O. Ortmann

Effects of LHRH-analogues on mitogenic signal transduction in cancer cells

The expression of luteinizing hormone-releasing hormone (LHRH) and its receptors has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Recent studies in our laboratory have demonstrated that the function of LHRH produced by ovarian cancer cells is the inhibition of their proliferation. Dose-dependent antiproliferative effects of LHRH-agonists have been observed by several laboratories in cell lines derived from the above cancers. Interestingly, also LHRH-antagonists have marked antiproliferative activity in most of the ovarian, breast and endometrial cancer cell lines tested so far, indicating that the dichotomy of LHRH-agonists/LHRH-antagonists is not valid for the LHRH-system in cancer cells. In addition, our data suggest that the classical LHRH receptor signal transduction mechanisms known from the pituitary (phospholipase-C, protein kinase C, adenylyl cyclase) are not involved in the mediation of LHRH effects in cancer cells. Data obtained by several groups, including ours, rather suggest that LHRH analogs interfere with the signal transduction of growth-factor receptors and related oncogene products associated with tyrosine-kinase activity. The mechanism of action is probably an LHRH-induced activation of a phosphotyrosine phosphatase, counteracting the effects of receptor associated tyrosine kinase. In our hands, LHRH analogs virtually blocked the EGF-induced MAP-kinase activity of ovarian and endometrial cancer cells. The pharmacological exploitation of this mechanism might provide promising new therapies for these cancers.

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.

Modulation of calcium signaling and LH secretion by progesterone in pituitary gonadotrophs and clonal pituitary cells

In estradiol-treated pituitary cells, progesterone enhances gonadotropin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells during short-term treatment but attenuates this response during prolonged treatment. In the present study, the effects of gonadal steroids on GnRH-induced cytoplasmic calcium ([Ca2+]i) responses in gonadotrophs were analyzed in rat pituitary cells and immortalized (alpha T3-1) murine gonadotrophs. Ca2+ responses were measured in cell suspensions and single gonadotrophs, loaded with Fura-2 or Indo-1, respectively, and pretreated for 48 h with 1 nM estradiol with or without 100 nM progesterone, or for 48 h with 1 nM estradiol and then for 3 h with 100 nM progesterone. In cells of the alpha T3-1 gonadotroph lineage, GnRH elicited biphasic Ca2+ signals composed of an initial peak response followed by a prolonged plateau phase. The amplitudes of both the extracellular Ca(2+)-independent spike phase and the extracellular Ca(2+)-dependent plateau phase were enhanced or inhibited by short- or long-term progesterone treatment, respectively. In single pituitary gonadotrophs, GnRH (0.5 nM) elicited oscillatory responses due to intermittent release and uptake of Ca2+ from intracellular stores. Treatment with progesterone shifted the oscillatory signal toward biphasic (3 h) or subthreshold (48 h) response profiles, revealing a steroid-induced change in the pattern of Ca2+ mobilization. In addition to these agonist-induced responses, the transient [Ca2+]i responses of pituitary cells and individual gonadotrophs to high K+ were enhanced or inhibited after short- or long-term progesterone treatment, respectively. These actions were correlated with the effects of progesterone on K(+)-induced LH secretion. The [Ca2+]i and LH secretory responses to phorbol ester treatment were also enhanced by short-term exposure of the cells to progesterone. The results demonstrate that the stimulatory and inhibitory effects of progesterone on agonist-induced Ca2+ signaling result from changes in Ca2+ mobilization and entry, and contribute to the modulatory actions of the steroid on GnRH-induced LH secretion.

The activation of an extracellular signal-regulated kinase by oestradiol interferes with the effects of trastuzumab on HER2 signalling in endometrial adenocarcinoma cell lines

Cellular response to oestradiol stimuli is mediated both by oestrogen receptor (ER) binding to oestrogen response elements (EREs) and by non-nuclear actions like activation of mitogen-activated protein kinase (MAPK) signal transduction. Therefore, oestradiol stimuli might be able to interfere with the action of antitumoral substances directed against receptor tyrosine kinase signalling. We investigated the effect of oestradiol on the inhibition of HER2 signalling by trastuzumab (Herceptin) in two human endometrial adenocarcinoma cell lines. Activation of the extracellular signal-regulated kinase (ERK-1/2), a major mediator of HER2 signalling, was measured by means of western blotting experiments and ERE activation was determined in transient reporter-gene assays. In endometrial Ishikawa and HEC-1A adenocarcinoma cells, HER2 signalling was inhibited by trastuzumab only in the absence of oestradiol. We were able to demonstrate that oestradiol counteracted the inhibitory effects of trastuzumab by rapid phosphorylation of ERK-1/2, a kinase downstream of the HER2 receptor. The pure anti-oestrogen ICI 182,780 was able to restore both the trastuzumab-triggered inhibition of the ERK-1/2 pathway and the antiproliferative action of this substance in Ishikawa cells. Our data suggest that combinations of trastuzumab with anti-oestrogens may be effective in the treatment of endometrial cancers with a positive ER and HER2 receptor status.

Ovarian steroids modulate gonadotropin-releasing hormone-induced biphasic luteinizing hormone secretory responses and inositol phosphate accumulation in rat anterior pituitary cells and αT3-1 gonadotrophs

The ovarian steroids estradiol and progesterone act as important modulators of GnRH-induced luteinizing hormone (LH) secretion from anterior pituitary cells. Recently, we demonstrated that the steroids are able to influence GnRH-stimulated Ca2+ mobilization from extra- and intracellular sources. Here we investigated the actions of estradiol and progesterone on GnRH-induced biphasic LH secretory responses in the model of perifused female rat pituitary cells. A 20 min GnRH stimulus elicited biphasic LH responses composed of an initial peak followed by a prolonged plateau phase. Both phases were equally enhanced by long-term (48 h) estradiol treatment. This action was facilitated by subsequent short-term progesterone treatment. In contrast, combined treatment with estradiol and progesterone for 48 h led to inhibited LH secretory profiles. To determine the steroid actions on the extracellular Ca2+ independent component of LH secretion we performed experiments using cells that were perifused with Ca2+ deficient medium. Under these conditions the cells responded exclusively with a single peak phase of LH secretion, which was augmented or inhibited by estradiol and progesterone treatment as described above. To test the hypothesis that an effect of estradiol and progesterone on GnRH-induced polyphophoinositide hydrolysis is responsible for their modulatory actions on Ca2+ signals and LH secretion we measured inositol phosphate (IP) accumulation after different steroid treatment paradigms in rat pituitary cells and alpha T3-1 immortalized gonadotrophs. GnRH-induced IP production was enhanced by long-term estradiol treatment. Short-term exposure of estradiol-primed cells to progesterone did not lead to significant changes of IP production. The long-term progesterone treatment paradigm enhanced GnRH-induced IP formation, while it decreased Ca2+ signals and LH secretion. Alpha T3-1 cells were used to perform more detailed analysis of IP formation. The actions of estradiol and progesterone on the production of inositol mono-, bis-, and trisphosphates were similar to those observed in the mixed cell population. It is concluded that estradiol and progesterone modulate both peak and plateau phases of GnRH-stimulated LH secretory responses, effects which are associated with their impact on Ca2+ signals. Our findings argue against a role of IP modulation in the mechanism of progesterone actions on Ca2+ signaling and LH secretion in gonadotrophs. Such a mechanism might be involved in the positive effects of estradiol in these cells.

Gonadotrophin-releasing hormone (GnRH) and GnRH agonists: mechanisms of action

The hypothalamic decapeptide gonadotrophin-releasing hormone (GnRH) binds to specific receptors on pituitary gonadotrophs. These receptors belong to the family of G protein-coupled receptors. Their activation leads to phosphoinositide breakdown with generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. These second messengers initiate Ca2+ release from intracellular stores and activation of protein kinase C, both of which are important for gonadotrophin secretion and synthesis. Prolonged activation of GnRH receptors by GnRH leads to desensitization and consequently to suppressed gonadotrophin secretion. This is the primary mechanism of action of agonistic GnRH analogues. By contrast, GnRH antagonists compete with GnRH for receptors on gonadotroph cell membranes, inhibit GnRH-induced signal transduction and consequently gonadotrophin secretion. These compounds are free of agonistic actions, which might be beneficial in certain clinical applications.

Effects of progesterone on gonadotropin-releasing hormone receptor concentration in cultured estrogen-primed female rat pituitary cells

Acute (0.5-4 h) treatment of estradiol (E)-primed female rat pituitary cells with progesterone (P) augments gonadotropin-releasing hormone (GnRH)-induced LH release, whereas chronic (48 h) P-treatment reduces pituitary responsiveness to the hypothalamic decapeptide. Dispersed E-primed (48 h, 1 nM) rat pituitary cells were cultured for 4 or 48 h in the presence of 100 nM P to assess the effects of the progestagen on GnRH receptors and on gonadotrope responsiveness to the decapeptide. P-treatment (4 h) significantly augmented GnRH-receptor concentrations (4.44 +/- 0.6 fmol/10(6) cells) as compared to cells treated only with E (2.6 +/- 0.5 fmol/10(6) cells). Parallel significant changes in GnRH-induced LH secretion were observed. The acute increase in GnRH-receptor number was nearly maximal (180% of receptor number in cells treated with E alone) within 30 min of P addition. Chronic P-treatment (48 h) significantly reduced pituitary responsiveness to GnRH as compared to E-treatment. The GnRH-receptor concentrations (3.9 +/- 0.6 fmol/10(6) cells), however, remained elevated above those in E-primed cells. GnRH-receptor affinity was not influenced by any of the different treatments. These results indicate that the acute facilitatory P-effect on GnRH-induced LH release is at least chronologically closely related to an increase in GnRH-receptor concentration. The chronic negative P-effect on pituitary responsiveness to GnRH, however, shows no relation to changes in available GnRH receptors.

