Aggregation of luteinizing hormone receptors in granulosa cells: a possible mechanism of desensitization to the hormone.

The temporal relationship between redistribution of receptors to lutropin (luteinizing hormone)/human chorionic gonadotropin in cultured rat ovarian granulosa cells and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin by indirect immunofluorescence, with hormone-specific antibodies after fixation with 2% formaldehyde, revealed the existence of small clusters around the entire cell circumference 5--20 min after exposure to the hormone at 37 degrees C. Such small receptor aggregates were also evident if hormone incubation was at 4 degrees C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incubation with the hormone (2--4 hr) at 37 degrees C. The later change coincided with diminished cyclic AMP accumulation in respose to challenge with fresh hormone. When the fixation step was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both receptor clustering and the desensitization process was observed. This maneuver also induced capping of the hormone receptors. In contrast, monovalent Fab' fragments of the antibodies were without effect. Internalization of the bound hormone in lysosomes, and subsequent degradation, was evident 8 hr after hormonal application and was not accelerated by the antibodies. It is suggested that clustering of the luteinizing hormone receptors may play a role in cellular responsiveness to the hormone. Massive aggregation of the receptors may desensitize the cell by interferring with coupling to adenylate cyclase.     

Toll-like receptors: lessons to learn from normal and malignant human B cells

The humoral immune system senses microbes via recognition of specific microbial molecular motifs by Toll-like receptors (TLRs). These encounters promote plasma cell differentiation and antibody production. Recent studies have demonstrated the importance of the TLR system in enhancing antibody-mediated defense against infections and maintaining memory B cells. These results have led the way to the design of vaccines that target B cells by engaging TLRs. In hematologic malignancies, cells often retain B cell-specific receptors and associated functions. Among these, TLRs are currently exploited to target different subclasses of B-cell leukemia, and TLR agonists are currently being evaluated in clinical trials. However, accumulating evidence suggests that endogenous TLR ligands or chronic infections promote tumor growth, thus providing a need for further investigations to decipher the exact function of TLRs in the B-cell lineage and in neoplastic B cells. The aim of this review is to present and discuss the latest advances with regard to the expression and function of TLRs in both healthy and malignant B cells. Special attention will be focused on the growth-promoting effects of TLR ligands on leukemic B cells and their potential clinical impact.     

Gonadal luteinizing hormone receptors and adenylate cyclase: transfer of functional ovarian luteinizing hormone receptors to adrenal fasciculata cells.

Luteinized rat ovaries contain a high concentration of particulate luteinizing hormone (lutropin, LH) receptors and a small quantity of lipid-associated receptors that float in the 360,000 X g supernatant fraction of ovarian homogenates. During fractionation of Lubrol-solubilized LH receptors and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from the ovary and testis, LH receptors and adenylate cyclase were coincident on gel filtration, but could be resolved during ion-exchange chromatography of soluble ovarian preparations and were completely separated by lectin-affinity chromatography on Sepharose-concanavalin A. For further analysis of receptor-adenylate cyclase coupling, the lipid-rich fraction of ovarian luteal cells was used to transfer gonadal LH receptors to isolated adrenal fasciculata cells. The lipid vesicles obtained from ovarian homogenates by flotation at 360,000 X g contained 5--10% of the ovarian LH receptors and were devoid of adenylate cyclase activity. During incubation of lipid-associated receptors with dispersed rat fasciculata cells at 16 degrees C, progressive incorporation of LH binding sites into the adrenal cells was observed. When adrenal cells bearing heterotopic LH receptors were incubated with 1 nM human choriogonadotropin, cyclic AMP production was consistently stimulated, with an accompanying increase in corticosterone production. These results indicate that LH receptors exist as separate entities from adenylate cyclase in the gonadal cell membrane and can become functionally coupled to adenylate cyclase to evoke cyclic AMP production and steroidogenesis in the host adrenal cells to which they are transferred.     

Gonadotropin-releasing hormone and its receptor in normal and malignant cells

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.     

