Dexamethasone can modulate glucose-regulated and heat shock protein synthesis

When L929 cells are cultured in the abssence of glucose the rate of synthesis of two polypeptides (95,000 and 82,000 M_r) is increased while that of an 85,000 M_r polypeptide is correspondingly decreased. It has been shown recently that the 85,000 M_r polypeptide is identical with the heat shock protein that is associated with the Rous sarcoma virus transforming gene product (pp60^src). The present study shows that dexamethasone completely inhibits the alterations in the pattern of protein synthesis that would normally ensue following glucose deprivation. In fact, synthesis of the 85,000 M_r polypeptide and another polypeptide (69,000 M,) that is not glucose regulated are dramatically increased. In contrast, when L929 cells are maintained in ample glucose the effects of dexamethasone are much less dramatic with only one major SDS polyacrylamide gel band (90,000 M_r) being affected. This effect is specific for glucocorticoids. One-dimensional peptide mapping did not demonstrate any obvious structural relatedness among the 95,000, 90,000, 85,000, and 82,000 M_r polypeptides. Therefore, the observed changes appear to result from alterations in the rate of protein synthesis rather than from changes in precursor-product relationships. It is suggested on the basis of this data that glucocorticoids may play a role in modulating the response of cultured cells to glucose deprivation by eliciting a heat shock-like response.     

Modulation of synthesis of specific proteins in endothelial cells by copper,cadmium, and disulfiram: An early response to an angiogenic inducer of cell migration

Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10^?3 M CuSO₄ or 10^?5 M CdCl₂ four cell-associated polypeptides (M_r = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell-associated protein (M_r = 32,000 and 40,000 daltons) was induced. Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO₄ (10^?6 M) enhanced the synthesis in Type 2 cells of a protein (M_r = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell?associated polypeptides, does not involve a sulphydryl?containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.     

Antagonistic effect of PDGF and NGF on transcription of ribosomal DNA and tumor cell proliferation

The molecular mechanism by which NGF and PDGF affect growth of tumor cells was tested in human melanoma WM 266-4 and colorectal carcinoma SW 707 cell lines. We present evidence that NGF translocated to the nucleus and bound to the chromatin of SW 707 cells, which express the cell surface and the chromatin receptor for NGF, inhibits ribosomal RNA synthesis which in consequence leads to inhibition of cell proliferation. In WM 266-4 cells, which do not express NGF receptor, NGF does not affect cell proliferation. In contrast, PDGF translocated to the nucleus of both SW 707 and WM 266-4 cells activates ribosomal RNA synthesis. We report here that NGF abolishes PDGF-activated ribosomal RNA synthesis and PDGF-stimulated growth of tumor cells.     

Affinity labeling of high-affinity α-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). ¹²⁵I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (K_d ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling ¹²⁵I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of M_r ≈ 150 000 revealed as a ¹²⁵I-α-thrombin cross-linked complex of M_r ≈ 180 000. Independent of chemical cross-linking, ¹²⁵I-α-thrombin also formed a covalent complex with a minor, 35 000 M_r, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of M_r 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 M_r binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.     

Nerve growth factor chromatin receptor and cell surface receptor-regulated growth of melanocytes and nevus cells.

RNA synthesis in melanocytes and nevus cells, and the proliferation of those cells in the presence of nerve growth factor (NGF) and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), were found to correlate with the amount of NGF bound to the chromatin versus the total internalized NGF (n/c NGF = nuclear/cellular ration). In nevus cells and in melanocytes of the early passages (n/c NGF = 0.16-0.18), NGF slightly activated RNA synthesis but was without any effect on cell growth. At passage 5-6 of melanocytes (n/c NGF = 0.88), NGF inhibited RNA synthesis, which led to inhibition of cell growth. Removal of TPA from the culture of nevus cells resulted in increased n/c NGF ratio and in a switch from activatory to inhibitory action of NGF. The possibility that the cell surface receptor mediated the stimulatory effect of NGF and may antagonize the chromatin receptor-mediated inhibitory effect of NGF of melanocyte and nevus cell growth is discussed.     

