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Biomedical subjects

R A Blake

Publications and source records attributed to R A Blake.

11 recordsLinked to original sources
PubMed2001

Cellular screening assays using fluorescence microscopy.

The recent development of automated fluorescence imaging systems has enabled fluorescence microscopy to be used for the purposes of compound screening. This information-rich technique has found various applications, including screening for the effect of kinase inhibitors on the cytoskeleton, agonist-stimulated receptor internalization, and protein phosphorylation and acetylation. The discovery of novel fluorescent proteins and new fluorescent dyes will find many applications in multichannel fluorescence imaging assays.

Animals↗
PubMed2000

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.

3T3 Cells↗
PubMed2000

SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling.

The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C delta) were Src family substrates whereas others (for example, phospholipase C-gamma) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

3T3 Cells↗
PubMed2000

Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent.

Angiogenesis, or the sprouting of new blood vessels, is a central process in the growth of solid tumors. For many cancers, the extent of vascularization of a tumor is a negative prognostic indicator signifying aggressive disease and increased potential for metastasis. Recent efforts to understand the molecular basis of tumor-associated angiogenesis have identified several potential therapeutic targets, including the receptor tyrosine kinases for the angiogenic factor vascular endothelial growth factor (VEGF). Here we review the approach taken at SUGEN, Inc. to discover and develop small molecule inhibitors of receptor tyrosine kinases as anti-angiogenic agents. We focus on SU5416, a selective inhibitor of VEGF receptors that is currently in clinical development for the treatment of advanced malignancies. Its biochemical, biological and pharmacological properties are reviewed and clinical implications discussed.

Angiogenesis Inhibitors↗
PubMed1999

Src promotes PKCdelta degradation.

Platelet-derived growth factor BB (PDGF) stimulates DNA synthesis through a mechanism that is at least partially dependent upon Src family tyrosine kinases, although the signal transduction pathway downstream of Src is poorly understood. We have studied the signaling between Src and different protein kinase C (PKC) isoforms and its possible role in the regulation of PDGF-stimulated DNA synthesis. We found that Src promoted the tyrosine phosphorylation of PKCdelta, and its subsequent degradation. Enforced expression of PKCdelta inhibited PDGF-stimulated DNA synthesis, whereas expression of PKCalpha and PKCepsilon did not, a finding consistent with a model in which PKCdelta negatively regulates G1-to-S-phase progression. We used mutagenesis to map a critical Src phosphorylation site on PKCdelta to tyrosine 311. A mutant form of PKCdelta in which tyrosine 311 was replaced with phenylalanine (Y311F) was more stable in the presence of Src, suggesting that Src-induced degradation was a direct result of PKCdelta tyrosine phosphorylation. We conclude that PKCdelta is downstream of Src but is unlikely to play a positive role in the signaling pathway by which Src promotes DNA synthesis.

3T3 Cells↗
PubMed1994

Collagen stimulates tyrosine phosphorylation of phospholipase C-gamma 2 but not phospholipase C-gamma 1 in human platelets.

Collagen is an important primary stimulus of platelets during the process of hemostasis. As with many other platelet stimuli, collagen signal transduction involves the hydrolysis of inositol phospholipids; however, the mechanisms which underlies this event is not well understood. Neither the collagen receptor nor the isoform of phospholipase C that is activated have been identified. We report that collagen-activation of platelets induces tyrosine phosphorylation of phospholipase C-gamma 2 but not phospholipase C-gamma 1. We also show that the platelet low affinity Fc receptor (Fc gamma RII), which mediates activation of platelets by immune complexes, and wheat germ agglutinin, which binds non-specifically to glycoprotein, stimulate phospholipase C-gamma 2 tyrosine phosphorylation. In contrast, we could not detect phospholipase C-gamma 2 tyrosine phosphorylation in platelets stimulated by either thrombin or a stable thromboxane A2 analogue, U46619.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Fc gamma receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor.

We report that activation of phospholipase C (PLC) by cross-linking of the platelet low-affinity Fc gamma receptor II (Fc gamma RII) is inhibited by two structurally distinct tyrosine kinase inhibitors, staurosporine and ST271. This contrasts with PLC activation induced by thrombin and U46619, a thromboxane mimetic, whose receptors have seven transmembrane domains characteristic of G-protein coupled receptors. Several proteins undergo phosphorylation on tyrosine on Fc gamma RII cross-linking upstream of protein kinase C (PKC), Ca2+ and aggregation, including the Fc gamma RII itself. The role of Fc gamma RII phosphorylation in the regulation of PLC is discussed.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase C gamma and formation of inositol phosphates.

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

Enzyme Activation↗
PubMed1976

A method of integrating family nursing in an undergraduate curriculum.

The authors describe integrating theoretical content of families into an undergraduate curriculum based on the biopsychosocial spheres. Using audiovisual materials, and printed media, the student is introduced to independent learning and applies content to clinical settings in the community.

Audiovisual Aids↗