Oxytocin- and vasopressin-induced growth of human small-cell lung cancer is mediated by the mitogen-activated protein kinase pathway.

Malignant growth of small-cell lung carcinoma is promoted by various neuroendocrine autocrine/paracrine loops. Therefore, to interfere with this mitogenic process, it is crucial to elucidate the mechanisms involved. It is known that the oxytocin (OT) and vasopressin (VP) genes, normally transcriptionally restricted in their expression, are activated in small-cell lung cancer (SCLC), concomitantly with expression of their receptors (OTR, V1aR, V1bR/V3R and V2R). The aim of the present study was to characterize, in concentrations close to physiological and pharmacological conditions, intracellular signalling events triggered by OT and VP binding to their specific receptors in SCLC cells and to identify factors mediating OT- and VP-induced mitogenic effects on SCLC. Known agonists for OTR ([Thr4,Gly7]OT) and V1aR (F180), in addition to OT and VP, were able to elicit increases in cytosolic Ca2+ levels and this effect could be blocked using an OTR antagonist (OVTA) or a V1aR antagonist (SR49059) respectively. There was no activation of the cAMP pathway detected after VP, dDAVP (a V2R agonist), or OT treatment. Stimulation of SCLC cells with OT and VP led to an increase of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, maximal at 5 min, and the subsequent phosphorylation of its downstream target p90 ribosomal S6 kinase (p90RSK). Pre-incubation with OVTA and SR49059, and with inhibitors of phospholipase C (PLC), protein kinase C (PKC), mitogen-activated protein kinase/ERK kinase (MEK) 1/2 and a Ca2+ chelator significantly reduced OT- and VP-induced ERK1/2 phosphorylations. OVTA, SR49059 as well as MEK1/2 and PKC inhibitors also downregulated OT- and VP-induced p90RSK phosphorylation. In [3H]thymidine-uptake experiments, we subsequently observed that PLC, Ca2+, PKC and ERK1/2 are absolutely required for the OT- and VP-stimulated SCLC cellular growth process. In conclusion, the results presented here indicate that OT- and VP-induced mitogenic effects on SCLC are respectively mediated by OTR and V1aR signalling and that this mitogenic signalling passes through the phosphorylation of ERK1/2 and p90RSK in a PLC-, Ca2+-, PKC- and MEK1/2-dependent pathway.

Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor.

Resistance to glucocorticoid therapy has been observed in patients with autoimmune/inflammatory diseases and may be related to the inflammatory process itself. The aim of this study was to examine the ability of tumor necrosis factor alpha (TNFalpha, a proinflammatory cytokine) and interleukin (IL)-10 (an anti-inflammatory cytokine) to differentially regulate the sensitivity of human monocytes/macrophages to glucocorticoids. To accomplish this, we first analyzed the pattern of TNFalpha and IL-10 inhibition by dexamethasone in LPS-stimulated whole-blood cell cultures. Second, we studied the modulation of the sensitivity of these cells to dexamethasone by preincubation with TNFalpha or IL-10 and measurement of LPS-stimulated IL-6 secretion. In addition, we evaluated the effect of dexamethasone on phorbolmyristate-acetate-stimulated IL-1 receptor antagonist secretion by the human monocytic cell line U937. Finally, we investigated whether the modulation of corticosensitivity in TNFalpha- and IL-10-pretreated U937 cells was related to a change of the glucocorticoid receptor concentration and affinity. Dexamethasone had different effects on LPS-induced TNFalpha and IL-10 secretion; whereas it suppressed TNFalpha in a dose-dependent fashion, its effect on IL-10 secretion was biphasic, producing stimulation at lower, and inhibition at higher doses. The concentration of LPS employed influenced the effect of dexamethasone on IL-10 secretion (P < 0.001). Pretreatment with TNFalpha diminished, and with IL-10 improved, the ability of dexamethasone to suppress IL-6 secretion in whole-blood cell cultures (P < 0.01 for both) and to enhance IL-1 receptor antagonist secretion by U937 cells (P < 0.05 for both). TNFalpha decreased (P < 0.001), while IL-10 increased (P < 0.001), the concentration of dexamethasone binding sites in these cells, with no discernible effect on their binding affinity. We conclude that glucocorticoids differentially modulate TNFalpha and IL-10 secretion by human monocytes in a LPS dose-dependent fashion and that the sensitivity of these cells to glucocorticoids is altered by TNFalpha or IL-10 pretreatment; TNFalpha blocks their effects, whereas IL-10 acts synergistically with glucocorticoids. This is accompanied by opposite glucocorticoid receptor changes, respectively opposing and favoring glucocorticoid actions. This study suggests that the pattern of pro-/antiinflammatory cytokine secretion may alter the response of patients to glucocorticoid therapy.

