Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.

Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.

The antiproliferative effects of tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in vitro and in nude mice.

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Inhibition of breast cancer cell growth in vitro by a tyrosine kinase inhibitor.

Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of breast cancer.

Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis.

Previous studies have shown that alpha-adrenergic activation reduces myocardial damages caused by ischemia/reperfusion. However, the molecular mechanisms of how alpha-adrenergic activation protects the myocardium are not completely understood. The objective of this study was to test the hypothesis that alpha-adrenergic activation protects the myocardium by, at least in part, inhibiting apoptosis in cardiomyocytes. The current data has shown that apoptosis in neonatal rat cardiomyocytes, induced by 24 h treatment with hypoxia (95% N2 and 5% CO2) and serum deprivation, was inhibited by co-treatment with phenylephrine. Pre-treatment with phenylephrine for 24 h also protected cardiomyocytes against subsequent 24 h treatment with hypoxia and serum deprivation. Exposure of cardiomyocytes to phenylephrine for up to 9 days under normoxic conditions did not cause apoptosis. The phenylephrine-mediated cytoprotection was blocked by an alpha-adrenergic antagonist, phentolamine. beta-adrenergic activation with isoproterenol did not protect cardiomyocytes against hypoxia and serum deprivation-induced apoptosis. Under hypoxic conditions, phenylephrine prevented the down-regulation of Bcl-2 and Bcl-X mRNA/protein and induced hypertrophic growth. Phenylephrine-mediated protection was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin and was mimicked by the caspase-9 peptidic inhibitor LEHD-fmk. These results suggest that alpha-adrenergic activation protects cardiomyocytes against hypoxia and serum deprivation-induced apoptosis through regulating the expression of mitochondrion-associated apoptosis regulatory genes, preventing activation of mitochondrial damage-induced apoptosis pathway (cytochrome C-caspase-9), and activating hypertrophic growth.

Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes. We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote. Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S. cerevisiae from the yeast ADHI promoter. The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K. pneumoniae. Estimates of Kp2 antigen and assays of beta-galactosidase activity specified by nifH'-'lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains. A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K. pneumoniae is not translated in yeast.

A new series of PDGF receptor tyrosine kinase inhibitors: 3-substituted quinoline derivatives.

A series of 63 3-substituted quinoline derivatives has been prepared and tested for inhibition of cell-free platelet derived growth factor receptor tyrosine kinase (PDGF-RTK) activity. The compounds were generally prepared either by a Friedlander condensation between an aryl-acetaldehyde and an o-aminobenzaldehyde or by a palladium-catalyzed coupling between an aryl bromide or triflate and an organostannane or organozinc chloride. The presence of 6,7-dimethoxy groups on the quinoline ring was found to be advantageous although not essential for potent inhibition of PDGF-RTK. A lipophilic group attached to the quinoline 3-position contributed substantially to activity. The lipophilic groups generally consisted of monocyclic aromatics or small alkynyl, alkenyl, and alkyl groups. Optimum activity of ca. < or = 20 nM (IC50) was observed when 6,7-dimethoxyquinoline was substituted in the 3-position with 4-methoxyphenyl (15d), 3-fluoro-4-methoxyphenyl (17m), 3-fluorophenyl (17b), 4-hydroxyphenyl (24), 6-methoxypyridin-3-yl (15o), 5-pyridin-2(1H)-one (23), trans-beta-styryl (15e), thiophene-3-yl (2e), 5-chlorothiophene-2-yl (15f), or cyclopentenyl (17n) groups. Most of the compounds in the series were tested for inhibition of cell-free epidermal growth factor receptor tyrosine kinase activity and found to be inactive.

Anionic- and lipophilic-mediated surface binding inhibitors of human leukocyte elastase.

We report the synthesis of a series of diphenylmethane-based oligomers containing anionic and lipophilic functionalities that are potent inhibitors of human leukocyte elastase (HLE). The enzyme inhibition is regulated by the size of the oligomer, as well as, the number of charges. Lipophilicity is an important element in determining potency and specificity against other basic enzymes. Compounds whose scaffolds contain three phenoxyacetic acid groups and three alkyl ethers are competitive and specific inhibitors of HLE with Ki = 20 nM. The mechanism of action of this class of compounds is believed to involve multidendate interactions with the surface of HLE near the active site which prevents substrate access to the catalytic site.

Inhibition kinetics and selectivity of the tyrosine kinase inhibitor erbstatin and a pyridone-based analogue.

