Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.

Expression of basic fibroblast growth factor in the rat ovary: detection of mRNA using reverse transcription-polymerase chain reaction amplification.

Development of the ovarian follicle and corpus luteum involves proliferation and differentiation of several cell types: granulosa cells, thecal cells, and various stromal cells, particularly the endothelial cells that compose the rich thecal and luteal vascular networks. Basic fibroblast growth factor (bFGF) is a potent mitogen for cells of mesodermal and neuroectodermal origin, including endothelial cells. With the use of reverse transcription-polymerase chain reaction (PCR), we have examined the expression of bFGF in the rat ovary. RNA was extracted from fetal bovine aortic endothelial cells, hypothalami of adult rats, and either whole ovaries or isolated granulosa cells from PMSG-primed immature rats. The RNA was reverse transcribed and then amplified by PCR using two oligonucleotide primers specific for both bovine and rat bFGF. A sample of the PCR solution was size fractionated by electrophoresis in an 8% polyacrylamide gel, which was then stained with ethidium bromide and examined under ultraviolet light. When reverse transcription-PCR was performed on RNA from bovine endothelial cells, rat hypothalamus, or whole rat ovary, a single major DNA band corresponding in length to the distance between the 5'-ends of the two bFGF-specific primers (354 base pairs) was obtained. The identity of this material with the bovine and rat bFGF sequences was confirmed by restriction enzyme analysis. When RNA from isolated granulosa cells was examined, however, no bFGF mRNA was detected. These results confirm that the bFGF gene is expressed in the ovary during follicular development. Furthermore, they demonstrate that ovarian bFGF expression is cell specific, since granulosa cells do not contain detectable bFGF mRNA.

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium.

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.

Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth.

In the uterus, estrogen causes a rapid increase in microvascular permeability, followed later by growth of the endometrium, including the richly vascular stroma. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF or VEG/PF) is an angiogenic protein that is not only a specific mitogen for endothelial cells, but also a potent stimulator of microvascular permeability. Because of these properties, it seems likely that VEG/PF might mediate estrogen-induced increases in uterine vascular permeability and blood vessel growth. Therefore, we determined whether the gene for VEG/PF is expressed in the rat uterus and if mRNA abundance is regulated by steroid hormones, using reverse transcription-polymerase chain reaction. The VEG/PF gene is alternatively spliced and gives rise to three transcripts coding for proteins of 188, 164, and 120 amino acids, which, in turn, form the active dimeric factors. Transcripts for VEG/PF mRNAs were detected in the uterus of the rat by reverse transcription-polymerase chain reaction. The mRNAs for the VEG/PF164 and VEG/PF120 subunits were the dominant forms expressed. Treatment with both estradiol (E2) and estriol (E3) rapidly induced an increase in the level of the two smaller transcripts. The increase was detectable as early as 0.5-1 h and peaked at 2 h. Levels of the two smaller transcripts then declined, but remained above control levels for 24 h. The degree of stimulation of VEG/PF mRNA levels was 8-fold at 2 h. VEG/PF188 mRNA levels were higher by 6 h compared to control values. The increase in VEG/PF mRNA levels in response to E2 was not contingent upon de novo protein synthesis, as it was not blocked by cycloheximide. The increase occurred as rapidly as that of the mRNA for Zif268, an estrogen-induced transcription factor. Progesterone also stimulated the expression (at 6 h) of VEG/PF164 and VEG/PF120, but not that of VEG/PF188. We conclude that the VEG/PF gene is expressed in the rat uterus, and that mRNA levels are rapidly enhanced by estrogen. This response suggests that VEG/PF may be involved in the estrogen-induced increase in permeability and proliferation of uterine blood vessels. The identification of VEG/PF as a primary response gene also suggests that VEG/PF expression may be a prerequisite for the subsequent expression or action of other growth factors in the uterus.

Production of 6-keto-prostaglandin F1 alpha by rat granulosa cells in vitro.

