The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. essential for the induction of aggregation. mitogen-activated protein kinase (MAPK) activation (Miranti Dunnett’s two-sided test was used for statistical evaluation of differences, as shown in legends to each physique. Cytofluorometric analysis Expression of cell surface molecules on U937 cells was determined by flow cytometry according to standard protocols. MTT assay mogroside IIIe (colorimetric assay) for measurement of cell viability Cell viability was measured by standard MTT assay. MTT answer (10 for 5 min at 4C. The supernatant was further centrifuged at 100,000 for 25 min at 4C to obtain the cytosolic fraction. The pellet was resuspended in 300 for 10 mogroside IIIe min at 4C. The supernatant was collected and used as membrane fraction. The protein concentration of each sample was determined by the Bradford method and 2 Laemmli sample buffer was added before boiling for 5 min. For preparing whole-cell lysates, cells (5 106 cells ml?1) were washed three times in cold PBS containing 1 mM sodium orthovanadate, and lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mogroside IIIe mM EDTA, 2 mM EGTA, 50 mM for 10 min at 4C. Western blotting Samples made up of equal amounts (40 or 80 (% and and (Cho Dunnett’s, Dunnett’s, Dunnett’s, protein synthesis, while staurosporine does not. PMA-induced aggregation was also blocked by antibodies to CD18 (Ikewaki and and novel isoforms by staurosporine has been reported previously (Kiley em et al /em ., 1992; Jones em et al /em ., 1997), mogroside IIIe but the activation of the conventional forms has not been reported, and may be a particular property of the cell line used in this study. Inhibitors showing selectivity for either conventional or novel PKC isoforms, both blocked staurosporine-induced aggregation, suggesting either that both classes of enzyme were required for aggregation or that this inhibitors were not selective under the conditions used in these assays. In addition to inducing translocation of PKCs, staurosporine induced rapid phosphorylation of the MAP kinase ERK, and a somewhat slower activation of p38. Furthermore, inhibitors of both these kinase pathways blocked aggregation, albeit partially. Inhibitors of both ERK and PKC blocked aggregation most effectively when added prior to staurosporine, and lost activity if added afterwards. ERK and PKC may therefore be required for the inductive phase of staurosporine-dependent aggregation, while protein tyrosine kinases may be involved in later phases, since aggregation remains sensitive to genistein up to 1 1.5 h after staurosporine addition. In conclusion, staurosporine is usually a potent inducer of homotypic aggregation in the promonocyte cell line, U937, which is a widely used model of myeloid cell function. The activity of the compound does not appear directly related to its known activity as a kinase inhibitor. The PROCR proaggregating activity is also not simply related to the agonistic activity of staurosporine on PKCs. Rather, staurosporine induces a rapid and nonspecific activation of multiple kinase pathways in U937 cells. This uncoordinated activation of several key signaling pathways may result in changes in cell surface properties of the cells, leading to their aggregation. The molecular basis of this staurosporine activity requires further study, and may provide useful information for the further rational design of staurosporine analogues for use in cancer. Abbreviations ATCCAmerican tissue culture collectionBSAbovine serum albuminERKextracellular-signal-regulated kinaseFCSfetal calf serumGF109203X((2-[1-(3-dimethylaminopropyl)-indol-3-yl-3-(indol-3-yl)]-maleimide)G?6976(12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5 em H /em -indolo(2,3-apyrrolo(3,4- em c /em )-carbazole)H-7(1-(5-isoquinolinesulfonyl)-2-methylpiperazine)H-89( em N /em -[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline)ICAMintercellular adhesion moleculeJNKc-JUN N-terminal kinaseK-252b(C26H19N3O5, CAS [99570-78-2]KT5720[9 em R /em ,10 em S /em ,12 em S /em ]-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1 em H /em -diindolo[1,2,3- em fg /em :3,2,1- em kl /em ]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid hexyl ester)MAPKmitogen-activated protein kinaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideODoptical densityPD98059(2-amino-3-methoxyflavone)PKCprotein kinase CPMAphorbol 12-myristate 13-acetatePMSFphenylmethylsulfonyl fluorideSB203580(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 em H /em -imidazole).