Detection of increased icb-1 transcript levels in maturing HL-60 cells: a novel marker for granulocytic and monocytic in vitro differentiation

Human gene icb-1 was initially described as a gene with increased expression in endometrial tumor cells differentiated in vitro by culture on a reconstituted basement membrane. We provide evidence for a more general involvement of icb-1 gene function in cellular differentiation processes. We report the up-regulation of icb-1 transcript levels in HL-60 promyelocytic leukemia cells during their in vitro differentiation induced by all-trans retinoic acid, vitamin D(3) or DMSO. Increased icb-1 mRNA levels could be observed both in monocytic and granulocytic differentiation. We also report the identification of the novel icb-1 splice variants icb-1beta and gamma, and the spleen-specific isoform icb-1delta. Expression of icb-1 can be used as a novel marker for in vitro differentiation processes of HL-60 cells.

Effect of the gonadotropin-releasing hormone antagonist ganirelix on cyclic adenosine monophosphate accumulation of human granulosa-lutein cells

OBJECTIVEnTo evaluate whether the GnRH antagonist ganirelix exerts an effect on cyclic adenosine monophosphate (cAMP) production of human granulosa-lutein (GL) cells in vitro.nnnDESIGNnIn vitro cell culture study.nnnSETTINGnResearch laboratory of a university hospital.nnnPATIENT(S)nMural GL and cumulus cells were obtained from 15 patients on whom controlled ovarian hyperstimulation was being performed for intracytoplasmic sperm injection treatment.nnnINTERVENTION(S)nMural GL and cumulus cells were cultured for 48 hours with and without 1 nM ganirelix or triptorelin. For the last 6 hours, the cells were either exposed to 1-5 IU hCG or left unstimulated.nnnMAIN OUTCOME MEASURE(S)nAt the end of the culturing period, the intracellular and extracellular cAMP accumulations were measured by an (125)I-scintillation proximity assay.nnnRESULT(S)nhCG induced dose-dependent increases in total cAMP accumulation. Stimulation with 1 IU/mL hCG resulted in 9-fold and 13-fold increases, and 5 IU/mL hCG resulted in 19-fold and 14-fold increases in total cAMP release from cumulus and mural GL cells, respectively. On the other hand, treatments with 1 nM GnRH antagonist ganirelix and 1 nM GnRH agonist triptorelin did not exert any significant changes on the basal and hCG-stimulated cAMP accumulation of mural GL cells and cumulus cells as compared with controls.nnnCONCLUSION(S)nGanirelix does not influence basal and hCG-stimulated cAMP accumulation of human GL cells in vitro. cAMP is apparently not involved in the mechanism of action of GnRH analogs in human ovary.

Modulatory actions of the new antiprogestins ZK 98.299 and ZK 98.734 and of RU 486 on luteinizing hormone secretion and progesterone effects in pituitary gonadotrophs

The effects of the antiprogestins (APs) ZK 98.299, ZK 98.734 and RU 486 on GnRH-stimulated LH secretion and their antagonistic activity on progesterone (P) actions were investigated in cultured pituitary cells from adult female Wistar rats. P (100 nM) was able to exert a facilitatory effect on GnRH (1 nM)-induced LH secretion after short-term (4 h) treatment of estradiol-primed (1 nM, 48 h) rat pituitary cells. When the APs (10 pM-10 microM) were introduced during the 4 h incubation period with P the facilitatory effect of P was totally abolished at concentrations greater than 10 nM (ZK 98.299, ZK 98.734) and greater than 1 nM (RU 486). Also the APs were shown to block the inhibitory action of P which occurs after long-term incubation of pituitary cells with this steroid. However at concentrations greater than 10 nM (ZK 98.734, RU 486) and greater than 100 nM (ZK 98.299) this antagonistic action of the APs was lost. To evaluate whether the APs have direct effects on GnRH-induced LH secretion in the absence of exogenous P pituitary cells cultivated for 48 h with or without 1 nM estradiol were incubated for 4 or 24 h with increasing concentrations of the APs (10 pM-10 microM). Four hour treatment of non-estradiol-primed cells with ZK 98.299 or ZK 98.734 was without any effect on the LH response to a 1 nM GnRH-stimulus. Only the highest concentration of RU 486 (10 microM) reduced the LH response. Twenty-four hour treatment of the cultures with the APs led to enhancement of GnRH-stimulated LH secretion by up to 113, 37 and 33% for ZK 98.734, ZK 98.299 and RU 486, respectively. When estradiol-primed cells were used for the same experiments we observed exclusively inhibitory effects on GnRH-induced LH secretion after 4 and 24 h treatment periods. It is concluded that these new APs are potent inhibitors of P-actions, but also per se they induce diverse effects on GnRH-stimulated LH secretion in cultured rat pituitary cells which have to be taken into account.