Characterization of Systemic Disease Development and Paw Inflammation in a Susceptible Mouse Model of Mayaro Virus Infection and Validation Using X-ray Synchrotron Microtomography.

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR-/-) to characterize the disease. MAYV inoculations in the hind paws of IFNAR-/- mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease.

Methamphetamine, smoking, and gestational hypertension affect norepinephrine levels in umbilical cord tissues.

BACKGROUND: These studies were undertaken to determine methamphetamine (METH) and smoking effects on umbilical vascular dynamics and pregnancy outcomes. MATERIALS AND METHODS: Umbilical cords (54) were collected prospectively at birth, washed of blood, and stored at -80°C. Cords were thawed and lysates prepared, then catecholamine levels quantified with enzyme-linked immunosorbent assay (ELISA). RESULTS: Catecholamine levels in umbilical cords were not associated with maternal or gestational age, gravidity, parity, neonatal or placental weight. Neither smoking nor METH affected dopamine or epinephrine. However, smoking (two-fold) and METH (four-fold) decreased norepinephrine and together a 60-fold reduction occurred (p = 0.025). Cesarean section and hypertension were both associated with lower norepinephrine levels (p < 0.001) regardless of drug status. In normotensive pregnancies, smoking and METH significantly decreased norepinephrine levels (two-fold and 3.5-fold each, respectively) with a 40-fold decrease for METH/smoking together. DISCUSSION: Depletion of norephinephrine by METH and smoking likely contributes to pregnancy complications, including the higher incidence of respiratory distress and postpartum hemorrhage in cesarean section.

The spin temperature of NH3 in Comet C/1999S4 (LINEAR).

A high-dispersion spectrum of Comet C/1999S4 (LINEAR) was obtained in the optical region with the high-dispersion spectrograph on the Subaru telescope when the comet was 0.863 astronomical units from the Sun before its disintegration. We obtained high signal-to-noise ratio emission lines of the cometary NH2 bands from which an ortho-to-para ratio (OPR) of 3.33 +/- 0.07 was derived on the basis of a fluorescence excitation model. Assuming that cometary NH2 mainly originates from ammonia through photodissociation, the derived OPR of NH2 molecules should reflect that of ammonia, which provides information on the environment of molecular formation or condensation and of the thermal history of cometary ices. Assuming that the OPR of ammonia in comets was unchanged in the nucleus, the derived spin temperature of ammonia (28 +/- 2 kelvin) suggests that a formation region of the cometary ammonia ice was between the orbit of Saturn and that of Uranus in the solar nebula.

Electron spin resonance studies of erythrocytes from patients with Duchenne muscular dystrophy.

The membrane organization of the erythrocytes from patients with Duchenne muscular dystrophy was studied by means of electron spin resonance. The fluidity of the membrane near the polar region of Duchenne muscular dystrophy erythrocytes was similar to that of normal erythrocytes. The membrane environment in the nonpolar region, however, was quite different from that of normal erythrocytes, judged by the spectra with 2-(14-carboxytetradecyl) - 2 - ethyl - 4,4 - dimethyl - 3 - oxazolidinyloxyl as probe. The temperature dependence of the ratio of the line height of central field to that at the low field showed two inflection points in normal erythrocytes at pH 7.4 (13.5 degrees -16.5 degrees and 37.5 degrees -40.5 degrees C, respectively) but the inflection point in the lower temperature range was not detected in Duchenne muscular dystrophy erythrocytes. When pH was varied, an abrupt decrease in the ratio was observed at pH 5.9-5.6 in normal erythrocytes whereas there was a gradual decrease over the range of pH from 6.6 to 5.0 in Duchenne muscular dystrophy erythrocytes. The rate of reduction of the radical 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl by ascorbate in normal erythrocytes was faster than that in Duchenne muscular dystrophy erythrocytes. Treatment of both erythrocytes with phloretin markedly reduced the rate of reduction by ascorbate and eliminated the difference in the two types of erythrocyte. These results indicate that in Duchenne muscular dystrophy the erythrocyte membrane is involved as well as the muscle cell.

Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.

Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.

Inhibition of murine transformed Leydig cell proliferation by leukotrienes in serum-free culture.