The production of 6-keto-prostaglandin (PG)-F1 alpha by rat granulosa cells in vitro was measured in order to determine if the precursor of this compound, prostacyclin (PGI2), is a potential mediator of preovulatory changes in follicular function. Granulosa cells were collected from immature rats (27-29 days old) 48 h after an injection of PMSG (20 IU). The cells were incubated in medium 199 containing 1% BSA with or without arachidonic acid and various treatments for up to 5 h. PGI2 synthesis was determined by extracting the combined cells and medium, purifying the extract by thin layer chromatography, and measuring 6-keto-PGF1 alpha using a sensitive RIA. PGE synthesis was also determined by RIA in order to compare and contrast effects of treatments on PGE synthesis with those on 6-keto-PGF1 alpha synthesis. Both exogenous arachidonic acid and LH stimulated 6-keto-PGF1 alpha synthesis approximately 4-fold in comparison to controls during 5-h incubations. Maximum stimulation was achieved by the combination of arachidonic acid and LH. The effect of arachidonic acid was evident as early as 1 h of incubation, but LH had no effect until 3 h of incubation. PGE synthesis was also stimulated by arachidonic acid within 1 h of incubation and by LH within 3 h of incubation. A potent LHRH agonist also significantly stimulated 6-keto-PGF1 alpha and PGE production during a 5-h incubation, whereas three vasoactive agents (histamine, bradykinin, and angiotensin II) had no stimulatory effect on the synthesis of either compound. Based on the measurement of 6-keto-PGF1 alpha, it is concluded that rat granulosa cells have the capability to synthesize PGI2 and that this synthesis is stimulated by LH and a potent LHRH agonist. Therefore, PGI2 is a potential mediator of hormone actions in the preovulatory follicle.

The effects of a gonadotropin-releasing hormone agonist on ovulation and steroidogenesis during perfusion of rabbit and rat ovaries in vitro.

The ability of a GnRH agonist (GnRHa) to exert direct effects on rat and rabbit ovaries was examined in vitro. Ovaries of estrous rabbits and immature, PMSG-primed rats were surgically removed and perfused with a defined medium via an aortic cannula. In this system, the ovary remains viable and capable of undergoing ovulation in response to LH. Samples of perfusion medium were taken for steroid measurements and the number of ovulations determined by direct observation (rabbit) or oocyte recovery (rat). Follicles of ovaries perfused with medium alone rarely ovulated. GnRHa (0.1 micrograms/ml) induced ovulations in 6 of 7 rat ovaries (4 to 22 ovulations per ovulating ovary) and this effect was blocked by a GnRH antagonist. In contrast, a much higher dose of the agonist (10 micrograms/ml) induced ovulations in only 7 of 15 rabbit ovaries. GnRHa caused small but significant increases in progesterone levels in the perfusion medium in both species in comparison to no treatment. Mean estradiol levels also tended to be higher in the GnRHa groups in comparison to controls but the differences were not significant. GnRHa appears to act directly on both the rabbit and rat ovary but the rat ovary is much more sensitive to its ovulation-inducing effects.

Detection of prolactin receptor (PRL-R) mRNA in the rat hypothalamus and pituitary gland.

Prolactin receptor (PRL-R) mRNAs exist in several tissues where prolactin is known to act including the liver, testes, prostate, ovary, mammary gland, adrenal gland and kidney. PRL also acts at the level of the hypothalamus and pituitary gland to feed back and regulate its own secretion and the secretion of other anterior pituitary hormones. Therefore, we hypothesized that PRL-R mRNA would exist in these target tissues as well. Total RNA was extracted from rat anterior and medial basal hypothalamus, anterior and posterior pituitary gland, cerebral cortex, skeletal muscle and liver. After reverse transcribing total RNA with Murine-MLV reverse transcriptase and random or oligo(dT) pmers, the polymerase chain reaction (PCR) was performed. PCR products were then analyzed by ethidium bromide staining. Using primers that flanked the coding region for the extracellular binding domain we detected PRL-R mRNA in the anterior and medial basal hypothalamus, anterior and posterior pituitary gland, as well as in the liver, but not in the cerebral cortex or skeletal muscle. In addition, when we used primers that distinguish the long and short forms of the PRL-R mRNA, both forms of the PRL-R mRNA were detectable in the same tissues. Our data suggest that PRL may feed back at the level of the hypothalamus and pituitary gland through the same short and/or long PRL-R mRNA that mediate PRL action in the peripheral tissues.

Developmental increase in low density lipoprotein receptor messenger ribonucleic acid levels in placental syncytiotrophoblasts during baboon pregnancy.