Distribution of complement receptors on human normal and malignant mononuclear cells

Mononuclear cells from normal human subjects and patients with chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (LCL), and hairy cell leukemia (HCL) were labeled with fluoresceinated, purified human C3b (FI-C3b) and analyzed using the fluorescence- activated cell sorter (FACS). FI-C3b labeled 17.6% +/- 6.0% of peripheral blood mononuclear cells (PBM) from 20 normal subjects, which, when separated by the FACS, consisted of B lymphocytes and approximately 5% monocytes. Analyses in which either monocytes or B lymphcoytes were excluded from consideration demonstrated that both these cell types were labeled by the FI-C3b with a heterogeneous distribution of fluorescence intensity, indicating either heterogeneity of CR density or variable avidity of individual CR for the FI-C3b. FACS profiles of PBM ( < 5% monocytes) from 14 of 15 patients with CLL showed a homogeneous distribution of very low fluorescence intensity, with > 60% of the cells being slightly more fluorescent than unlabeled controls. This low, homogeneous distribution of fluorescence is strikingly similar to profiles of CLL cells labeled with anti-Ig reagents and suggests homogeneity of low CR density and/or avidity. Similarly, CR+ mononuclear cells from five patients with HCL and three patients with LCL displayed more homogeneous FI-C3b labeling than normal CR+ PBM. Homogeneity of FI-C3b binding to CLL, LCL, and HCL cells further supports the concept for a clonal origin for these disorders.     

Homologous desensitization of cultured chick kidney cells to parathyroid hormone

The development of refractoriness of the cAMP response to PTH in primary cultures of chick kidney cells and recovery from the refractory state was investigated. When cells were preincubated with bovine PTH1-34, complete refractoriness to a subsequent challenge with the hormone developed within 2 h and at hormone concentrations as low as 5 ng/ml. The ability of PTH to stimulate activation of cAMP-dependent protein kinase was also abolished by preincubation with the hormone. When cells were desensitized and then incubated in hormone-free medium, recovery of the cAMP response began within an hour and was maximal, but not complete (80%) after 16 h. Cycloheximide did not affect either desensitization or the rate or extent of recovery from the refractory state. Low concentrations of forskolin (2.5 X 10(-7) M) greatly enhanced cAMP production stimulated by PTH and higher concentrations (10(-6) - 10(-4) M) stimulated rates of cAMP production 50 times those obtained with PTH alone. Preincubation with forskolin did not bring about desensitization to PTH nor did preincubation with PTH affect the subsequent response to forskolin. The half-life of biologically active bovine PTH1-34 in chick kidney cell culture was approximately 12 h and the rate of its removal was not significantly altered during a 20-h incubation period. The results suggest that desensitization of chick kidney cells to PTH is not suggest that desensitization of chick kidney cells to PTH is not brought about by cAMP generation itself, is not primarily dependent on protein synthesis, and does not involve a change in the rate of removal of biologically active hormone from the medium. In addition, recovery of the cAMP response to PTH also does not require new protein synthesis. These results are compatible with a mechanism of desensitization which occurs at the level of the receptor or hormone-receptor coupling to adenyl cyclase.     

Differential protection of normal and malignant human myeloid progenitors (CFU-GM) from Ara-C toxicity using cycloheximide

Cycloheximide, a reversible protein synthesis inhibitor, is thought to block DNA replication in normal cells by preventing synthesis of a labile protein. In animal systems, cycloheximide protects normal cells from cytotoxic S-phase specific agents, such as cytosine arabinoside (Ara-C). Malignant cells appear not to be susceptible to cycloheximide- induced cycle arrest and, subsequently, may not be protected from Ara-C cytotoxicity. The effect of cycloheximide on granulocyte/macrophage progenitors (CFU-GM) after in vitro Ara-C exposure was examined using normal human bone marrow, malignant progenitors from patients with chronic myelogenous leukemia (CML), and clonogenic cells from the human acute nonlymphocytic leukemia cell lines HL-60 and KG-1. Mononuclear or clonogenic cells were incubated for one hour with cycloheximide, followed by the addition, for three or 17 hours, of Ara-C before being plated in a methylcellulose culture system. CFU-GM survival was significantly increase if normal cells were treated with cycloheximide before Ara-C exposure. Similar cycloheximide pretreatment of CML progenitors and clonogenic HL-60 and KG-1 cells failed to protect CFU- GM from Ara-C-induced cytotoxicity.     

Mechanisms of desensitization to parathyroid hormone in human osteoblast-like SaOS-2 cells.