We have previously shown that estrogen-dependent growth enhancement of murine transformed Leydig cells (B-1 F) is mediated through inhibition of arachidonic acid metabolite formation. In the present study, the growth-inhibitory ability of leukotrienes (LTs) on B-1 F cells in serum-free culture was directly addressed. All peptidyl LTs (LTC4, LTD4, and LTE4) inhibited B-1 F cell growth in a dose-dependent manner and exhibited maximum inhibition of DNA synthesis (60-80%) compared with that of untreated cells in a range of 10(-9) to 10(-8) M. To examine the mechanism of this LT-dependent inhibition, binding studies of LTD4 toward plasma membrane were conducted. Specific binding sites for LTD4 were identified. Scatchard analyses indicated the presence of a single class of high-affinity sites (Kd = 0.9 +/- 0.2 nM; maximum binding sites, 61 +/- 18 fmol/mg protein). This binding of LTD4 to the high-affinity site was markedly inhibited by ICI 198615, a specific inhibitor for LTD4. These results would suggest that inhibitory effects of LTs, at least LTD4, are elicited as a receptor-mediated event. In addition, this LT-dependent growth inhibition could not be blocked by simultaneous exposure of cells to estrogen, whereas estrogen partially protected arachidonic acid-dependent growth inhibition. Furthermore, treatment of cells with estrogen resulted in marked suppression of 5-lipoxygenase activity. Collectively, the present data clearly show that LTs play an important role as intermediates in an autocrine loop for B-1 F cells to exhibit estrogen-dependent growth.

Effects of thyroid hormone on androgen- or basic fibroblast growth factor-induced proliferation of Shionogi carcinoma 115 mouse mammary carcinoma cells in serum-free culture.

DNA synthesis of SC-3 cells cloned from mouse mammary carcinoma (Shionogi carcinoma 115) was remarkably enhanced by androgen as well as basic fibroblast growth factor (bFGF) at the early phase (days 1-3) of stimulation in serum-free culture condition. However, bFGF-induced DNA synthesis could not be observed at the late phase (days 4-6) of stimulation while androgen was able to continuously elicit DNA synthesis. When the effect of androgen on cell yield was examined, the cell number was increased while bFGF could not enhance cell growth. Androgen-induced heparin-binding growth factor partially purified from conditioned medium behaved like bFGF in terms of DNA synthesis and replication in SC-3 cells. SC-3 cells were found to contain the high-affinity binding site toward triiodothyronine. The dissociation constant and the maximum number of the binding sites were 7 x 10(-10) M and 1800/cell, respectively. Triiodothyronine significantly blunted the testosterone-induced DNA synthesis. On the other hand, bFGF-enhanced DNA synthesis was not substantially inhibited by triiodothyronine. These results suggest that androgen, but not bFGF, has unique action site(s) which might be important for SC-3 cell replication and might be antagonized by thyroid hormone.

Biological characteristics and estrogen and progesterone receptors in mammary carcinoma induced in male Sprague-Dawley rats by a series of intragastric intubations of 7,12-dimethylbenz(a)anthracene.

Biological characteristics and estrogen (ER) and progesterone (PR) receptors were studied in male mammary carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) in male inbred Sprague-Dawley rats (MM). DMBA-induced carcinomas in females (MF) were used as controls. In 36 of 44 female rats given 20 mg DMBA once by gastric intubation at 50 days of age, MF with microscopic characteristics of cystic papillary adenocarcinoma developed 124 +/- 49 (S.D.) days after challenge. In all of the 42 male rats given 10 mg DMBA at 14-day intervals for 14 weeks starting from 28 days of age. MM with microscopic characteristics of medullary adenocarcinoma developed 106 +/- 21 days after the first intubation of DMBA. The growth of primary MM was unaffected by orchiectomy or estrogen. Eighty to 100% of the MM transplanted in the four groups could grow in intact female rats, ovariectomized female rats, intact male rats, and castrated male rats, while the transplanted MF could grow only in intact female rats. The histology of MM was unchanged in primary and transplanted tumors under various hormonal conditions. ER were present in almost all of the hormone-independent primary and transplanted MM, although the levels for cytosol ER in MM were significantly lower than those in MF. Injection of 10 micrograms 17 beta-estradiol induced marked synthesis of PR in primary and transplanted MM, even 24 and 48 hr after the 17 beta-estradiol injection. These findings show that MM are hormone independent but, like hormone-dependent female tumors, contain ER and estrogen-dependent PR.

