Biomedical subjects

H S Earp

Publications and source records attributed to H S Earp.

At least 73 records · Page 4Linked to original sources

PubMed1986

Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors.

A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.

Dexamethasone↗

PubMed1986

Epidermal growth factor (EGF) stimulates EGF receptor synthesis.

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.

Animals↗

PubMed1986

The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells.

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.

Alkaline Phosphatase↗

PubMed1986

Lipoprotein modulation of the intracellular localization of protein kinase C and alteration of phorbol ester-stimulated differentiation in the human monoblastic U937 cell line.

The subcellular localization of protein kinase C and the ability of phorbol esters to alter cell phenotype were examined in the U937 monoblastic cell line. Protein kinase C activity was evaluated using an in vitro assay measuring histone phosphorylation in the cytosolic and detergent extracted particulate fractions obtained after disrupting cells that had been cultured previously under varying conditions. Depriving cells of serum for 2-3 days resulted in a time-dependent decrease in protein kinase C activity of the particulate fraction. The addition of as little as 0.5-1% fetal bovine serum to serum-deprived cells increased protein kinase C in the particulate fraction by up to 2- to 3-fold. In contrast lipoprotein-deficient serum did not mimic the effect of whole serum. However addition of high or low density lipoproteins to cells grown in lipoprotein-deficient serum or serum-free medium produced a concentration-dependent 2- to 3-fold increase in particulate protein C kinase activity. The maximal lipoprotein effect was similar to that observed with 5% fetal bovine serum and the concentrations of lipoproteins needed to increase protein kinase C activity were in the physiological range. Adherence to plastic was used as a marker of the differentiated phenotype. Cells cultured in lipoprotein-deficient serum did not differentiate in response to phorbol ester stimulation as well as cells cultured in 5% fetal bovine serum. These results suggest that serum lipoproteins modulate protein kinase C localization and the response to phorbol ester stimulation in the U937 cell.

Blood Physiological Phenomena↗

PubMed1985

Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes.

Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 degrees C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.

B-Lymphocytes↗

PubMed1985

Gradation of carcinogen-induced capacity for anchorage-independent growth in cultured rat liver epithelial cells.

The effect of epidermal growth factor (EGF) on the capacity for anchorage-independent growth of chemically treated rat hepatic epithelial cells has been investigated. We have performed the studies using 16 clonally derived cell strains which represented single cell-derived subpopulations of a heterogeneous rat hepatic epithelial cell line that had been tumorigenically transformed by 11 repeated treatments with N-methyl-N'-nitro-N-nitrosoguanidine. The results can be summarized as follows. (a) Secondary clonal subpopulations isolated from the colonies formed by these strains in soft agar subsequently and invariably acquired markedly enhanced colony-forming efficiencies as compared to their parental strains. (b) EGF could enhance or induce colony-forming ability in soft agar in all of these cell strains. (c) The magnitudes of enhancement of the colony-forming efficiencies by EGF in soft agar could not be correlated with the absolute EGF-binding capacity of these cell strains. (d) The enhancement or induction of the colony-forming ability by EGF was either reversible or irreversible, partially correlating with the expression of gamma-glutamyl transpeptidase activity by the strains. These findings indicate that the cellular capacity of liver epithelial cells to grow anchorage independently in soft agar medium can be graded according to the pattern of response of EGF induction of colony-forming ability. These grades may reflect the level of neoplastic transformation of these cells. Moreover during the multistep transformation of rat hepatic epithelial cells by a chemical carcinogen, EGF can be used to reveal the presence of altered cells which have acquired partial capacity for the anchor-age-independent growth property. This property may constitute an additional identifiable early step of the neoplastic transformation of these cells.

Animals↗

PubMed1984

Dimethyl sulfoxide decreases specific EGF binding.

Dimethyl sulfoxide (DMSO) stimulates tyrosine phosphorylation of the hepatic EGF receptor in isolated membrane preparations. To determine whether DMSO affects EGF binding, primary cultures of rat hepatocytes were incubated with 1-10% DMSO for 30 min prior to the addition of 125I-EGF. DMSO (1-2%) reduced specific 125I-EGF binding; the effect was maximal (a 40-60% reduction) at 5-7.5% DMSO and was reversed by removing the DMSO. Scatchard analysis showed that the reduction in binding was due to a change in receptor affinity. The decrease in binding was not seen when other, slightly less polar, solvents (eg, acetone and ethanol) were tested. DMSO also reduced 125I-EGF binding to purified rat liver plasma membranes. This reduction was seen in the absence of added ATP and in membranes that had been pretreated with TLCK, a tyrosine kinase inhibitor. Thus, completion of the receptor autophosphorylation reaction was not necessary to effect the change. The data are consistent with a DMSO-induced alteration of receptor conformation that reversibly reduces receptor affinity.