We have previously shown that there was an estrogen-regulated developmental increase in low density lipoprotein (LDL) uptake by placental syncytiotrophoblasts during baboon pregnancy. To determine whether this reflected enhanced expression of the LDL receptor, the levels of LDL receptor messenger RNA (mRNA) were determined by Northern blot and reverse transcription-polymerase chain reaction in placental tissue obtained from baboons (Papio anubis) in early (days 58-64; pooled to yield 5 samples), mid- (days 97-110; pooled to yield 12 samples), and late (days 161-175; pooled to yield 15 samples) gestation (term = 184 days). Whole villous tissue and a trophoblast cell fraction isolated by 50% Percoll gradient centrifugation were analyzed. The latter cell fraction was equally comprised predominantly of syncytiotrophoblasts at early, mid-, and late gestation as determined by extensive immunocytochemical reactivity with antisera to syncytiotrophoblast-specific peptides. Tissues were extracted with guanidine isothiocyanate and 5 micrograms polyadenylated-enriched RNA hybridized to a 32P-labeled human LDL receptor complementary DNA (cDNA). A major 6.2-kilobase LDL receptor mRNA transcript was expressed in syncytiotrophoblasts and whole villous tissue, as determined by Northern blot. In the syncytiotrophoblast-rich cell fraction, LDL receptor mRNA levels, analyzed by Northern blot and autoradiodensitometry and expressed as a ratio of beta-actin, were similarly low in early (0.66 +/- 0.12 arbitrary units) and mid- (1.15 +/- 0.23) gestation, then increased to a level in late gestation (2.71 +/- 0.33) that was over 4- and 2-fold greater (P < 0.01) than that in early or midgestation, respectively. In contrast, in whole villous tissue, LDL receptor and beta-actin mRNA levels exhibited no consistent change or decreased slightly with advancing pregnancy, so that when corrected for beta-actin, LDL receptor mRNA levels were similar in early (1.53 +/- 0.33), mid- (1.44 +/- 0.16), and late (2.32 +/- 0.29) gestation. The unchanged levels of LDL receptor mRNA in whole placental villous tissue with advancing primate gestation may reflect potential villous tissue with advancing primate gestation may reflect potential confounding effects that nontrophoblast, e.g. vascular, components of the developing placenta may have on assessing trophoblast endocrine function in whole villous tissue. Amplification of trophoblast RNA by reverse transcription-polymerase chain reaction with LDL receptor primers generated a single cDNA product of approximately 258 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitors of mammalian tissue collagenase and metalloproteinases suppress ovulation in the perfused rat ovary.

We have examined the effects of a new synthetic inhibitor of mammalian tissue collagenase, CI-1 (N-[3-N-(benzyloxycarbonyl)amino-1-(R)carboxypropyl]L-leucyl-O-methyl-L- tyrosine N-methylamide; G. D. Searle SC 40827), and a general metalloproteinase inhibitor, 1,10-phenanthroline, on ovulation, as judged by the observation of follicular rupture, and on progesterone production of the perfused rat ovary. Ovaries of PMSG (20 IU)-primed rats were perfused for 21 h, and samples of medium were taken for analysis of progesterone concentration. The number of ovulations was estimated by counting the number of oocytes released into the perfusion chamber. Ovaries were stimulated with LH (0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM), and this treatment resulted in a mean of 17.2 ovulations/treated ovary. 1,10-Phenanthroline dose-dependently inhibited ovulation, with 0, 0.2, and 12.5 ovulations/treated ovary at 1.0, 0.1, and 0.01 mM, respectively. This inhibition of ovulation closely paralleled the inhibition of extracted collagenase from uterus and ovary. However, 1,10-phenanthroline also suppressed progesterone release in a dose-dependent manner. Addition of the collagenase inhibitor (CI-1; 25 microM) 1 h after LH plus IBMX inhibited ovulation (6.3 ovulations/treated ovary). Its relatively inactive stereoisomer (CI-2; 25 microM) did not suppress ovulation (20.0 ovulations/treated ovary). CI-1 inhibited extracted uterine collagenase 50% at a concentration of 2 microM, whereas CI-2 was only 1/15th as effective. There was an 80% loss of CI-1 from the medium during the perfusions. Neither CI-1 nor CI-2 had any effect on LH plus IBMX-stimulated progesterone release. These data demonstrate that the general metalloproteinase inhibitor 1,10-phenanthroline is able to inhibit ovulation, but also inhibits steroidogenesis. The more specific inhibitor of collagenase, CI-1, can inhibit ovulation without affecting steroid production. These data indicate an important role for collagenase in the ovulatory process.

Relaxin stimulates uterine edema via activation of estrogen receptors: blockade of its effects using ICI 182,780, a specific estrogen receptor antagonist.