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. II. Inhibin and aromatase inhibitor activity.

The inhibin content and aromatase inhibitor activity (AIA) of 72 follicular fluids (FF) obtained from 42 women undergoing in vitro fertilization (IVF) and embryo transfer (ET) were studied as a function of IVF ET outcome. Inhibin levels were determined by bioassay (BA) and RIA; AIA was measured by BA. The inhibin content of follicles characterized as immature by their estradiol (E2) levels and E2/progesterone (P) ratios was significantly lower (P less than 0.05) than that of mature follicles (i.e. leading to pregnancy). The mean AIA for mature follicles were significantly lower than AIA in groups where pregnancy was not obtained. AIA for follicles from which a pregnancy was obtained for each ET was also significantly lower than that in FF characterized as immature of hypermature. The highest E2/AIA and inhibin BA/AIA ratios were associated with the highest incidence of successful IVF ET outcome. No correlation was found between AIA and inhibin, on the one hand, and E2, delta 4-androstenedione, E2/P, and PRL, on the other. However, a positive correlation was found between inhibin (RIA and BA) and P, reflecting the production of inhibin by granulosa cells during luteinization. These studies allowed us to conclude that FF inhibin levels do not differ according to IVF ET outcome, but are an index of follicular maturation. AIA not only constitutes an index of follicular maturation and granulosa cell luteinization, but is of predictive value for IVF ET outcome as E2/AIA and inhibin BA/AIA ratios.

Characterization of the insulin-like growth factor axis in the human thymus.

The components of the insulin-like growth factor (IGF) axis have been investigated in the normal human thymus. Using ribonuclease protection assays (RPA), IGF-II transcripts were detected in the normal human thymus. By reverse transcriptase polymerase chain reaction (RT-PCR) analyses, promoters P3 and P4 were found to be active in the transcription of IGF2 gene within human thymic epithelial cells (TEC). No IGF-II mRNA could be detected in human lymphoid Jurkat T cells with 30 cycles of RT-PCR. By Northern blot analyses, IGFBP-2 to -6 (but not IGFBP-1) were found to be expressed in TEC with a predominance of IGFBP-4. Interestingly, Jurkat T cells only express IGFBP-2 but at high levels. The type 1 IGF receptor was detected in Jurkat T cells but not in human TEC. The identification of the components of the IGF axis within separate compartments of the human thymus adds further evidence for a role of this axis in the control of T-cell development. The precise influence of thymic IGF axis upon T-cell differentiation and immunological self-tolerance however needs to be further investigated.

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. I. Sex steroids.

The steroid content of 72 follicular fluids (FF) obtained from 42 women subjected to ovulation induction with the luteinizing hormone-releasing hormone analogue D-Trp6 and human menopausal gonadotropin was studied in terms of the evolution of in vitro fertilization (IVF) and embryo transfer (ET) results. The FF were classified into several categories based on oocyte evolution. Individual values of FF estrone and estradiol (E2), as well as androstenedione and testosterone could not be correlated with ET outcome. However, FF progesterone (P) levels for follicles leading to pregnancy were significantly lower when compared with those in the other categories. The correlation between the E2/P ratio and E2 permitted the definition of a band wherein IVF-ET outcome was successful and enabled the characterization of different functional follicular maturational states.

[Urinary excretion of 6-sulphatoxymelatonin in normal subjects: statistical approach to the influence of age and sex]. / Approche statistique de l'influence de l'âge et du sexe sur l'excrétion de 6-sulfatoxymélatonine urinaire (a-MT6s) chez l'individu normal.