Signal transduction by the PTH receptor is now known to involve generation of multiple second messengers. Desensitization of the adenylate cyclase response to PTH is a common feature of bone- and kidney-derived target cells; however, no single mechanism appears to explain desensitization in the different cell types studied. To examine the role of protein kinase-A (PKA) in homologous desensitization to PTH, we employed human SaOS-2 osteoblast-like cells and a mutant subclone (Ca 4A), which expresses an inducible cAMP-resistant form of PKA. Pretreatment of SaOS-2 cells with PTH for 4 h reduced by 60-80% the cAMP response to subsequent rechallenge with the hormone. This homologous desensitization was significantly, but not completely, inhibited in Ca 4A cells. Desensitization was not mimicked by pretreatment of the cells with forskolin. PTH binding to its receptor was reduced 50% in both SaOS-2 and Ca 4A cells after 4-h incubation with PTH (homologous down-regulation), whereas forskolin did not cause receptor down-regulation. Pretreatment with the ionophore ionomycin for 4-24 h did not mimic desensitization to PTH. Both desensitization to PTH and receptor down-regulation were induced, however, by pretreatment with a phorbol ester (12-O-tetradecanoyl phorbol-13-acetate), and these effects were blocked completely by staurosporine. PTH-induced desensitization was not blocked by staurosporine, and receptor down-regulation was enhanced by the drug. Pertussis toxin did not prevent desensitization induced by either PTH or 12-O-tetradecanoyl phorbol-13-acetate. We conclude that homologous desensitization to PTH in SaOS-2 cells involves both cAMP-dependent and -independent mechanisms. Homologous PTH receptor down-regulation apparently is mediated by mechanisms independent of PKA activation. Neither pathway of homologous desensitization to PTH involves the action of pertussis toxin-sensitive G-proteins.     

Adenovirus-directed expression of functional luteinizing hormone (LH) receptors in undifferentiated rat granulosa cells: evidence for differential signaling through follicle-stimulating hormone and LH receptors

This study was conducted to determine the feasibility of using replication-defective adenovirus vectors to express receptors for LH. Two vectors were constructed, one that directs the expression of wild-type human LH receptor (LHr; AdRSVLHrwt) and another that directs the expression of the constitutively activated D578H mutant human LH receptor (AdRSVD578HLHr). When infected with AdRSVwtLHr and AdRSVD578HLHr, COS-1 cells expressed LH/hCG-binding sites as reflected by specific binding of [(125)I]hCG. To determine the ability of the vectors to confer LH responsiveness, undifferentiated rat granulosa cells, which possess only FSH receptors, were infected with AdRSVwtLHr and AdRSVD578HLHR: Expression of the constitutively activated D578H LHr increased basal (gonadotropin-independent) estrogen and progesterone production. Expression of the wild-type LHr in granulosa cells did not stimulate basal steroid production, but conferred responsiveness to exogenous LH. For both wild-type LHr and D578HLHr, the absolute levels of steroid production were dependent upon the input of viral titers. Using these vectors, we compared effects of FSH and LH receptor activation in undifferentiated granulosa cells. Stimulation of undifferentiated granulosa cells by FSH and D578HLHr, as well as activation of wild-type LHr with LH resulted in comparable production of progesterone. In contrast, estradiol production in cells stimulated with FSH was greater than that in cells that expressed either D578H receptors or wild-type LHr in the presence of LH. Analysis of messenger RNAs (mRNAs) revealed that activations of FSH and the LH receptors were comparable in the induction of alpha-inhibin and 3betahydroxysteroid dehydrogenase mRNAS: However, activation of FSH receptor led to significantly greater expression of P450 aromatase and LHr mRNAs than did activation of LHR: These results suggest that activation of FSH and LH receptors in granulosa cells may differ with respect to activating intracellular signaling pathways and stimulating gene expression.     

Remission maintenance in acute myeloid leukemia: impact of functional histamine H2 receptors expressed by leukemic cells

Post-consolidation immunotherapy with histamine dihydrochloride and interleukin-2 has been shown to improve leukemia-free survival in acute myeloid leukemia in a phase III trial. For this study, treatment efficacy was determined among 145 trial patients with morphological forms of acute myeloid leukemia as defined by the French-American-British classification. Leukemia-free survival was strongly improved in M4/M5 (myelomonocytic/monocytic) leukemia but not in M2 (myeloblastic) leukemia. We also analyzed histamine H₂ receptor expression by leukemic cells recovered from 26 newly diagnosed patients. H₂ receptors were typically absent from M2 cells but frequently expressed by M4/M5 cells. M4/M5 cells, but not M2 cells, produced reactive oxygen species that triggered apoptosis in adjacent natural killer cells. These events were significantly inhibited by histamine dihydrochloride. Our data demonstrate the presence of functional histamine H₂ receptors on human AML cells and suggest that expression of these receptors by leukemic cells may impact on the effectiveness of histamine-based immunotherapy.Key words: acute myeloid leukemia, remission maintenance, histamine H₂ receptors     

