Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro.

Catecholamine treatment of isolated rat adipocytes decreases insulin binding and inhibits insulin stimulation of the glucose-transport system. There is increasing evidence that the insulin signal is transmitted after insulin is bound to the receptor via a tyrosine kinase, which is an intrinsic part of the receptor. To find whether the receptor kinase is modified by catecholamines, we solubilized and partially purified the insulin receptor of isoprenaline-treated adipocytes and studied the effect of insulin on its kinase activity. (1) Insulin increased the tyrosine autophosphorylation of the insulin receptor kinase from catecholamine-treated cells only 4-fold, compared with a 12-fold stimulation in control cells. (2) The rate of insulin-stimulated 32P incorporation into the receptor of isoprenaline-treated cells at non-saturating [32P]ATP concentrations (5 muM) was decreased to 5-8% of the values for receptor from control cells. (3) 125I-insulin binding to the partially purified receptor from catecholamine-treated cells was also markedly decreased. The insulin receptor from catecholamine treated cells bound 25-50% of the amount of insulin bound by the receptor from control cells at insulin concentrations of 10 pM-0.1 muM. Part of the impaired insulin-responsiveness of the receptor kinase of catecholamine-treated cells is therefore explained by impaired binding properties; however, an additional inhibition of the kinase activity of the insulin receptor from catecholamine-treated cells is evident. (4) This inhibition of kinase activity decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis revealed that the Km for ATP of the receptor kinase from isoprenaline-treated cells was increased to approx. 100 muM, compared with approx. 25 muM for receptor of control cells. (5) We conclude from the data that catecholamine treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of the mechanism leading to insulin-resistance in these cells.     

Inhibition of the biologic response to insulin-like growth factor I in MCF-7 breast cancer cells by a new monoclonal antibody to the insulin-like growth factor-I receptor. The importance of receptor down-regulation.

We developed a mouse monoclonal antibody (4G11) against insulin-like growth factor I receptor by immunizing mice with mouse embryo fibroblasts overexpressing the human insulin-like growth factor-I receptor. Not only did the 4G11 antibody inhibit the binding of [ (125)I]insulin-like growth factor-I to the fibroblast receptor, but 4G11 antibody also potently down-regulated the insulin-like growth factor-I receptor. 4G11 Fab fragment inhibited ligand binding, but did not down-regulate the receptor, suggesting that receptor aggregation is required for down-regulation. 4G11 antibody also down-regulated the receptor in MCF-7 breast cancer cells, a panel of colon cancer cells and MG-63 osteosarcoma cells. Receptor recovery in MCF-7 cells after down-regulation by 4G11 antibody was slow, requiring 32 - 48 h for full recovery. Receptor down-regulation in MCF-7 cells by 4G11 antibody was confirmed by FACS analysis of intact and permeabilized cells. In contrast to 4G11 antibody, insulin-like growth factor-I did not down-regulate the receptor in MCF-7 cells. Down-regulation of the receptor by 4G11 antibody in MCF-7 cells resulted in inhibition of Akt and MAPK activation by insulin-like growth factor-I. We conclude that the ability of a monoclonal antibody to down-regulate the receptor may be an important antibody property in targeting the insulin-like growth factor-I receptor for the treatment of certain cancers.     

Analysis of the role of glycosylation of the human fibronectin receptor

1-Deoxymannojirimycin (MNJ), an inhibitor of Golgi alpha-mannosidase IA and IB, was used to assess the possible roles of asparagine-linked oligosaccharides in the structure and function of the integrin fibronectin receptor from cultured human fibroblasts. These cells normally attach well to fibronectin substrates and have only mature forms of the fibronectin receptor on their surfaces. MNJ inhibits the intracellular trimming of high mannose oligosaccharides, and cells treated with 0.2 mg/ml MNJ synthesize only immature precursor forms of both the alpha and beta subunits of the fibronectin receptor. The immature receptor polypeptides were found to be nonfunctional by two criteria: 1) cells treated with MNJ attached poorly to fibronectin substrates; and 2) receptor from the treated cells was defective in binding to fibronectin affinity columns. The precursor forms of the fibronectin receptor subunits were found on the surfaces of cells treated with MNJ, demonstrating that processing of receptor carbohydrates to mature forms was not necessary for receptor insertion into the plasma membrane. A monoclonal antibody that specifically bound the alpha subunit of the fibronectin receptor immunoprecipitated both alpha and beta subunit polypeptides from both control cells and cells treated with MNJ. Similarly, a monoclonal antibody that specifically bound only the beta subunit also immunoprecipitated both alpha and beta subunit polypeptides of the receptor from extracts of both control and MNJ-treated cells. These results indicate that receptor assembly can occur in the absence of complete oligosaccharide processing. Thus, oligosaccharide processing to the mature form of the fibronectin receptor is important for its binding function but not for receptor assembly or insertion into the plasma membrane.     

Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses.

Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage.     

Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause.

To obtain more insight into the relationship between cyclic and regional changes in steroid receptor expression and function-related changes in the various types of cell of the normal human uterus, we performed an immunocytochemical study on paraffin-embedded sections. The distribution and intensity of immunostaining for the oestrogen receptor and the progesterone receptor in the various types of cell were semiquantitatively scored. The data were statistically compared for the different phases of the menstrual cycle and after the menopause, and for the different regions of the corpus and (endo)cervix uteri. During the menstrual cycle, significant changes in oestrogen receptor score were observed in glandular and stromal cells of endometrium basalis and functionalis and in smooth muscle cells of the myometrium. In all types of cell, oestrogen receptor expression reached a maximum in the late proliferative phase. During the early secretory phase, oestrogen receptor staining declined sharply in stromal and smooth muscle cells, whereas, in glandular epithelium, oestrogen receptor expression decreased more gradually. During mid- and late-secretory phases, an increase in oestrogen receptor staining was also observed in predecidualizing stromal cells and smooth muscle cells. Progesterone receptor numbers changed significantly in glandular epithelium but not in stromal and smooth muscle cells. Glandular progesterone receptor expression reached a maximum in the early secretory phase and was then drastically reduced. During mid- and late-secretory phases stromal cells were moderately stained for progesterone receptor in contrast to epithelial gland cells which showed no or very weak staining. No regional variations in steroid receptor distribution in endometrium and myometrium were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase.

The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.     

Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone

We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.     

Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation

Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor beta with Src, activates the Src/Raf-1/Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar findings for oestradiol receptor alpha were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK(-) abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor alpha or beta is detected using glutathione S:-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor alpha and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor alpha with Src and consequent activation of Src in intact Cos cells.     

Expression of low density lipoprotein receptor in cultured human granulosa cells: regulation by human chorionic gonadotropin, cyclic AMP, and sterol

Steroidogenic cells utilize lipoprotein-delivered cholesterol as a primary substrate for hormone synthesis. We studied low density lipoprotein (LDL) receptors in cultured human granulosa cells to determine what factors regulate receptor expression. Granulosa cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) for 1.5 to 14 hr. The LDL receptor content of cells increased by approximately twofold within 6 hr of hCG treatment, and the content continued to increase for at least 14 hr, as determined by immunoblotting. The rate of LDL receptor synthesis was also demonstrated to increase within 2.5 to 3.5 hr of hCG treatment by immunoisolation of LDL receptor from cells metabolically labeled with a pulse of [35S]methionine. The cyclic AMP analogue, 8-bromo-cAMP, was also found to increase LDL receptor synthesis. This increased rate of synthesis was shown to be dependent on ongoing RNA synthesis, since actinomycin D abolished hCG- or 8-bromo-cAMP-stimulated LDL receptor synthesis. We also demonstrated that hCG- and 8-bromo-cAMP-mediated regulation of LDL receptor synthesis in granulosa cells supersedes the classical cholesterol-mediated regulation of the receptor described in fibroblasts. Although 25-hydroxycholesterol induced a decrease in LDL receptor content and synthesis within 6 hr, this action was overridden by simultaneous exposure to hCG. Our findings demonstrate the existence of a novel cAMP-mediated mechanism for regulation of LDL receptor synthesis in steroidogenic cells.     

Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.     

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort     

Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.     

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The migratory behavior of avian embryonic cells does not require phosphorylation of the fibronectin-receptor complex

When locomotory embryonic cells become stationary, they acquire new substratum-adhesion properties. In particular, the distribution of fibronectin receptors shifts from diffuse and highly mobile on the cell membrane to immobilized in close association with fibronectin molecules and cytoskeletal elements in focal contacts. Receptor phosphorylation has been proposed as a possible regulator of the interaction between the receptor and its intracellular and extracellular ligands. In the present study, we have compared the phosphorylation state of the fibronectin receptor in motile neural crest and somitic cells, in stationary somitic cells, and in Rous-sarcoma virus transformed-chick embryo fibroblasts, using immunoprecipitation following metabolic labeling. While no receptor phosphorylation was detected in motile embryonic cells, the beta subunit of the receptor was phosphorylated in stationary cells. This subunit was also highly phosphorylated in Rous-sarcoma virus-transformed chicken cells. These results suggest that phosphorylation of the fibronectin receptor cannot account for its distribution in the cell membrane and for the nature of the interactions between this receptor and its ligands in embryonic cells.