Stimulative effects of estrogens on tumor growth and 5 alpha-steroid production in a mouse Leydig cell tumor line (T 124958-R).

The effects of estrogens on the growth and enzyme activities for androgen synthesis in a mouse Leydig cell tumor line (T 124958-R) were studied. The s.c. implantation of a diethylstilbestrol pellet resulted in a marked enhancement of the tumor growth. 5 alpha-Reductase activity (nmol/g/h) in tumors rapidly grown in the presence of diethylstilbestrol pellet was 4 times higher than that in tumors slowly grown in the absence of diethylstilbestrol, whereas an inverse relation was found for 17 beta-hydroxysteroid oxidoreductase activity. 17-Hydroxylase activities were similar in both tumors. The major C21- and C19-steroids formed from progesterone by the tumors grown in the presence of estrogen were 5 alpha-steroids such as 3 alpha- or 3 beta-hydroxy-5 alpha-pregnan-20-one, 3 alpha, 17-dihydroxy-5 alpha-pregnan-20-one, androsterone, and 5 alpha-androstane-3 alpha, 17 beta-diol, whereas the major steroids formed by the tumors in the absence of estrogen were 4-ene-3-ketosteroids such as 20 alpha-hydroxy-4-pregnen-3-one, 17-hydroxy-4-pregnene-3,20-dione, and testosterone. Furthermore, 10(-8) M of 17 beta-estradiol added in serum-free medium for 10 days significantly enhanced 5 alpha-reductase activities per 10(6) cells but significantly inhibited 17 beta-hydroxysteroid oxidoreductase activity in primary cell culture. These results indicate that estrogens stimulate the growth of T 124958-R in vivo and that estrogens may directly enhance 5 alpha-reductase activity but inhibit 17 beta-hydroxysteroid oxidoreductase activity in T 124958-R cells.

Hyperlipidemia enhancement of renal preneoplasia but not tumor formation is due to elevated apoptosis in the Eker rat.

The influence of a high-fat diet on the appearance of renal tumors was assessed in the Eker rat model of hereditary renal carcinoma. Examination of H&E-stained sections showed a significant increase in the number of microscopic solid adenomas in the high-fat group compared with the low-fat group, whereas there was no significant difference in the number of macroscopic tumors between the two groups. Where had the tumor buds gone? Staining for apoptotic bodies occasionally revealed apoptosis in and around the microscopic adenomas. In addition, an Eker rat renal tumor-derived cell line showed apoptosis when it was cultured with high concentrations of native and acetylated low-density lipoprotein. These findings suggested that tumor buds repeatedly appeared and disappeared in Eker rats on a high-fat diet.

Effect of retinoic acid on proliferation of estrogen-responsive transformed murine Leydig cells in serum-free culture.

B-1 F cells, one of the sublines established from mouse Leydig cell tumor, have been found to be maintained as an estrogen-responsive cell line under the serum-free culture conditions. Reported results that retinoids have action mechanisms similar to those of estrogen prompted us to examine the effect of retinoids on the proliferation of B-1 F cells. Stimulation of B-1 F cell growth by retinoic acid in a dose-dependent manner was observed, whereas retinoic acid did not promote but inhibited the proliferation of MCF-7 cells (estrogen- and retinoic acid receptor-positive human breast cancer cells). To elucidate the mechanism of retinoic acid-dependent cell growth, simultaneous treatment with retinoic acid and estradiol was carried out. The result did not show the additive effect on B-1 F cell growth. Hydroxytamoxifen, a potent antiestrogen, inhibited not only estradiol-dependent but also retinoic acid-dependent cell growth. However, retinoic acid failed to be associated with estrogen receptor, suggesting that retinoic acid induced enhancement of B-1 cell growth through its interaction with retinoic acid receptor. Northern blot analyses of polyadenylated RNA with complementary DNA probes for human retinoic acid receptor alpha, beta, and gamma revealed the presence of transcripts encoded by retinoic acid receptor alpha gene in B-1 F cells. These results would suggest that enhancement of the B-1 F cell growth is mediated through interaction of retinoic acid with retinoic acid receptor alpha. This stimulatory activity is inhibited by estrogen receptor complexed with hydroxytamoxifen.