A selective endothelin ETA antagonist,BQ-123, inhibits125I-endothelin-1 (125I-ET-1) binding to human meningiomas and antagonizes ET-1-induced proliferation of meningioma cells

Summary 1. We studied the effects of BQ-123, a selective ET_A receptor antagonist, on¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding to cell surface receptors in surgically excised human meningiomas and on ET-1-induced DNA synthesis in cultured human meningioma cellsin vitro, using a quantitative receptor autoradiographic technique with radioluminography and³H-thymidine incorporation, respectively.2. All of the human meningiomas expressed high-affinity binding sites for¹²⁵I-ET-1, regardless of differences in histological subtypes (K _d=2.6±0.2 nM,B _max=374±93 fmol/mg; mean ± SE;n=9).3. BQ-123 competed for¹²⁵I-ET-1 binding to sections of meningiomas with IC₅₀s of 3.2±0.9×10^–7 M, and 10^–4 M BQ-123 displaced 80% of the binding.4. ET-1 significantly stimulated DNA synthesis in cultured human meningioma cells, up to 170% of the basal level in the presence of 10^–9 M ET-1. BQ-123 inhibited ET-1 (10^–9 M)-induced DNA synthesis in meningioma cells, in a dose-dependent manner, and 10^–5 M BQ-123 reduced it to 120% of the basal level.5. The number of meningioma cells determined after 4 days in culture was dose dependently increased in the presence of ET-1 (10^–9 and 10^–7 M). The growth rate of meningioma cells, incubated with 10^–9 M ET-1, was reduced by 50% in the presence of 10^–7 M BQ-123.6. Our data suggest that (a) human meningioma cells express a large number of ET_A endothelin receptors, with a small proportion of non-ET_A receptors linked to proliferation of the cells, and (b) ET receptor antagonists, including BQ-123, might prove to be effective treatment for patients with meningioma.     

Demonstration of Type I Insulin-Like Growth Factor Receptors on Human Platelets

Abstract

Human platelets, freshly isolated from healthy human adults, express receptors for insulin-like growth factor I. The IC₅₀ for displacement of ¹²⁵I-IGF-I binding by unlabeled IGF-I was 0.2 nM, by IGF-II 32 nM and by insulin 160 nM. Scatchard analysis of IGF-I binding demonstrates dissociation constants of 0.14 ± 0.08 nM for high affinity binding site and 54 ± 18 nM for low affinty binding site. The presence of the α-subunit of type I IGF receptor, as high affinity binding site, was verified by affinity crosslinking of ¹²⁵I-IGF-I to platelet surface membranes. Under reducing con-conditions a M_r= 135,000 band was preferentially labeled. The complete type I IGF receptor complex, which revealed under nonreducing conditions, has an approximately molecular mass of M_r > 400,000. The immunoprecipitation of the ¹²⁵I-IGF-I cross-linked type I receptor with αIR-3 confirmed the results achieved by affinity crosslinking.     

Phosphorylation of the IgE receptor from ionophore A23187 stimulated intact rat mast cells

Purified rat serosal mast cells were labeled either with [³²P]orthophosphate or [³⁵S]methionine and their receptors for immunoglobulin E were isolated by repetitive affinity chromatography. In ³⁵S-labeled receptor preparations SDS polyacrylamide gels revealed a broad receptor band, M_r 45,000 to 53,000, and two other bands, M_r 30,000 and 16,000, which apparently represent receptor-associated proteins. Only the receptor band was labeled by ³²P. Phosphorylation of receptor was markedly stimulated by the divalent cation ionophore A23187, a known stimulator of histamine release, with changes occuring as early as 15 seconds. This early increase in receptor phosphorylation may be involved in the control of mediator secretion.     

Characterization of the Hepatic Glucagon Receptor

Abstract

The hepatic glucagon receptor was covalently labeled with [¹²⁵I-Tyr¹⁰]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccini-midyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradio-gram of the gel showed one band at M_r=63,000 that was sensitive to excess unlabeled glucagon and GTP. The labeled receptor was solubilized with Lubrol-PX and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are S_{20, w} = 4.3 ± 0.1, Stokes radius = 6.3 ± 0.1 nm, frictional coefficient f/f° = 1.8 and a calculated M_r = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a M_r = 24,000 fragment that binds hormone in a GTP-sensitive manner. The M_r = 24,000 fragment is contained within the M_r = 33,000 fragment. The M_r = 63,000 receptor upon treatment with endo-β-N-acetylglucosamine F for 4 h yields four fragments of apparent M_r = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither M_r = 33,000 and 24,000 fragment appear to be substrates for endo-β-N-acetylglucosaminidase F.