Relaxin's ability to stimulate uterine growth is well established. The mechanisms by which relaxin exerts this effect, however, remain unclear. In light of previous work demonstrating peptide growth factor activation of estrogen receptors (ERs), the present study was conducted to determine if relaxin similarly stimulates ERs. Twenty-five day-old female Sprague-Dawley rats were bilaterally ovariectomized and treated with estradiol or porcine relaxin alone or in combination with the ER antagonist ICI 182,780. Following treatment with 17beta-estradiol or relaxin alone, the uterine weight/body weight ratio (UtW/BW) increased significantly over control values (+98% and +77% respectively, p<0.0003). Pre-treatment of animals with ICI 182,780 (3 microg/g BW) prior to either estradiol or relaxin treatment completely inhibited the hormone-induced increases in uterine weight (p<0.0005). ICI 182,780 alone had no significant effect. Histological analysis of uterine cross-sections revealed that the edema present in the endometrium of animals treated with estradiol or relaxin alone was completely absent in the uteri of animals pre-treated with ICI 182,780. These data indicate that relaxin-induced uterine edema and growth is mediated by ERs.

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Acute effect of basic fibroblast growth factor on secretion of prolactin as assessed by the reverse hemolytic plaque assay.

The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 micrograms/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P less than 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 x 10(-6) M, while lesser reductions were observed at 1.68 x 10(-6) and 5.60 x 10(-7) M. A dose of 3.36 x 10(-6) M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimulation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P less than 0.01) by 10(-7) M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P less than 0.001) by 10(-5) M dopamine. Coincubation of bFGF with dopamine reduced (P less than 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRH-induced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion.

The regulation of granulosa cell proopiomelanocortin messenger ribonucleic acid by androgens and gonadotropins.

We have previously demonstrated the presence of proopiomelanocortin (POMC) messenger RNA (mRNA) in rat granulosa cells. This study examines the unique, tissue-specific regulation of granulosa cell POMC mRNA levels by hormones with established regulatory effects on these cells. Northern blot analysis of immature rat ovarian RNA revealed the presence of a single mRNA of approximately 900 base pairs in length. The levels of ovarian POMC mRNA increased approximately 6-fold 48 h after priming with PMSG when expressed per microgram of total RNA. Granulosa cells were isolated from immature PMSG-primed rats and cultured under serum-free conditions in the presence and absence of various hormones to determine their effect on POMC mRNA levels. Treatment of the cells for 48 h with LH (100 ng/ml), androstenedione (10(-7) M), or LH plus androstenedione elevated POMC mRNA levels. LH alone elicited a 5-fold increase in POMC mRNA, and androstenedione elicited a 10-fold increase; the combination treatment led to a 47-fold increase. The effects of the nonaromatizable androgen dihydrotestosterone (DHT) were also evaluated. Treatment for 48 h with DHT (10(-7) M) elicited a 40-fold increase in POMC mRNA levels. In vivo experiments measuring ovarian POMC mRNA from immature female rats corroborated the in vitro results. Animals injected sc with DHT (500 micrograms) demonstrated 5-fold increases in ovarian POMC mRNA when expressed per microgram of total RNA. These studies provide both in vitro and in vivo evidence that granulosa cell POMC mRNA is under unique hormonal regulation by both androgens and gonadotropins.

Detection of aromatase and keratinocyte growth factor expression in breast tumors using reverse transcription-polymerase chain reaction.

It has been proposed that local production of estrogen may contribute to breast tumor growth, in part by regulating growth factor production. In view of this, we studied the expression of mRNAs for aromatase cytochrome P-450, the enzyme which catalyzes estrogen synthesis, and keratinocyte growth factor (KGF), a heparin-binding growth factor specific for epithelial cells, in breast tumors. In order to detect mRNAs of low-abundance, reverse transcription-polymerase chain reaction (RT-PCR) was used. RNA from breast tumors and normal breast tissue was reverse transcribed and then amplified using oligonucleotide primers for human aromatase or KGF. Twelve of 15 breast tumor samples yielded variable amounts of aromatase PCR product, but consistently strong KGF PCR signals. Of the three aromatase mRNA-negative samples, two gave weak KGF signals while one was negative for KGF. Both aromatase and KGF transcripts were also detected in all five normal breast tissue samples examined. These results indicate that a high proportion of breast tumors have the potential to produce aromatase and KGF, both of which could play important roles in their growth. The results also suggest that RT-PCR can be used to evaluate local expression of growth mediators in tumors.

Aromatase and other inhibitors in breast and prostatic cancer.