Effect of androgen on proliferation of estrogen-responsive transformed mouse Leydig cells in serum-free culture.

We examined the effects of steroid hormones on the proliferation of transformed mouse Leydig cells (B-1) in serum-free culture condition. Among hormones examined, androgen as well as estrogen enhanced the cell proliferation rate. Hormone binding studies revealed that B-1 cells contained both androgen and estrogen receptors. In addition, androgen-enhanced cell growth was inhibited by antiandrogen, but not by antiestrogen, while estrogen-stimulated cell growth was suppressed by antiestrogen. However, the simultaneous addition of androgen and estrogen did not show an additive effect. Dose-response study on androgen-dependent cell growth revealed that relatively high concentrations (10(-7)-10(-6) M) of dihydrotestosterone were required to obtain the maximum response. This was at least partly explained by the finding that B-1 cells could metabolize dihydrotestosterone into the less active steroids. Finally, B-1 cells were found to grow more rapidly in normal than in castrated male mice. These results clearly indicate that the proliferation of B-1 cells is stimulated by both androgen and estrogen, which utilize the different receptor systems.

Effects of tamoxifen on estrogen-induced enhancement or inhibition of tumor growth in mouse Leydig cell tumor lines.

In order to avoid the complex dual effects of estrogen and antiestrogen, the attempt was made to establish the tumor lines in which estrogens show either stimulatory or inhibitory property in terms of the tumor growth. The administration of estrogen to host mice bearing one of the mouse Leydig cell tumor lines, called T 124958-R, resulted in marked enhancement of the tumor growth even at pharmacological levels of estrogen. On the other hand, estrogenization of host mice almost completely inhibited the growth of the other tumor line (T 22137) without detectable stimulatory effects. The physiocochemical properties of the cytosol estrogen receptor in both sublines were found to be similar in relation to the affinity toward ligands, steroid specificity, sedimentation profile, and the dissociation rate kinetics. Using these tumor lines, the action mechanism of tamoxifen on the tumor growth was examined. Daily administration of this compound (30 micrograms/mouse) led to enhanced tumor growth in T 124958-R, while the growth of T 22137 was inhibited by the same procedure. In the combination experiments, tamoxifen was found to be unable to antagonize estrogen-induced enhancement or inhibition of the growth in these tumors. In addition, both tumors contained similar levels of the antiestrogen binding sites. These results suggest that tamoxifen modulated the tumor growth through its estrogenic potency.

Growth-stimulative effect of estrogen on androgen-dependent Shionogi carcinoma 115.

The stimulative effect of 17 beta-estradiol on the growth of androgen-dependent Shionogi carcinoma 115 and estrogen receptor in the tumor were studied. The incorporation of 17 beta-[3H]estradiol following a single injection of 17 beta-[3H]estradiol into tumor-bearing animals was 5- to 20-fold higher in the tumor than in the spleen and blood. Scatchard plot analyses showed that the tumor cytosol possessed a 17 beta-estradiol-binding site having a high affinity for 17 beta-estradiol [Kd 1.1 +/- 0.1 nM (S.E.)]. Competition experiments demonstrated that the 17 beta-estradiol binding was specific only for estrogenic compounds. Using sedimentation coefficient obtained by high-salt sucrose gradient (4.0S) and Stokes radius obtained by gel chromatography on Sephadex G-200 (46 A), the molecular weight of 17 beta-estradiol-binding component in the tumor cytosol was estimated to be 76,400. In castrated DS mice, a slight but significant increase in growth of s.c. grafted tumors was found by daily s.c. injections of either 17 beta-estradiol or 10 micrograms per mouse of testosterone propionate. Growth of the tumor maintained by 10 micrograms of testosterone propionate was augmented markedly by the addition of 4 micrograms of 17 beta-estradiol, and the growth approached the level induced by 100 micrograms of testosterone propionate. Simultaneous injections of bromocryptine inhibited an increase in 17 beta-estradiol-induced prolactin secretion but had no effect on the 17 beta-estradiol-enhanced tumor growth. These results demonstrate for the first time the stimulative effect of estrogen on the growth of androgen-dependent Shionogi carcinoma 115. The tumor contains typical estrogen receptor, which might be able to transmit estrogen signal to tumor cell nuclei with regard to tumor growth.