Animals↗

PubMed1984

Insulin inhibits the glucocorticoid-mediated increase in hepatocyte EGF binding.

Hydrocortisone and dexamethasone produced a time-dependent increase [125I]epidermal growth factor [( 125I]EGF) binding in primary cultures of isolated rat hepatocytes. Maximally effective doses of glucocorticoids resulted in a 70-100% increase in binding. The effect was similar when hepatocytes were maintained on collagen-coated plates or directly on culture dishes. The glucocorticoid-mediated increase in [125I]EGF binding could be detected after 4 h exposure to glucocorticoid and was substantial by 8 h. The major effect of glucocorticoid appeared to be to increase the number of EGF receptors. While insulin (100 nM) had no effect on basal [125I]EGF binding, it significantly inhibited the increase produced by the glucocorticoid. Since the inhibitory effect of insulin was only observed when insulin was added with the inducing glucocorticoid, insulin appears to inhibit an early hydrocortisone-mediated event.

Animals↗

PubMed1984

Phosphorylation of the hepatic EGF receptor with cAMP-dependent protein kinase.

The 170 000 dalton hepatic epidermal growth factor (EGF) receptor is phosphorylated on serine and tyrosine residues. The evidence indicates that distinct protein kinases are involved. Since EGF and agents that elevate cAMP are believed to participate in the regulation of liver regeneration, we tested whether or not the catalytic subunit of cAMP-dependent protein kinase (catalytic subunit), a known serine kinase, would utilize the EGF receptor as a substrate. The catalytic subunit increased phosphorylation of the EGF receptor in purified rat liver plasma membranes. The serine specificity of the catalytic subunit was established by phosphoamino acid analysis of electrophoretically purified EGF receptor. The result was confirmed by catalytic subunit phosphorylation of affinity purified preparations of the EGF receptor. The rates of dephosphorylation of the membrane-associated EGF receptor phosphorylated on different residues were compared. Dephosphorylation of serine residues (after catalytic subunit phosphorylation) was considerably slower (t1/2 greater than 120 sec) than the removal of phosphotyrosine after stimulation with EGF (t1/2 less than 30 sec).

Animals↗

PubMed1984

Membranes from T and B lymphocytes have different patterns of tyrosine phosphorylation.

Membrane fractions isolated from mouse and rat spleen expressed substantial tyrosine-specific protein kinase activity. Phosphotyrosine (P-Tyr) accumulation in endogenous membrane substrates was stimulated by vanadate or nonionic detergents. When in vitro phosphorylation was carried out at 0 degree C in the presence of 1 mM Mn2+ and Triton X-100, P-Tyr constituted up to 40-50% of the total phospho amino acid. Polyacrylamide gel electrophoresis showed that membranes from mixed lymphocyte populations have four major P-Tyr-containing proteins. Whereas nonionic detergents were potent stimuli for P-Tyr accumulation in all four substrates, tyrosine phosphorylation of two of these (p61 and p55) was markedly dependent on vanadate. These two substrates were present in membranes from surface Ig-bearing splenic lymphocytes purified by affinity chromatography and Raji, a human B-lymphoblastoid cell line. P-Tyr accumulation in the two other substrates observed in splenocyte membranes (p64 and p58) was much less dependent on vanadate. p64 and p58 were phosphorylated in membranes from mouse thymocytes and human and mouse T-lymphoma cell lines, while p61 and p55 were not. Thus it appears that in both murine and human lymphocytes, p64 and p58 served as T-cell-specific substrates, while p61 and p55 were specifically associated with B lymphocytes. Moreover, these distinct P-Tyr substrate patterns were conserved in some neoplastic cell lines derived from B and T cells.

Animals↗

PubMed1984

Interferon inhibits agonist-induced cyclic AMP accumulation in human lymphocytes.