These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of ～ 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only ～ 21,000 daltons that does not contain any N-linked glycans.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Pancreatic CCK receptors: Characterization of covalently labeled subunits

¹²⁵I-CCK was crosslinked with ultraviolet light to its receptor on pancreatic plasma membranes. The predominant labeled species following polyacrylamide gel electrophoresis had a molecular weight of 120,000 in the absence, and 80,000 in the presence of the reducing agent dithiothreitol. The M_r = 120,000 labeled band could be extracted, reduced and converted to M_r = 80,000. Moreover, peptide mapping with Staph aureus V8 protease showed a similar pattern for the 120,000 and 80,000 dalton bands. The crosslinked receptor could be solubilized with Triton X-100, absorbed to wheat germ agglutinin and eluted with N-acetylglucosamine. The results indicate, therefore, that the CCK receptor is a glycoprotein with subunits coupled by disulfide bonds.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Receptors for a cytotoxic lectin, abrin and their role in cell intoxication

The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [¹²⁵I]abrin bound to CHO cells at 37°C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [¹²⁵I]abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3·10³ abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an M_r of about 45 000 may be responsible for the lactose-resistant binding of abrin.     

Identification and characterization of a mouse cell surface antigen with alternative molecular forms

We present the characterization of a new mouse cell surface protein, recognized by the 3138-specific monoclonal antibody. The expression of this antigen is predominantly restricted to the hematopoietic and lymphoid tissues: bone marrow, spleen, lymph node, and thymus. Immunoblot analyses show that the 3138 determinant is present on molecules with different apparent relative masses. The 3138 antigen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of M _r 115 000 for normal nonstimulated spleen cells and thymocytes and as two bands of M _r 115 000 and M _r 125 000 for bone marrow cells and mitogen-stimulated spleen cells. The multiple sizes of the 3138 antigens (isoforms) found on various cell lines are not due to allelic polymorphism, but instead may reflect the specific cell type or reflect the cell's state of activation or maturation. Results from lectin chromatography and N-glycanase and neuraminidase studies suggest that the 3138 antigen is a heavily sialylated O-linked glycoprotein. The unusual features of this antigen indicate that it may be the mouse homologue of the rat W3/13 antigen and the human leukosialin/sialophorin antigens.Abbreviations used in this paper Con A concanavalin A - Gal galactose - Ga1Nac N-acetyl galactosamine - Ig immunoglobulin - IL-2 interleukin 2 - 2-ME 2-mercapto-ethanol - M _r relative mass - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - Th T helper - TX-100 Triton X-100 - TTS 0.3% TX-100, 0.01 M Tris, pH 7.4, 0.15 M NaCl     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Expression of human p140trk receptors in p140trk-deficient,PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140^trk. These biological effects of NGF depend upon the signal-mediating function of p140^trk substrates which are likely to differ from cell to cell. To define p140^trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140^trk cDNA sequence. Two stably transfected clones, one expressing p140^trk with higher affinity (PC12EN-trk3; K_d 57.4 pM, B_max 9.7 pmole/mg) and one expressing p140^trk with a lower affinity (PC12EN-trk1; K_d 392.4 pM, B_max 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140^trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140^trk receptors. NGF stimulates p140^trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70^S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [³H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140^trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140^trk receptor substrates. J. Cell. Biochem. 66:229-244. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.     

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of ¹²⁵I-GM-CSF was incubated. Scatchard analysis of ¹²⁵I-GM-CSF binding to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the β-chain, M_r 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existng specifically on hemopoietic cells.     

Purification and characteristics of DOPA-decarboxylase from the integument of Calliphora vicina larve.

A method is described for the purification to homogeneity of the DOPA-decarboxylase present in the integument of blowfly larve. The enzyme has a M_r of 90,000–96,000 and is composed of two subunits of M_r 50,000 and 46,000. The enzyme decarboxylates DOPA and is practically inactive towards 5-hydroxytryptophan, tryptophan, tyrosine, and phenylalanine. Enzyme activity is enhanced by Al³⁺ and Mn²⁺ and is depressed by Cu²⁺ and Hg²⁺. N-Acetyldopamine is a competitive inhibitor of the decarboxylase.