Partial characterization of protease(s) in human breast cancer cytosols that can degrade estrogen and progesterone receptors selectively.

Proteolytic activity in human breast cancer cytosols was studied using hormone receptors from rats as the substrates. Under the conditions tested, limited proteolysis of both the estrogen and the progesterone receptors in uterine cytosol was observed, but not proteolysis of the glucocorticoid or androgen receptors in liver or prostate cytosols, respectively. Although both the nonactivated and activated uterine estrogen receptors were attacked by the enzyme(s), molybdate-stabilized receptors were resistant to proteolysis. The product of estrogen receptor cleavage sedimented at approximately 4S in low-salt gradients and at 3 to 4S in high-salt gradients. This fragment retained both the steroid-binding and DNA-binding domains. The marked decrease in its DNA-binding ability, compared with the salt-dissociated but non-proteolyzed receptors, may be attributable to interactions of the fragment with dialyzable modulator(s) in cytosol. The proteolytic activity in tumor cytosol was leupeptin sensitive and was precipitated by (NH4)2SO4 at 30 to 60% saturation. Its sedimentation coefficient was 4 to 5S. The proteolytic activity was identified in 70% of estrogen receptor-negative tumors but in only 40% of estrogen receptor-positive tumors.

Proliferation of Shionogi carcinoma 115 cells by glucocorticoid-induced autocrine heparin-binding growth factor(s) in serum-free medium.

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. Recently, the growth of the tumor was also found to be stimulated by pharmacological, but not physiological, doses of glucocorticoid. In a serum-free culture system [Ham's F-12:Eagle's minimal essential medium (1:1, v/v) containing 0.1% bovine serum albumin], we have established that 10(-8) M testosterone, or 10(-6) M dexamethasone significantly stimulates the growth of SC-3 cells (a cloned cell line from a SC115 tumor) via androgen and glucocorticoid receptors, respectively. Recently, we demonstrated that the testosterone-induced growth of SC-3 cells is mediated through autocrine fibroblast growth factor (FGF)-like peptide(s). In the present study, mechanisms of glucocorticoid-induced growth of SC-3 cells were investigated. Serum-free conditioned medium obtained from 10(-6) M dexamethasone-stimulated SC-3 cells was fractionated by heparin-Sepharose affinity chromatography; one sharp peak of growth-stimulatory activity for SC-3 cells, eluted at 1.3 M NaCl, was identified. When the peak fraction was added to serum-free medium, the shape of SC-3 cells changed from an epithelial to a fibroblast-like appearance, similar to that induced with testosterone or basic (b)FGF. Furthermore, the growth-stimulatory activity induced with the peak fraction as well as testosterone or bFGF was markedly inhibited by anti-bFGF antibody immunoglobulin G (75 to 90% inhibition was obtained), and the specific binding of 125I-bFGF on SC-3 cells was significantly inhibited by the peak fraction. These results suggest that the glucocorticoid-induced growth of SC-3 cells is also mediated through FGF-like peptide(s) in an autocrine mechanism, which is very similar to that induced by testosterone, if not identical.

Unsaturated fatty acids are required for continuous proliferation of transformed androgen-dependent cells by fibroblast growth factor family proteins.