Estrogens have an important role in the growth of breast and other hormone-sensitive cancers. We have shown that 4-hydroxyandrostenedione (4-OHA) selectively blocks estrogen synthesis by inhibiting aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. In postmenopausal men and women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either by daily oral or parenteral weekly treatment with 4-OHA, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. We recently studied the effects of 4-OHA and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione (PED) and imidazo[1,5-alpha]3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile) (CGS 16949A) as well as 5 alpha-reductase inhibitors, N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxyamide (4-MA) and 17 beta-hydroxy-4-aza-4-methyl-19norandrost-5-en-3-one (L651190) in prostatic tissue from 11 patients with prostatic cancer and six patients with benign prostatic hypertrophy (BPH), and from normal men at autopsy. We attempted to measure aromatase activity in tissue incubation by quantitating 3H2O released during aromatization of androstenedione or testosterone labeled at the C-1 position. The amount of 3H2O released from all samples was at least twice that of the heat inactivated tissue samples. The 3H2O release was significantly inhibited by 4-OHA and 4-MA, but not by the other aromatase inhibitors. However, when HPLC and TLC were used to isolate steroid products, no estrone or estradiol was detected in the incubates. Furthermore, no aromatase mRNA was detected following amplification by PCR. The 4-OHA was found to inhibit 5 alpha-reductase in both BPH and cancer tissue, although to a lesser extent than 4-MA. The other aromatase inhibitors were without effect. Although a mechanism involving intraprostatic aromatase is not likely, inhibitors may act to reduce peripherally-formed estrogens. In postmenopausal breast cancer, the results indicate that 4-OHA is of significant benefit.

Pseudoaromatase in circulating lymphocytes.

A variety of data suggesting a relationship between estrogens and the immune system prompted a study of aromatase activity in blood lymphocytes. A tritiated water aromatase assay detected activity of 1.9 to 25.1 pmol/g protein/h in 14 samples of human lymphocytes. To confirm these results, additional tritiated water and product isolation assays were performed on a large pool of lymphocytes obtained from 4 U of blood. An assay using [1 beta-3H]androstenedione generated high apparent aromatase activity, 943 pmol/g protein/h, but this activity could not be blocked by the aromatase inhibitor, CGS 16949A. More direct methods of evaluation yielded the following results: (1) PCR demonstrated no aromatase mRNA production in lymphocytes; (2) direct product isolation using [1,2,6,7-3H]androstenedione yielded insignificant production of estrone and estradiol; (3) immunostaining of fixed lymphocyte smears with a polyclonal antibody to aromatase yielded equivocal results. These data suggest the presence of pseudoaromatase in blood lymphocytes. Since circulating lymphocyte pseudoaromatase levels can be correlated with various factors in patients, such as age, menopausal status, and glucose ingestion, further studies of this activity are warranted.

Pregnant mare serum and human chorionic gonadotropin stimulate ovarian delta5-3beta-hydroxysteroid dehydrogenase in aged mice.

Aged (12- to 14-month-old) estrous and diestrous C57BL mice exhibited lower histochemically demonstrable ovarian delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in thecal, luteal, and interstitial cells, and lower (P less than 0.01) ovarian 3beta-HSD concentration and total content than did young (3-month-old) estrous animals. Administration of pregnant mare serum (PMS, 10 IU subcutaneously), followed in 40 hours by human chorionic gonadotropin (HCG, 5 IU subcutaneously) or HCG (2 IU daily for 4 days) alone, restored luteal and interstitial 3beta-HSD in aged mice. Follicular, lutea, and interstitial 3beta-HSD activity was increased in aged mice by a single PMS injection (10 IU). The total ovarian dehydrogenase concentration was increased 100% in aged animals by PMS and/or HCG administration. Restoration of histochemically demonstrable ovarian 3beta-HSD and total enzyme content in aged mice by PMS and/or HCG indicates ovarian sensitivity to gonadotropin and subnormal tropic hormone stimulation of the ovary in situ.

The effect of reduced oxygen tension on progesterone accumulation in rat granulosa cell cultures.

Studies were carried out on the effect of oxygen tension on progesterone (P) accumulation in rat granulosa cell cultures. At 1-2% oxygen, basal, luteinizing hormone (LH)-stimulated, and follicle stimulating hormone (FSH)-stimulated P accumulations were 20, 18, and 11%, respectively, of P levels at 20% oxygen. Basal P accumulation was also inhibited at 5% oxygen, but LH- and FSH-stimulated P levels were 50% and 40% higher, respectively, than at 20% oxygen. P levels at 10% oxygen were intermediate between those at 5% and 20% oxygen. The inhibitory effect of 1-2% oxygen on P accumulation was reversible: LH-stimulated P accumulation was inhibited in cultures incubated in 1-2% oxygen for 24 h, but rebounded during a subsequent 24 h period in 20% oxygen to the same level as that in cultures maintained continuously in 20% oxygen. We conclude that oxygen tension does influence granulosa cell steroidogenesis in vitro. Changes in blood flow and oxygen delivery to the ovary before and after ovulation could, therefore, effect the pattern of steroidogenesis during this period.

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required.

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans.

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.