Wheezing episodes often accompany viral respiratory infections. Viral pathogens are also known to induce interferon production. Cyclic AMP (cAMP) levels affect bronchial reactivity; therefore, we analyzed cAMP accumulation in human lymphocytes as a model to determine if interferon might influence the accumulation of this important intracellular mediator. After an overnight exposure to interferon in whole blood, a reduction in agonist-stimulated cAMP accumulation was demonstrated. Basal cAMP concentrations were equivalent in control and interferon-exposed lymphocytes from the same donor, but the response to isoproterenol and prostaglandin E1 was significantly reduced by 61 and 30%, respectively. The decreased responsiveness persisted in the presence of RO 20-1724, a phosphodiesterase inhibitor, indicating that the effect was in part due to a decrease in cAMP synthesis in intact cells. These results suggest that interferon may play a role in inducing or potentiating bronchospasm.

Cyclic AMP↗

PubMed1983

Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes.

A membrane fraction from Raji human lymphoblastoid cells exhibited tyrosine-specific kinase activity. Vanadate increased tyrosine phosphorylation up to 5-fold; serine and threonine phosphorylation were unchanged. The stimulation was detectable within 15 s at 0 degrees C and at concentrations of vanadate (0.3 and 1.0 microM) present in normal tissues and blood. The tyrosine phosphorylation of two substrates, M1 61 000 and 55 000, was dependent upon vanadate and incorporation into these substrates represented the majority of the vanadate-sensitive tyrosine phosphorylation.

Burkitt Lymphoma↗

PubMed1983

Somatomedin-C stimulates the phosphorylation of the beta-subunit of its own receptor.

Phosphorylation of the somatomedin-C receptor was investigated both in intact IM-9 cells and in IM-9 cells that had been solubilized with Triton X-100. Intact IM-9 cells were incubated with [32P]H3PO4 for 1 h and for an additional 5 min in the absence or presence of insulin or somatomedin-C. The cells were then solubilized and subjected to wheat germ agglutinin Sepharose chromatography. The extent of phosphorylation of insulin and somatomedin-C receptors was assessed by immunoprecipitating the wheat germ agglutinin Sepharose eluates with monoclonal antibodies specific for each receptor and analyzing the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The beta-subunits of both receptors were phosphorylated in the absence of hormone, and the extent of phosphorylation of each receptor was enhanced by both hormones. However, each hormone was more potent than the other in enhancing phosphorylation of its own receptor. The beta-subunit of the somatomedin-C receptor was also phosphorylated when solubilized IM-9 cells that had been purified on wheat germ agglutinin Sepharose were incubated with [gamma-32P]ATP. In this soluble preparation, phosphorylation occurred on tyrosyl residues and was enhanced by concentrations of somatomedin-C in the range of 2.5 to 250 ng/ml, which is consistent with its receptor affinity. Tyrosyl phosphorylation of the somatomedin-C receptor also occurred when highly purified receptor, prepared by wheat germ agglutinin Sepharose affinity chromatography followed by immunoprecipitation, was incubated with [gamma-32P]ATP. This indicates that the responsible tyrosyl kinase activity is intrinsic to the receptor or tightly associated with it.

Cell Line↗

PubMed1983

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells.

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.

Animals↗

PubMed1983

Solubilization of EGF receptor with Triton X-100 alters stimulation of tyrosine residue phosphorylation by EGF and dimethyl sulfoxide.

In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation.

Animals↗

PubMed1983

Dimethyl sulfoxide stimulates tyrosine residue phosphorylation of rat liver epidermal growth factor receptor.

Epidermal growth factor, a potent mitogen, stimulates phosphorylation of its 170,000-dalton plasma membrane receptor. Dimethyl sulfoxide selectively increased phosphorylation of the epidermal growth factor receptor in rat liver microsomal fraction. Maximal stimulation occurred at 15 to 25 percent dimethyl sulfoxide and resembled the effect of epidermal growth factor in magnitude and rapidity. Like epidermal growth factor, dimethyl sulfoxide selectively stimulated tyrosine residue phosphorylation of this protein.

Animals↗

PubMed1982

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells.

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of medium supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin., The addition of cholera toxin plus 30 microM isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1982

Alteration of epidermal growth factor-dependent phosphorylation during rat liver regeneration.

Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.

Animals↗