A radioimmunoassay of urinary 6-sulphatoxymelatonin (a-MT6s) was performed in 90 normal subjects: 44 males and 46 females (17-67 years). Patients treated with betablokers or antidepressants were not included in this study. Urine samples were collected over three periods of time: 7 to 11 p.m., 11 p.m. to 7 a.m., and 7 to 11 a.m. Between 11 p.m. and 7 a.m., the subjects slept in their normal environment and had not ingested alcohol for 24 hours. We searched for a possible relation between urinary a-MT6s excretion (expressed in ng/l/h) and age. From 7 to 11 p.m. and from 7 to 11 a.m. no significant relation could be found. On the contrary, between 11 p.m. and 7 a.m. there was a significant relation indicating decrease of a-MT6s secretion with increasing age. Several linear or non-linear curve patters were tested: Boltzmann sigmoid (1(st), 2(nd), and 3(rd) degree), polynomial curves. The Boltzmann sigmoid showed the best fit judging by the r-squared value (0.152) and the runs test (p=0.64). On this curve the inflection point was located at 53 4 years (SDM, standard deviation of the mean). From 19 to 45 years, the upper sigmoid plateau was located at 1381 91 ng/l/h (SDM). The decrease was found between 45 and 60 years and the lower sigmoid plateau then stabilized at 467 370 ng/l/h (\SDM). In the study group, there was no significant difference between men and women according to the Mann-Withney test. Finally, use of oral contraceptives did not affect urinary a-MT6s (Mann-Withney).

[Inhibin: new gonadal hormone (author's transl)]. / Inhibine: nouvelle hormone gonadique.

There are many convincing arguments to accept the existence of inhibin. This hormone is produced inside the seminiferous tubules by the Sertoli cells in males and by the granulosa cells of the follicule in females. The biological, immunological and chemical characteristics of testicular and ovarian inhibin are identical so that it could be speculated the same molecule is secreted by both organs. This hormone is not a knownsteroid but is a protein substance. Thus, its biological activity is destroyed by trypsin and pepsin digestion and by heating at 60 degrees for 30 minutes. Furthermore, immunization with inhibin from rete testis fluid induces antibodies capable of neutralizing endogenous inhibin of adult male and female rats. This polypeptide hormone is not identical neither to ABP nor to a fragment of gonadotrophins. The molecular weight is not yet exactly defined and the possibility exists that two forms of inhibin are present in RTF: one of high (greater than 10,000 Daltons) and the other of low molecular weight. The high M.W. species could be a polymer or alternatively the combination of native inhibin and a carrier substance or unique precursor molecule. Inhibin preparations selectively depress the synthesis and the release of FSH in pituitary cell culture. The threshold dose to affect the LH production is higher than that active on FSH secretion. Furthermore, they reduce LH-RH content of hypothalamus maintained in organ culture. In animals, inhibin induced effects are depending on both hypothalamus and pituitary actions according to the functions of these two structures. In that sense, apparently contradictory results are obtained in short and long term castrated animals. Inhibin does not modify TSH, GH and prolactin in vivo and in vitro. This substance displays an inhibition on the synthesis of DNA in the testis of pubertal male rats and depresses the maturation of follicle in female.

Novel plasma extraction procedure and development of a specific enzyme-immunoassay of oxytocin: application to clinical and biological investigations of small cell carcinoma of the lung.

Paraneoplastic secretion of the lactation-inducing hormone oxytocin (OT) has been reported in about 30% of cases of small cell carcinoma of the lung (SCCL). In order to investigate the role of OT in the biology of SCCL tumours, a specific enzyme-immunoassay (EIA) for OT, which can be applied to both human plasma and culture medium, has been developed. OT EIA is performed on 96-well microtiter plates coated with a rabbit polyclonal antibody (Ab) anti-OT (04). This antibody does not exhibit any significant cross-reactivity either with vasopressin (VP) or with vasotocin (VT). The immunological reaction involving Ab anti-OT is a competition between the tracer (biotinylated OT) and synthetic OT (standard curve) or OT present in biological samples. In order to limit interference induced by plasma proteins, plasma samples are filtrated by a one-step centrifugation on centricon YM-3 (cut-off 3000 Da). After plasma filtration, 90.7 +/- 5.1 (SD) % (n = 22) immunoreactive (IR) OT is recovered. The sensitivity of OT EIA is 1 pmol/L, while intra- and inter-assay coefficients of variation (CV) are around 3.41% and 2.84%, respectively. In healthy volunteers, plasma IR OT is 7.28 +/- 4.49 (SD) pmol/L (n = 32) with no gender difference. As shown by the data both from plasma of SCCL patients and from supernatants and cell contents of SCCL cell lines, this EIA procedure offers a novel, reproducible, specific and sensitive method for the measurement of IR OT.

