ag-213 and erbstatin

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial d

Topics: Alkaline Phosphatase; Caco-2 Cells; Catechols; Cell Differentiation; Cell Division; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Humans; Hydroquinones; Microvilli; Nitriles; Phosphorylation; Protein-Tyrosine Kinases; Second Messenger Systems; Tyrosine; Tyrphostins; Vanadates

The role of tyrosine kinase(s) in the regulation of cGMP accumulation in rat pinealocytes was investigated using three tyrosine kinase inhibitors. Treatment with genistein, erbstatin or the active analogues of tyrphostin selectively increased basal cGMP accumulation in a dose-dependent manner without a concomitant increase in cAMP. In contrast to the norepinephrine- and vasoactive intestinal peptide-stimulated cGMP responses, the stimulatory effect of genistein was not blocked by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Furthermore, in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, neither genistein nor erbstatin had an effect on cGMP accumulation. These results indicate that tyrosine kinase inhibitors increase pineal cGMP accumulation through inhibition of the metabolism of cGMP.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Oxidoreductases; Animals; Arginine; Catechols; Cyclic AMP; Cyclic GMP; Genistein; Hydroquinones; Isoflavones; Isoproterenol; Male; Nitric Oxide Synthase; Nitriles; Norepinephrine; omega-N-Methylarginine; Pineal Gland; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Tyrphostins

1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.6. Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in beta-cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Analysis of Variance; Animals; Benzoquinones; Calcium; Catechols; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Epinephrine; Female; Genistein; Hydroquinones; Inositol Phosphates; Insulin; Insulin Secretion; Islets of Langerhans; Isoflavones; Lactams, Macrocyclic; Mice; Nitriles; Patch-Clamp Techniques; Potassium Channels; Protein-Tyrosine Kinases; Quinones; Rifabutin; Rubidium; Tyrphostins

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

Topics: Amino Acid Sequence; Animals; Apoptosis; Binding Sites; Catechols; Cell Line; Cricetinae; Genistein; Hydroquinones; Isoflavones; Mice; Molecular Sequence Data; Nitriles; Protein-Tyrosine Kinases; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Tyrphostins

We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N-cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein-dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.

Topics: 3T3 Cells; Animals; Calcium Channels; Catechols; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Cerebellum; Cholera Toxin; Genistein; GTP-Binding Proteins; Hydroquinones; Integrins; Isoflavones; Leukocyte L1 Antigen Complex; Mice; Neurites; Neurons; Nitriles; Phenols; Potassium Chloride; Protein-Tyrosine Kinases; Rats; Tyrphostins

1. The differential effects of inhibitors of protein kinase (PK) or tyrosine kinase (TK) on phosphatidylcholine (PC) biosynthesis in monocyte-like U937 cells were compared in pulse-chase-studies in which the cells prelabelled with [3H]choline for 30 min were chased in the absence or presence of kinase inhibitors. 2. PKA inhibitor (H-89) decreased the label incorporation into PC, while PKA activator (8-BrcAMP) had no effect. 3. PKC inhibitors (chelerythrine and hypericin) inhibited PC biosynthesis; on the other hand, PKC activator (SC-10) was stimulatory. 4. The inhibition of PC biosynthesis by H-89 and chelerythrine was accompanied by the inactivation of CTP: cholinephosphate cytidylyltransferase (CT). 5. In contrast, TK inhibitor (genistein) markedly stimulated CT and PC biosynthesis, while erbstatin and tyrphostin No. 25 showed no effect.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Catechols; Cyclic AMP-Dependent Protein Kinases; Genistein; Humans; Hydroquinones; Isoflavones; Isoquinolines; Monocytes; Nitriles; Phosphatidylcholines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Sulfonamides; Tumor Cells, Cultured; Tyrphostins

The kinetics of inhibition of the epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase (TK) activity by erbstatin, tyrphostins, and lavendustin derivatives were studied in a system that employs poly(Glu6Ala3Tyr) (GAT) and ATP as substrates, after preactivation with EGF. All data were analyzed for computer best-fit curves by a program that was written for this purpose and is available upon request to those interested. The inhibition kinetics followed a sequential, Bi-Bi, rapid equilibrium, random mechanism, the mechanism of the EGFR-TK. Erbstatin and a few tyrphostins that contain a 3,4-dihydroxy-(cis)-cinnamonitrile [1-(3',4'-dihydroxyphenyl)-2-nitriloethene] group were found to be pure competitive inhibitors with respect to both substrates of the kinase reaction, i.e., GAT and ATP. Two tyrphostins, each containing an additional dihydroxyphenyl group in the alpha-position, were found to be pure competitive inhibitors with respect to GAT and noncompetitive (or mixed-competitive) inhibitors with respect to ATP. A lavendustin derivative with a 2,5-dihydroxyphenyl ring and a lavendustin derivative with a 3,4-dihydroyphenyl ring were also found to be competitive inhibitors with respect to both ATP and GAT. Various possible modes of binding at the EGFR-TK active center for the tyrphostins studied are proposed and the significance of the present findings, as well as the interpretations of computer analyses of kinetic data, is discussed.

Topics: Catechols; ErbB Receptors; Humans; Hydroquinones; In Vitro Techniques; Kinetics; Nitriles; Receptor Protein-Tyrosine Kinases; Software; Substrate Specificity; Tyrphostins

Tyrosine kinase inhibitors have been widely used to probe the role of tyrosine phosphorylation in cellular signalling. These inhibitors exhibit an apparent specificity for tyrosine kinases over the serine/threonine kinases but little is known about their effects on other enzymes or biological systems. We demonstrate that genistein, erbstatin and alpha-cyanocinnamamides (tyrphostins) have inhibitory effects on fatty acid synthesis, lactate transport, mitochondrial oxidative phosphorylation and aldehyde dehydrogenase. We propose, therefore, that results obtained using tyrosine kinase inhibitors should be interpreted with caution, particularly if used at concentrations sufficient to inhibit these non-protein kinase-dependent events.

Topics: Aldehyde Dehydrogenase; Animals; Biological Transport; Catechols; Fatty Acids; Genistein; Hydroquinones; Isoflavones; Lactates; Mitochondria, Liver; Nitriles; Oxidative Phosphorylation; Protein-Tyrosine Kinases; Pyruvates; Rats; Rats, Wistar; Tyrphostins; Uncoupling Agents