ag-213 and Lung-Neoplasms

We describe nonspecific and moderate activation of cellular protein tyrosine phosphorylation by a chemical compound, vesnarinone, which results in enhanced synthesis and/or assembly of cytoskeletal proteins and morphological alterations in several transformed cells. In A431 cells, vesnarinone induced tyrosine phosphorylation of the overexpressed epidermal growth factor receptors (EGFR) as well as other unidentified proteins, increased the synthesis of cytokeratins, and caused amplification of the intermediate filament networks and cell flattening. The drug effects were abolished by tyrphostin, a protein tyrosine kinase inhibitor. Two other cell lines responded to the drug with increased synthesis of a cell type-specific cytoskeletal protein: vimentin in QG56 human lung carcinoma cells and alpha-tubulin in NIH3T3 cells transformed with v-src. In all cell lines tested, the drug-induced tyrosine phosphorylation was localized in cell-cell and cell-substrate contacts as detected by immunofluorescent staining. Responsive protein substrates and their sensitivity to the drug varied from one cell line to another as observed by immunoblot analysis. Vesnarinone exerted neither activating nor inhibitory effect on in vitro enzyme reactions including EGFR tyrosine kinase, v-src kinase, and protein tyrosine phosphatases. This suggests that vesnarinone indirectly activates tyrosine phosphorylation of membrane proteins related to cell adhesion, which influences a signaling pathway linked to the stress fiber assembly in certain cell lines. The possible mechanism by which vesnarinone induces the cellular responses is discussed.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Carcinoma, Squamous Cell; Cardiotonic Agents; Catechols; Cell Communication; Cell Line; Cell Line, Transformed; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Genes, src; Humans; Immunoblotting; Lung Neoplasms; Mice; Nitriles; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Pyrazines; Quinolines; Tubulin; Tumor Cells, Cultured; Tyrosine; Tyrphostins; Vimentin

Inhibition of growth factor-stimulated DNA synthesis carried out in defined medium is often compared with inhibition of serum-stimulated DNA synthesis so as to assess the selectivity of growth-factor-receptor tyrosine kinase inhibitors such as tyrphostins. We investigated whether protein binding may influence the interpretation of these experiments. Protein binding of tyrphostins was determined by ultrafiltration, equilibrium dialysis or spectrophotometer, and was quantitated by high-performance liquid chromatography (HPLC). For growth factor-stimulated DNA synthesis, we used the non-small-cell lung cancer cell line L23/P stimulated by transforming growth factor alpha (TGF alpha). The epidermal growth factor (EGF)-receptor kinase was assayed by phosphorylation of a peptide substrate or by receptor autophosphorylation. Protein binding of a number of tyrphostins ranged from 64% to 98%. There was a positive correlation (r = 0.995) between the degree of protein binding and the hydrophobicity. Inhibition of the EGF-receptor tyrosine kinase activity by the highly protein-bound tyrphostin B56 [N-(4-phenylbutyl)-3,4-dihydroxybenzylidene cyanoacet-amide] was reduced by bovine serum albumin (BSA), but BSA had less of an effect on inhibition of the EGF-receptor kinase by the weakly protein-bound tyrphostin A47 (RG 50864: 3,4-dihydroxybenzylidene cyanothioacetamide). Tyrphostins B46 [N-(3-phenylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both highly protein-bound) inhibited DNA synthesis of L23/P cells with approximately 3-fold greater potency in 0.5% serum than in 10% serum, but the inhibition of DNA synthesis in 0.5% serum was reduced by the addition of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated by TGF alpha in defined medium to a greater extent than DNA synthesis stimulated by serum. However, this apparent selectivity for inhibition of TGF alpha-stimulated DNA synthesis was lost when the protein concentration in the defined medium was made equivalent to that in the serum-containing medium. By contrast, BSA enhanced the selective inhibition of TGF alpha-stimulated DNA synthesis by tyrphostin A47. These results demonstrate that protein binding accounts for the apparent selectivity of some highly protein-bound tyrphostins for TGF alpha-stimulated DNA synthesis of L23/P cells. Therefore, protein binding should be taken into consideration in assessments of the selectivity of tyrphostins.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Catechols; Chromatography, High Pressure Liquid; DNA, Neoplasm; ErbB Receptors; Growth Inhibitors; Humans; Lung Neoplasms; Nitriles; Phosphorylation; Protein Binding; Protein-Tyrosine Kinases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins