidative (E 2 → E 1) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E 1 to E 2. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E 2; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E 1 S to E 2. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease. 相似文献
5.
An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C 18 steroids such as 17β-estradiol (E 2) and estrone (E 1). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E 2 and E 1. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E 2/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties. 相似文献
6.
In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E 2) as precursor resulted in: (1) elevated formation (up to 90%) of E 2-sulphate (E 2-S), using lower precursor concentrations; (2) very limited conversion to estrone (E 1) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E 2-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E 1 production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E 1 (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E 2 metabolism could be minor in Ishikawa cells as a consequence of the high rate of E 2-S formation encountered is contradicted by the evidence that conversion to E 1 also remains limited in the presence of much lower E 2-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17β-hydroxysteroid dehydrogenase (17β-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E 2) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17β-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types. 相似文献
8.
It is well recognized that estradiol (E 2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E 1S: 5 × 10 −9 M), nomegestrol acetate blocked very significantly the conversion of E 1S to E 2. In the MCF-7 cells, using concentrations of 5 × 10 −6 M and 5 × 10 −5 M of nomegestrol acetate, the decrease of E 1S to E 2 was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E 1S at 2 × 10 −6 M and nomegestrol acetate at 10 −5 M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E 1: 5 × 10 −9 M) this estrogen is converted in a great proportion to E 2. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10 −7 M and 5 × 10 −5 M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10 −5 M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E 1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E 2 can open new clinical possibilities in breast cancer therapy. 相似文献
9.
Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P 4) and estradiol (E 2) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or androstenedione were subsequently added as substrates for the 3β-hydroxysteroid dehydrogenase (3β-HSD) or P450-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependant manner E 2 and P 4 secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E 2 secretion, but not on P 4. These results show that calcitriol is a physiological regulator of placental E 2 and P 4 production and suggest a novel role for calcitriol upon placental steroidogenesis. 相似文献
10.
Effects of cannabinoid on expression of β-type transforming growth factors (TGF-β1, -β2 and -β3), insulin-like growth factor-I (IGF-I) and c- myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17β (E 2) and/or progesterone (P 4), and uteri were analyzed at various times thereafter. TGF-β isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E 2 caused a modest increase in TGF-β1 and -β3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E 2 by changing the timing for the peak of TGF-β3 mRNA levels to 12 h. In comparison, E 2 treatment substantially elevated the levels of TGF-β2 mRNA at 6 h, and THC potentiated this E 2 response without affecting the timing for the response. Imposition of P 4 treatment did not antagonize any of these responses. P 4 treatment alone or with THC had insignificant effects on mRNA levels for these TGF-β isoforms. Uterine levels of IGF-I and c- myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E 2 treatment induced a rapid, but transient, increase in IGF-I and c- myc mRNAs, and THC antagonized the rapid c- myc mRNA response and altered the timing of the IGF-I mRNA response. P 4 treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P 4 plus E 2 resulted in further modest increases in IGF-I and c- myc mRNA levels as compared to E 2 or P 4 treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression. 相似文献
11.
In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E 1), estradiol (E 2), 16-OHE 1, and estriol (E 3), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β- 3H]testosterone (1.24 × 10 3 dpm 3H/ng, 35–150 nM) or [1β- 3H,4- 14C]androstenedione (3.05 × 10 3 dpm 3H/ng, 3H/ 14C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E 1, E 2, 16-OHE 1, and E 3 for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the Km of aromatase was 61.8 ± 2.0 nM ( n = 5) for testosterone. The Ki of E 1, E 2, 16-OHE 1, and E 3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the Km of aromatase was 35.4 ± 4.1 nM ( n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase. 相似文献
12.
The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE 2) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE 2 metabolites were more pronounced than that of estradiol-17β(E 2). 相似文献
13.
The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E 2). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [ 3H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E 2. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E 2-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E 2-treated cells relative to control values. The present data show that E 2 exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones. 相似文献
14.
Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E 1) and 17 β-estradiol (E 2) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E 1 and E 2 after activation are able to bind DNA. This paper reports that after DMDO activation, [ 3H]E 1 and [ 3H]E 2 were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E 1–DNA and E 2–DNA adducts was detected by 32P-postlabeling analysis. 相似文献
15.
The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E 2) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17β-hydroxysteroid dehydrogenase, aromatase involved in the last steps of E 2 bioformation. Sulfotransferases which convert estrogens into the biologically inactive estrogen sulfates are also present in this tissue. Quantitative data show that the ‘sulfatase pathway’, which transforms estrogen sulfates into the bioactive unconjugated E 2, is 100–500 times higher than the ‘aromatase pathway’, which converts androgens into estrogens. The treatment of breast cancer patients with anti-aromatases is largely developed with very positive results. However, the formation of E2 via the ‘sulfatase pathway’ is very important in the breast cancer tissue. In recent years it was found that antiestrogens (e.g. tamoxifen, 4-hydroxytamoxifen), various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. In another series of studies, it was found that E2 itself has a strong anti-sulfatase action. This paradoxical effect of E2 adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Interesting information is that high expression of steroid sulfatase mRNA predicts a poor prognosis in patients with +ER. These progestins, as well as tibolone, can also block the conversion of estrone to estradiol by the inhibition of the 17β-hydroxysteroid dehydrogenase type I (17β-HSD-1). High expressison of 17β-HSD-1 can be an indicator of adverse prognosis in ER-positive patients. It was shown that nomegestrol acetate, medrogestone, promegestone or tibolone, could stimulate the sulfotransferase activity for the local production of estrogen sulfates. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. A possible correlation between this stimulatory effect on sulfotransferase activity and breast cancer cell proliferation is presented. In agreement with all this information, we have proposed the concept of selective estrogen enzyme modulators (SEEM). In conclusion, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity in combination with anti-aromatases can open interesting and new possibilities in clinical applications in breast cancer. 相似文献
16.
Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E 1) and estradiol (E 2) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E 1(E 2)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol- O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE 1(E 2)-1-N3Ade and 4-OHE 1(E 2)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E 2 and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E 2 metabolism to 4-OHE 1(E 2) and 4-OCH 3E 1(E 2). Inclusion of Ro41-0960 and E 2 in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E 2, whereas without the inhibitor, no increases in adducts were detected with E 2 ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer. 相似文献
17.
It is well accepted that estradiol (E 2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E 1S) ‘via sulfatase’ is a much more likely precursor for E 2 than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E 2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [ 3H]-E 1S (5 × 10 −9 M) alone or in the presence of E 2 (5 × 10 −5 to 5 × 10 −7 M) during 30 min or 3 h. E 1S, E 1 and E 2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E 1 was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10 −7 M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10 −5 M after 30 min incubation. The values were 24% and 18% for 5 × 10 −7 M and 49% and 42% for 5 × 10 −5 M, respectively after 3 h incubation. It was observed that [ 3H]-E 1S is only converted to [ 3H]-E 1 and not to [ 3H]-E 2 in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone. 相似文献
18.
Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E 1), estradiol (E 2) and estrone sulfate (E 1S) levels in the ultra-low range. Following incubation with [ 3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E 1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E 1, E 2) were separated by chromatography (LH-20). Estrone and E 1S (following hydrolysis) were converted into E 2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[ 125I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E 1, E 2 and E 1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E 1, 0.67 pmol/L for E 2, and 0.55 pmol/L for E 1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E 1, 5.1% for E 2 and 6.1% for E 1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E 1, E 2, and E 1S, respectively. Considering normal plasma levels for E 2 (15 pmol/L), E 1 (80 pmol/L) and E 1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E 2, E 1 and E 1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels. 相似文献
19.
We have demonstrated previously that rat adipose tissue showed sex and depot-specific responses to gonadal steroids. The epididymal fat pad in males responded exclusively to androgens by increased specific activity of the brain type isozyme of creatine kinase (CK). In females, the parametrial adipose tissue responded exclusively to estrogens. The present study was undertaken to follow the responsiveness to steroid hormones, and the presence of estrogen receptors (ER), in 3T3L1 cells during their differentiation from pre-adipocytes to adipocytes. In pre-adipocytes in which the basal CK specific activity is low, there was no CK response to 17β estradiol (E 2) or dihydrotestosterone (DHT). Differentiation of the cells into adipocytes was accompanied by increased basal CK activity which was stimulated by E 2, but not by DHT. Responsiveness to E 2 began 5 days after switching pre-adipocytes to differentiation medium. Upon differentiation, ER became demonstrable in the cell nuclei by staining with FITC labeled anti-idiotypic antibody (clone 1D5) directed against the steroid binding domain of ER. The response to E 2 was time-dependent and blocked completely by cycloheximide or actinomycin D. 1D5 itself, which has an estrogen mimetic effect, stimulated CK activity in the cells similarly to E 2. The antiestrogen tamoxifen which also stimulated CK activity in the adipocytes, completely blocked E 2 action. The ‘pure’ antagonist of E 2, ICI 164,384 and the tissue-selective antiestrogens, raloxifene or tamoxifen methiodide were also complete antagonists with no agonistic effects. The response of the 3T3L1 adipocytes to E 2 was upregulated by 1,25(OH) 2D 3. Moreover, IGF1 was also a potent stimulator of CK in these cells, and therefore may mediate partially the stimulation by E 2. Transient transfection of the pre-adipocytes with ER permitted E 2 induction of CK. Thus, the appearance of ER and concomitant responsiveness to E 2 is another hormone-related change occurring in 3T3L1 cells during differentiation, in addition to changes such as development of insulin responsiveness. The interactions in this system provide a useful in vitro model for investigating the development of responsiveness to E 2. 相似文献
20.
The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an -proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E 2 site), but is in a different location in two other family members (the E 1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E 2 to the E 1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme ( Km = 0.6 and 2.0 μM, and Vmax = 2.8 and 0.3 s −1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis—abstraction of the -H R from the substrate followed by transfer of the β-H R to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E 1 and E 2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform γ-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the -H R but is far removed from the γ-Hs. These findings provide further support for a one base mechanism of - and γ-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases. 相似文献
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