Pituitary and gonadal desensitization after continuous luteinizing hormone-releasing hormone infusion in normal females

Three normal females were studied during the early follicular phase of the menstrual cycle employing a continuous infusion of LHRH (1.4 micrograms/min) for 72 h. Blood samples were taken every 4 h. LH concentrations climbed from 9.3 mIU/ml basally to peak values of 120 mIU/ml at 12 h, then fell to a plateau between 23-31 mIU/ml from 36-72 h. FSH levels rose from 5.4 to 36 mIU/ml at 4 h of infusion and then returned to baseline. 17 beta-Estradiol increased from 48 to 184 pg/ml at 32 h, but subsequently fell toward basal concentrations. 17-Hydroxyprogesterone increased from 0.5 to 1.23 ng/ml at 12 h and remained elevated for the remainder of the infusion period. Serum progesterone levels remained constant. Three females with premature ovarian failure were studied, and the pattern of gonadotropin release was similar to that of normal subjects. Studies demonstrate that continuous LHRH infusion in normal females causes pituitary and gonadal desensitization. The desensitization of the pituitary is independent of gonadal activity.     

A novel method to modulate desensitization and truncation of luteinizing hormone receptors using antisense oligodeoxynucleotides.

We report a novel method to study the mechanisms of luteinizing hormone (LH) receptor desensitization and truncation, using antisense oligodeoxynucleotides that code for regions of the NH2-terminus, the third extracellular loop and the C-terminus of the LH receptor. Mouse tumour (MA10) Leydig cells were incubated for 48 h with the addition of 2.5 microM antisense oligodeoxynucleotides at time 0 and 24 h. It was found that the NH2-terminus oligodeoxynucleotide completely inhibited synthesis of the LH receptor. Pretreatment with the third extracellular loop oligodeoxynucleotide inhibited LH-, dibutyrylcyclic AMP (db-cAMP)- and phorbol 12-myristate 13-acetate (PMA)-induced desensitization and truncation of LH receptors. Truncation but not desensitization, of the LH receptor was prevented in cells pretreated with the C-terminus oligodeoxynucleotide. These results indicate that different sites of the C-terminal intracellular tail of the LH receptor are involved in the regulation of desensitization and truncation of the LH receptor.     

Differential expression of retinoic acid receptors and p53 protein in normal, premalignant, and malignant esophageal tissues

Purpose: Esophageal cancer remains a significant health problem worldwide. The very low 5-year survival rates and rapid increase in the incidence of adenocarcinoma indicate the urgent need for early identification of and new approaches to the prevention and treatment of this cancer. Methods: To find biomarkers for early identification of the disease, we analyzed nuclear retinoic retinoid receptor mRNAs, p53 protein, and the proliferation marker Ki 67 in surgical specimens of normal, mildly, and severely dysplastic and malignant esophageal tissues. Results: Nuclear retinoid receptors (RAR-α, RAR-γ, and RXR-α) were expressed in most (79%–100%) normal, dysplastic, and malignant esophageal mucosae, whereas expression of RAR-β was progressively lost from normal esophagus to carcinoma (84%–54%). In contrast, expression of p53 protein and Ki 67 were dramatically increased in severely dysplastic and cancerous tissues of the esophagus (from 5% to 62%). Conclusions: Loss of RAR-β expression and accumulation of p53 and Ki 67 proteins may serve as biomarkers for esophageal cancer. Received: 17 April 2000 / Accepted: 12 July 2000     

Differential sensitivity of functional subsets of t cells to the cytotoxicity of natural t-lymphocytotoxic autoantibody of systemic lupus erythematosus