The inhibition mechanisms of the epidermal growth factor (EGF) receptor tyrosine kinase and the cAMP-dependent kinase activities by erbstatin and its analogue, RG 14921, were studied by kinetic analysis. Both compounds were slow-binding inhibitors of the EGF receptor kinase. Erbstatin inhibited the EGF receptor kinase as a partial competitive inhibitor with respect to both ATP and the peptide substrate, suggesting that it binds at a site distinct from the ATP and peptide binding sites of the enzyme, and thus lowers the binding affinities of the enzyme for both substrates. In contrast, the analogue RG 14921 inhibited EGF receptor kinase activity as a non-competitive inhibitor with respect to both ATP and the peptide substrate. The distinct modes of inhibition by structurally related compounds suggest a dynamic and possibly extended structure of the catalytic center of the kinase domain of the receptor. Erbstatin and RG 14921 exerted similar effects on cAMP-dependent protein kinase activity. In this system, both compounds displayed potent inhibition and acted by a mode of competitive inhibition with respect to ATP and non-competitive with the peptide substrate.

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells.

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Digestion of delta-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC.

The present study describes the correlation between gut protease activity of lepidopteran larvae of different instars, the inactivation of Bacillus thuringiensis delta-endotoxins in crystalline and noncrystalline forms, and the reduced susceptibility of advanced larval instars of Spodoptera littoralis to the toxin. The original assembly of delta-endotoxins in a crystal structure is essential for causing efficient larval mortality. Denaturation and renaturation (D/R) of delta-endotoxin crystals increased the vulnerability of the toxin molecules to proteolysis, reduced their capability to kill neonate larvae of S. littoralis, but sustained most of their larval growth-inhibition activity. E. coli-produced CryIC delta-endotoxin applied as a fraction of inclusion bodies exerted a growth inhibition effect, similar to the molecules released from the crystals by denaturation and subsequent renaturation. Incubation of CryIC with gut juice of 1st or 2nd instar larvae, left part of the CryIC toxin intact, while the toxin was completely degraded when incubated with gut juice of 5th instar larvae. The degradation rate was consistent with the increase of protease specific activity of the gut juice during larval development. This increase in toxin degradation may account for the loss of sensitivity of 5th instar larvae to CryIC. Specific protease inhibitors such as PMSF and Leupeptin were shown to inhibit gut proteases activity in all instar larvae, while, 1,10 phenanthroline, TLCK and TPCK were effective only in young instar larvae. The differential effect of protease inhibitors on proteases obtained from different larval instars indicated that gut juice protease profiles change with larval age. The observed quantitative and qualitative differences in degradation of delta-endotoxin by larval gut proteases that occur during larval maturation may account for the difference in susceptibility to the delta-endotoxin. This finding should be taken into consideration when designing strategies for the development of transgenic crops expressing delta-endotoxins as potent insecticidal proteins.

Elucidation of Septoria tritici x Wheat Interactions Using GUS-Expressing Isolates.

ABSTRACT Isolate ISR398 of Septoria tritici (which produces none to few pycnidia on the wheat cv. Seri 82 and high coverage on cv. Shafir) and isolate ISR8036 (which is virulent on both cultivars) were genetically cotrans-formed using the selectable marker gene hph, which confers resistance to hygromycin B (hygB), and the reporter gene uidA, encoding beta-glucuronidase (GUS). Most of the genetically transformed isolates (98.8%) produced similar pycnidial coverage on seedlings of 'Seri 82' and 'Shafir' as the two wild-type isolates. Southern analysis of 25 randomly selected hygB(R)GUS(+) transformants probed with the uidA sequence revealed multiple insertion sites. GUS activity was determined fluorimetrically by measuring the conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone (MU). The high GUS-expressing transformants 398D97 and 8036E27 were used to elucidate fungal development within inoculated leaf tissue by using GUS activity to estimate the fungal proteins content in planta. Increase in fungal biomass was recorded in 'Shafir' inoculated with the GUS-expressing transformants 398D97 and 8036E27 following a 12-day latent period. A 15-day latent period was recorded in 'Seri 82' inoculated with 8036E27, whereas an 18-day latent period was recorded on 'Seri 82' inoculated with 398D97 and the two mixtures 398D97 + ISR8036 and ISR398 + 8036E27. The rate of fungal development and the estimated level of fungal proteins at the pycnidia maturation stage was high in leaves of 'Shafir' and moderate to low on 'Seri 82', even in cases in which no significant differences were recorded in pycnidial coverage. An endogenous capacity to hydrolyze beta-1,4-D-glucuronidase was recorded in leaves inoculated with wild-type isolates. The latent periods in MU production of the uidA-expressing transformants mimicked those recorded for the wild-type isolates. However, at all stages, the levels of MU produced in wheat inoculated with wild-type isolates were markedly lower than those produced by GUS-expressing transformants. The mode of interaction (compatible or incompatible) determined the onset of the induction, rate, and level of enzyme production.