Effect of mouse epidermal growth factor on DNA and protein synthesis, progesterone and inhibin production by bovine granulosa cells in culture.

The effect of mouse epidermal growth factor (EGF) was investigated on DNA and protein synthesis, progesterone and inhibin production by bovine antral granulosa cells. When incubated for the whole period of culture, EGF inhibited inhibin production the second day of culture, progesterone the third and the fourth days whereas it stimulated DNA and protein synthesis only the fourth day of culture. Inhibition of progesterone and stimulation of DNA and protein were dose-dependent when treatment with EGF (pre-incubation) is followed by 24 h without EGF, a stimulatory effect on DNA and protein synthesis was observed after 48 and 72-h pre-incubation. Progesterone was reduced after 3 day pre-incubation and inhibin only after 2-day pre-incubation. Effects observed after 3-day pre-incubation were dose-dependent. These experiments demonstrated the stimulatory effect of EGF on growth of granulosa cells and its inhibitory action on hormonal production by these cells in vitro. The inhibitory effect on progesterone and inhibin production is more precocious than stimulatory effect on DNA and protein synthesis. The inhibitory action of EGF on granulosa cell production of progesterone and inhibin could thus be not directly dependent on its stimulatory action on DNA synthesis.

Inhibin and related peptides. Mechanisms of action and regulation of secretion.

The structure of inhibin is known: it consists in a heterodimer constituted by one alpha and one beta subunits. The homodimer of beta A or the heterodimer beta A or the heterodimer beta A-beta B called activin A and B stimulates the release and the synthesis of FSH by gonadotrophs. Inhibin displays actions at hypophyseal, hypothalamic and gonadal levels. Produced by granulosa cells in female and by Sertoli cells in male, inhibin synthesis is stimulated by FSH, and reduced by hypophysectomy and progesterone. At the present time, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves with follicular maturation as aromatase activity whereas luteinization arrests its production. Nevertheless it seems to exist large difference in the regulation of inhibin secretion from one species to the other. Sperm inhibin levels are correlated with spermatozoa number. Its administration to the sheep induce either an anovulation or an increase of ovulation rate according to the scheme of treatment.

Inhibin and related peptides: mechanisms of action and regulation of secretion.

The structure of inhibin is known; it consists of a heterodimer composed of one alpha and one beta subunit. The homodimer of beta A (beta A-beta A) and the heterodimer beta A-beta B, called activin A and B, respectively, stimulate the release and synthesis of FSH by gonadotrophs. Inhibin exerts effects at the hypophyseal, hypothalamic, and gonadal levels. Produced by granulosa cells in the female and by Sertoli cells in the male, inhibin synthesis is stimulated by FSH and reduced by hypophysectomy and progesterone. At present, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves in parallel with follicular maturation and aromatase activity, whereas luteinization arrests its production. Nevertheless, important differences in the regulation of inhibin secretion seem to exist from one species to another. Sperm inhibin levels can be correlated with spermatozoa number. Administration of inhibin to sheep induces either anovulation or an increase in the rate of ovulation depending on the scheme of treatment.

[Endocrine, paracrine and autocrine mechanisms of aromatase activity]. / Mécanismes endocrines, paracrines et autocrines de l'activité aromatasique.

The endocrine, paracrine and autocrine mechanisms involved in aromatase activity. Development of a single follicle during the menstrual cycle is under control of hormones stimulating follicular maturation, ovulation and luteogenesis. Several factors intervene locally to avoid other follicles developing at the same time as the dominant follicle. These other follicles remain quiescent or go on to atresia. Atresia results from the action of several endocrine, paracrine and autocrine mechanisms which synergistically inhibit aromatase activity. The subsequent lack of estrogens reduces granulosa cell multiplication.

Identification in human seminal fluid of an inhibin-like factor which selectively regulates FSH secretion.

