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Biomedical subjects

N B Lydon

Publications and source records attributed to N B Lydon.

36 records · Page 2Linked to original sources
PubMed1995

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

3T3 Cells↗
PubMed1995

Paeciloquinones A, B, C, D, E and F: new potent inhibitors of protein tyrosine kinases produced by Paecilomyces carneus. I. Taxonomy, fermentation, isolation and biological activity.

Paeciloquinones A to F as well as versiconol have been isolated as inhibitors of protein tyrosine kinase from the culture broth of the fungus Paecilomyces carneus P-177. The novel anthraquinones inhibit epidermal growth factor receptor protein tyrosine kinase in the micromolar range. Two compounds, paeciloquinones A and C, are potent and selective inhibitors of the v-abl protein tyrosine kinase with an IC50 of 0.4 microM. Dependent on the fermentation conditions, partially different sets of paeciloquinones may be produced. An HPLC method allows separation of all major active components.

Animals↗
PubMed1994

Cdc2-mediated modulation of pp60c-src activity.

Following complete dephosphorylation, purified p60c-src was rephosphorylated with Csk. The Csk-phosphorylated form was isolated and found to be essentially inactive. Both the dephosphorylated p60c-src (Src A) and the inactive, phosphorylated pp60c-src (Src B) were then used to explore the regulatory role of other kinases and phosphatases. Phosphorylation by Cdc2 partially reactivated Csk-inactivated pp60c-src. This reactivation occurred in the absence of Tyr-527 dephosphorylation. Moreover, phosphorylation of Csk-treated pp60c-src by Cdc2 also facilitated complete reactivation by the protein-tyrosine phosphatase CD45 or by a synthetic phosphopeptide corresponding to the C-terminal, regulatory phosphorylation site (Tyr-527). These data indicate that the Src homology 2 domain of Csk-phosphorylated pp60c-src was more accessible for intermolecular interactions and that Tyr-527 was more readily dephosphorylated after treatment with Cdc2. In conjunction with in vivo studies, these data suggest that Cdc2 is involved in the regulation of pp60c-src during mitosis.

Amino Acid Sequence↗
PubMed1994

4,5-Dianilinophthalimide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth factor receptor signal transduction pathway and potent in vivo antitumor activity.

Deregulated signal transduction via the epidermal growth factor receptor (EGF-R) family of protein-tyrosine kinase growth factor receptors is associated with proliferative diseases. We describe a class of compounds (4,5-dianilinophthalimides) that inhibit the EGF-R protein-tyrosine kinase in vitro with high selectivity. In cells, 4,5-dianilinophthalmide selectively inhibited both ligand-induced EGF-R and p185c-erbB2 autophosphorylation and c-fos mRNA induction. Antitumor activity could be demonstrated in vivo against xenografts of the A431 and SK-OV-3 tumors, which overexpress the EGF-R and p185c-erbB2, respectively. In contrast, a platelet-derived growth factor-driven tumor was not inhibited by 4,5-dianilinophthalimide, which is compatible with its cellular selectivity and hypothesized mechanism of action. No overt cumulative toxicity was observed during treatment even though high efficacy was observed, indicating a good therapeutic window. 4,5-Dianilinophthalimides may offer therapeutic agents for the treatment of hyperproliferative diseases that overexpress EGF-R family protein-tyrosine kinases or their ligands.

3T3 Cells↗
PubMed1993

Cellular and in vivo characterization of the MCR rat mammary tumor model.

The slowly growing, transplantable MCR-83 rat mammary tumor is estrogen-dependent and non-metastasizing. A rapidly growing, estrogen-independent, metastasizing subline (MCR-86) was subsequently isolated in vivo. We have established and characterized cell lines from both MCR rat mammary tumors. MCR cell lines and tumors were studied in vivo and in vitro. Analysis of DNA from tumors and cell lines showed that mutations had not occurred in codons 12, 13 and 61 of the Ha-ras and Ki-ras genes. Additionally, dominant transforming activity could not be detected by DNA transfection using NIH 3T3 focus-forming assay. No gene amplification was detected for either the EGF-receptor or c-erbB-2 genes. Differences in the tyrosine phosphorylation patterns were found between the 2 MCR cell lines. Addition of serum to starved cells resulted in the tyrosine phosphorylation of a 120-kDa protein, which was elevated in the MCR-86. The lack of ras activation in the MCR tumors differentiates this model from the widely studied, chemically induced rodent mammary tumors. In addition, the differences in the cellular phosphotyrosine patterns between MCR-83 and MCR-86 suggests the occurrence of alterations in signalling pathways that involve tyrosine protein kinases.

3T3 Cells↗
PubMed1992

Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

Amino Acid Sequence↗
PubMed1992

Benzopyranones and benzothiopyranones: a class of tyrosine protein kinase inhibitors with selectivity for the v-abl kinase.

Abelson murine leukemia virus is an acutely transforming replication-defective virus which encodes a transforming protein with tyrosine-specific protein kinase activity. A variety of benzopyranone and benzothiopyranone derivatives have been identified which selectively inhibit the v-abl tyrosine protein kinase with 50% inhibitory concentrations ranging from 1 to 30 microM. The most active derivative inhibited v-abl with a Ki value of 0.9 microM. Active derivatives showed selectivity for the v-abl tyrosine protein kinase relative to the epidermal growth factor receptor tyrosine protein kinase (50% inhibitory concentration greater than 100 microM). Protein kinase C and protein kinase A, two members of the serine/threonine protein kinase family, were not inhibited by benzopyranones or benzothiopyranones (50% inhibitory concentration greater than 100 microM). Kinetically, a representative derivative (compound 2) showed competitivity with respect to ATP and noncompetitive behavior with respect to the exogenous peptide substrate. Autophosphorylation of p120v-abl and recombinant p70v-abl tyrosine protein kinases were also inhibited by benzopyranones and benzothiopyranones in vitro. When tested in Abelson murine leukemia virus-transformed BALB/c cell, active benzopyranone and benzothiopyranone derivatives inhibited tyrosine phosphorylation of cellular proteins by the v-abl tyrosine protein kinase.

Animals↗
PubMed1992

Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.

Angiotensin II↗
PubMed1992

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus.

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.

Alkaloids↗
PubMed1992

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗
PubMed1990

Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.

Animals↗
PubMed1990

An E. coli expression system for the rapid purification and characterization of a v-abl tyrosine protein kinase.

A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S. aureus protein A. pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC. The Km of the pabl HP kinase for ATP varied with [Val5]-angiotensin II concentration and was 21.2 microM at saturating concentrations of [Val5]-angiotensin II. The Km for [Val5]-angiotensin II at saturating concentrations of ATP was 3.8 mM. The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1. Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase. The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides. This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.

Abelson murine leukemia virus↗
PubMed1985

Immunochemical mapping of alpha-2 interferon.

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.

Amino Acids↗
PubMed1981

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF.

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ;burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ;lag' phase with p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.

Adenylyl Cyclases↗