A naturally occurring T-lymphocytotoxic autoantibody (Hu-NTA) in serum from a patient with systemic lupus erythematosus (SLE) showed a differential cytotoxic effect on functionally different T cell subsets as did natural thymocytotoxic autoantibody (NTA) of NZB mice. When the normal peripheral blood lymphocytes were treated, in the presence of complement, with Hu-NTA at a dilution that eliminated 25 to 30% of Hu-NTA-sensitive T cells, there was a marked reduction or a total depletion in the ability of resultant cells to show Con A-activated suppression on the proliferative response of responder cells in mixed lymphocyte reaction. The treatment of PBL in the same manner also resulted in a marked reduction in its responsiveness to Con A and PHA. However, the responder cells to allogeneic stimulator cells in MLR were found to be much more resistant to the cytotoxicity of Hu-NTA than other functional T cell subsets tested. These results suggest that Hu-NTA is responsible for the selective loss of certain functional T cell subsets including suppressor T cells in patients with SLE.     

Functional characterization of luteinizing hormone responsiveness and desensitization in perifused interstitial cells

The characteristics of the steroidogenic response of isolated rat testis interstitial cells to repeated 10 min pulses of ovine LH at 2 h intervals were examined in a Bio-gel perifusion system. Maximal responsiveness of the perifused interstitial cells could be maintained for 6-8 h. Thereafter, both basal and LH-stimulated testosterone production declined gradually despite supplementation of the perifusion medium with 1% and 5% foetal calf serum or 0.5 microgram/ml insulin. In contrast to the time-related steroidogenic decline, a dose-dependent refractoriness of the interstitial cells could be induced by repeated exposure to LH pulses from 0.01 to 10 ng/ml during the first 6 h of perifusion. The higher the stimulating dose of LH, the greater was the rate and magnitude of the resultant desensitization. With lower doses (0.01 and 0.1 ng/ml) of LH, an initial sensitization or priming effect was also observed. These changes in steroidogenic response occurred in the absence of any significant alterations in LH/hCG receptor binding of the perifused interstitial cells, nor could the refractory state be overcome by stimulation with analogues of cAMP. The perifused interstitial cells, when desensitized with low doses of LH (0.1 ng/ml), were capable of increasing or maintaining testosterone production in response to further stimulation with higher doses of LH (1 and 10 ng/ml). The mechanism(s) underlying the in vitro desensitization of perifused interstitial cells by LH may best be explained on the basis of the interaction between the negative effects of substrate depletion and the positive influence of mobilization of substrate(s) into the metabolically active pool for cholesterol side-chain cleavage. It was concluded that the dose of LH used in the pulsatile stimulation of perifused interstitial cells is critically important not only in determining the total amount of testosterone produced, but also in the pattern of response in terms of the degree of sensitization and desensitization induced.     

Comparative effects of G-CSF, GM-CSF and IL-3 on cytosine arabinoside- and daunorubicin-mediated cytotoxicity of acute myeloid leukemia cells and normal myeloid progenitors.

We carried out an in vitro study on the combined effects of three CSF (G-CSF, GM-CSF and IL-3) plus the cycle-specific chemotherapeutic drugs [cytosine arabinoside (Ara-C) and daunorubicin (DNR)] on the proliferation and cytotoxicity of blasts and clonogenic cells (CFU-AML) in the AML-193 cell line, in AML patients and in normal bone marrow CFU-GM. The number of surviving blasts and/or DNA synthesis in blasts treated with CSF plus Ara-C or DNR was greater than those treated without CSF in the AML-193 cell line, and in some AML patients. On the other hand, the Ara-C- and DNR-mediated cytotoxicity of CFU-AML was not abrogated by CSF in any instance, but rather, it was significantly enhanced by all the CSF in the majority of instances. Although the enhancement was clearer when Ara-C was used, compared with DNR, there were no significant differences among the enhancing effects of the CSF. Under the same culture conditions as those for CFU-AML, all of the CSF significantly enhanced the Ara-C-mediated cytotoxicity of day 7 normal CFU-GM, although to a lesser extent than in CFU-AML. However, none of the CSF significantly affected the Ara-C-mediated cytotoxicity of day 14 normal CFU-GM or the DNR-mediated cytotoxicity of day 7 or day 14 normal CFU-GM. These results suggest that in the selection of a strategy entailing combined use of cycle-specific drugs plus CSF to increase the antileukemic effectiveness of chemotherapy in AML, G-CSF is preferable to GM-CSF or IL-3, since it has fewer potential clinical side effects, and that, furthermore, DNR may be as useful as Ara-C.