Increase in dietary fat intake has been reported to be associated with progression of hormone-dependent cancers. To explore its mechanism, we examined the effects of fatty acids on the growth of androgen-dependent SC-3 cells cloned from mouse mammary cancer (Shionogi carcinoma 115). Their androgen-dependent growth was potentiated by linoleic acid in the defined medium. The effect of linoleic acid on fibroblast growth factor (FGF)-dependent growth was also addressed because androgen had been demonstrated to exert its mitogenic activity on SC-3 cells through an induction of the unique FGF family protein termed as androgen-induced growth factor. Exposure of SC-3 cells to basic FGF or androgen-induced growth factor exhibited only transient growth response. However, simultaneous addition of linoleic acid to the medium sustained the proliferation of FGF-stimulated, but not FGF-unstimulated, cells, although linoleic acid did not exert the significant effect on the process of S-phase entry of basic FGF-stimulated cells. Palmitoleic acid and oleic acid appeared to exert the actions similar to linoleic acid, while stearic acid was without any effect. Neither cyclooxygenase inhibitor nor 5-lipoxygenase inhibitor could block the growth-promoting ability of linoleic acid. Linoleic acid also enhanced their anchorage-independent growth in the presence of basic FGF. These results indicate that these unsaturated fatty acids play a role in sustaining the proliferation of FGF-stimulated SC-3 cells.

Effects of estrogen on cell proliferation and leukotriene formation in transformed mouse Leydig cells cultured under serum-free conditions.

B-1 F cells from mouse Leydig cell tumor (T 124958-R) were maintained in serum-free culture. Estrogen enhanced the growth of the cells, and this growth was suppressed by antiestrogens such as 4-hydroxytamoxifen or TAT, a newly developed antiestrogen. Since the growth of B-1 F cells was recently found to be inhibited by the metabolites of arachidonic acid, we examined the relationship between this metabolism and the enhancement of cell growth by estrogen. Among the modulators affecting the metabolism of arachidonic acid, 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoqu inone, an inhibitor of 5-lipoxygenase, reproducibly stimulated the growth of the cells, whereas the cyclooxygenase inhibitor indomethacin had only the marginal growth-stimulatory effects. Phorbol ester had no growth-modulating effect. 17 beta-Estradiol and 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoqu inone had some additive effects especially in terms of restoration of antiestrogen-induced inhibition. Moreover, the inhibition of DNA synthesis elicited by the addition of arachidonic acid in concentrations of 0.05 to 0.5 micrograms/ml was partly blocked by estrogen. Analyses of extracts of media and cells by high-pressure liquid chromatography and radioimmunoassay showed that 17 beta-estradiol inhibited the synthesis of leukotrienes B4, C4, D4, and E4 and this inhibition could be restored by antiestrogen. These results suggest that the enhancement of B-1 F cell growth by estrogen is at least partly mediated through its ability to inhibit leukotriene synthesis.

Characterization of a unique nuclear estrogen-binding component in an estrogen-responsive mouse Leydig cell tumor.

An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction. This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity. In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei. Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol. The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol. These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization. The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites. Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures. These Mg2+ and temperature effects were reversible. In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei. However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s). These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.

Stimulative effect of physiological doses of androgen or pharmacological doses of estrogen on growth of Shionogi carcinoma 115 in mice.

It was generally accepted for 20 yr that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen. In the present study, the growth-stimulative effect of estrogen alone on SC115 tumors was examined in castrated mice. Daily injections of physiological doses of 17 beta-estradiol did not enhance the tumor growth. However, high doses of 17 beta-estradiol (10-100 micrograms/mouse/day) significantly stimulated the growth of tumors in a dose-dependent manner. Since high doses of diethylstilbestrol (10-50 micrograms/mouse/day), which does not bind to androgen receptor, could markedly stimulate the growth of tumors and since antiandrogen (cyproterone acetate) failed to inhibit the growth stimulation induced by high doses of 17 beta-estradiol, it is concluded that high doses of 17 beta-estradiol, which binds to androgen receptor with relatively low but significant affinity, enhance the tumor growth not via the androgen receptor system. The growth speed, histological type, content, and affinity of androgen, estrogen, and progesterone receptors and pattern of newly synthesized proteins labeled in vitro with [35S]methionine of tumors grown by high doses of estrogen were not significantly different from those of the original SC115 tumors grown in normal males. Furthermore, seed tumors from one to six generations grown by pharmacological doses of estrogen alone could rapidly grow only in normal males and not in castrated males. The present findings demonstrate that the growth of SC115 tumors in vivo is stimulated by physiological doses of androgen or pharmacological doses of estrogen.