Human seminal plasma obtained by centrifugation of human semen contains a factor capable of selectively inhibiting the secretion of FSH both in vivo (reduction of the levels of FSH in rats 24 h after castration) and in vitro (reduction of the FSH released by LH-RH in rat pituitary cell culture). This effect is not due to testosterone, oestradiol-17 beta or progesterone present in the active fractions. The factor has the characteristics of a protein in that its biological activity is destroyed by heat and trypsin digestion. It does not resemble androgen-binding protein. The biological action is not completely specific for FSH as inhibition of LH can be seen with doses usually higher than those which produce inhibition of FSH alone. There is no effect on TSH or prolactin levels in vitro. The factor clearly acts on the release and synthesis of gonadotrophins by gonadotrophs but an effect on the hypothalamus is not excluded. This factor fits the definition of inhibin.

[Effects of leucine-enkephalin and methionine-enkephalin on prolactin and gonadotropin secretion and synthesis in vitro]. / Action de la leucine-enképhaline et de la méthionine-enképhaline sur la libération et la synthèse des gonadotrophines et de la prolactine in vitro.

In vivo, Enkephalins, stimulate PRL, inhibit LH and are inactive on FSH. However, in monolayer pituitary cell cultures, PRL, LH and FSH secretions and synthesis are not modified by Met-Enk. (5 microgram/ml) or Leu-Enk. (5 and 10 microgram/ml). But the simultaneous presence of LHRH and Enk. induces an increase in LH secretion and synthesis without modifying FSH and PRL. In conclusion 1) Enk do not act by themself at the pituitary level but 2) they are able to modify the responses induced by hypothalamic hormones.

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.

[Intracellular mechanism for the action of inhibin on the secretion of the follicular-stimulating hormone and luteinizing hormone induced by LH-RH in vitro]. / Un mécanisme intracellulaire de l'action de l'inhibine sur la sécrétion de l'hormone folliculo-stimulante et de l'hormone lutéinisante induite par le LH-RH in vitro.

The FSH secretion-inhibiting action of inhibin in vitro under basal conditions and also in the presence of LH-RH is suppressed by the addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH, inhibin reduces significantly the intracellular level of cAMP in isolated pituitary cells. In contrast, the simultaneous addition of MIX and inhibin raises the cAMP level, and this stimulation is comparable to the increase observed when MIX is added alone. These observations suggest that one mode of action of inhibin could be mediated by a reduction in cAMP within the pituitary gonadotropic cell.

Effect of neuraminidase on inhibin activity in vivo and in vitro.

Matrex gel red A purified follicular fluid has been used to study whether or not this material contains sialic acid residues and their importance in maintaining the biological activity of inhibin both in vitro and in vivo. It appears that sialic acid is present in these preparations and can be released either by neuraminidase treatment of acid hydrolysis. The addition of intact and desialylated inhibin-containing material to isolated rat pituitary cells in culture gives similar inhibition of LHRH-induced FSH release of these cells indicating that sialic acid is not required for inhibin activity in vitro. The injection of intact inhibin preparations leads to a reduction of the uterine weight increase seen in immature female mice primed with human chorionic gonadotropin. By contrast, the inhibition of this uterine weight increment by 80% desialylated inhibin-containing material is significantly reduced, suggesting that sialic acid residues play an important role in maintaining the biological activity of inhibin in vivo.

Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium.

BACKGROUND: The elucidation of the molecular mechanisms by which the embryo contributes to its implantation is an area of extensive research. The main objective of this study was to investigate the pattern of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) secretion by human endometrial epithelium, and their regulation by human chorionic gonadotropin (hCG) and other growth factors present at the embryonic-endometrial interface. METHODS: Endometrial epithelial cells (EEC) were isolated from biopsies collected at both proliferative and secretory phases of fertile women. RESULTS: HCG (1-50 IU/ml) increased LIF secretion by EEC cultures derived from follicular phase (up to 285+/-75%) or from secretory phase (up to 212+/-16%). In contrast, hCG reduced IL-6 secretion by EEC in both phases. The hCG/LH receptor gene was transcribed by EEC as evidenced by RT-PCR. Insulin-like growth factors 1 and 2 increased LIF secretion by EEC. Transforming growth factor beta1 stimulated LIF and reduced IL-6 secretion. CONCLUSIONS: Through hCG, the blastocyst may be involved in the control of its implantation (via an increase of proimplantatory LIF) and tolerance (via an inhibition of proinflammatory IL-6). Other growth factors present at the embryonic-endometrial interface are also involved in the control of LIF and IL-6 endometrial secretion.