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A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.

Activation of cytoplasmic serine/threonine kinase Raf-1, an important effector of Ras, requires direct binding to Ras. The yeast two-hybrid screening system used for identification of inhibitors of Ras/Raf-1 interaction showed radicicol to be an inhibitor. Radicicol has been shown to induce morphological reversion of transformed cells. Immunoprecipitation with an anti-Ras antibody revealed that the in vivo Ras/Raf-1 binding in v-Ha-ras-transformed cells was also blocked by low concentrations of radicicol (0.1 approximately 1 microg/ml), while degradation of Raf-1 was induced at concentrations higher than 2 microg/ml. However, in vitro binding of glutathion S-transferase-fused Ras to a maltose binding protein-fused RIP3 containing the Ras-binding domain (RBD) of Raf-1 was not inhibited by radicicol. Similar two-hybrid assays with several truncated forms of Raf-1 showed that both the conserved serine/threonine-rich domain (CR2) and the C-terminal protein kinase domain (CR3) were required for the full inhibition by radicicol. These results suggest that radicicol interacts directly or indirectly with the region except with RBD of Raf-1, thereby inhibiting a conformational change of Raf-1 prerequisite for binding to Ras.

Radicicol, an inhibitor of Src-family protein-tyrosine kinases, causes morphological reversion of v-src- and v-Ha-ras-transformed fibroblasts and arrest of the cell cycle at both the G1 and the G2 phases. Radicicol was found to inhibit the growth of several other oncogene-transformed cell lines and human carcinoma cell lines and to revert their cell morphology to be flat. In the radicicol-treated flat cells, actin stress fiber bundles were reorganized. Since this effect of radicicol on these cell lines was inhibited by cycloheximide, de novo protein synthesis is required for the morphological reversion. Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis suggested that the amount of gelsolin, an actin regulatory protein, was distinctly increased upon radicicol treatment. Western blot and Northern blot analyses showed that radicicol enhanced transcription of the gelsolin gene in human carcinoma cell lines, as a result of which the amount of gelsolin was increased several folds. Injection with an anti-gelsolin antibody into cells and successive treatment with radicicol resulted in approximately 80% reduction of the number of flat cells with stress fibers in comparison with controls treated with an irrelevant antibody. These results show that elevated expression of gelsolin is associated, at least in part, with the suppression of transformation and the restoration of actin stress fibers in human carcinoma cells by radicicol.

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: a constitutively expressed COX-1 and mitogen-inducible COX-2, which is selectively expressed in response to various inflammatory stimuli. Thus, COX-2 instead of COX-1 is implicated to produce prostanoids mediating inflammatory responses. Major efforts have been focused on identifying nonsteroidal anti-inflammatory drugs (NSAIDS) which can selectively inhibit the enzyme activity of COX-2. Such NSAIDS would be more desirable anti-inflammatory agents in comparison to NSAIDS which inhibit both COX-1 and COX-2. Other than glucocorticoids, pharmacological agents which can selectively suppress the expression of COX-2 without affecting that of COX-1 have not been identified. We report here that radicicol, a fungal antibiotic, is a potent protein tyrosine kinase inhibitor, and that it inhibits the expression of COX-2 without affecting COX-1 expression in lipopolysaccharide (LPS)-stimulated macrophages with the IC50 value of 27 nM. Radicicol inhibited tyrosine phosphorylation of p53/56lyn, a Src family tyrosine kinase and one of the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol also inhibited COX-2 expression in vivo in glomeruli of rats with experimental glomerulonephritis induced by the anti-glomerular basement membrane antibodies, in which COX-2 expression is known to be enhanced. The enzyme activity of COX-1 or COX-2 was not affected by radicicol in macrophages. Radiciciol also suppressed the COX-2 expression induced by IL-1 beta in rat smooth muscle cells. Other protein tyrosine kinase inhibitors suppressed the LPS-induced COX-2 expression in macrophages but at much higher concentrations than needed for radicicol. Radicicol did not inhibit the COX-2 expression induced by phorbol 12-myristate 13-acetate in macrophages. These results suggest that the activation of tyrosine-specific protein kinases is the proximal obligatory step in the LPS-induced signal transduction pathway leading to the induction of COX-2 expression in macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. These results suggest that radicicol inhibits COX-2 expression mainly at post-transcriptional steps.

Radicicol, an inhibitor of src or src-like kinases, causes hypophosphorylation and down-regulation of the RB retinoblastoma tumor suppressor protein in HL-60 human promyelocytic leukemia cells. Both of these changes in the RB protein typically are associated with myeloid or monocytic differentiation of these cells induced by retinoic acid or 1,25-dihydroxy vitamin D3. When added with either inducer, radicicol caused the typically induced myeloid or monocytic differentiation of these cells to be accelerated. By itself radicicol caused a transient inhibition of G1 to S transit, but did not cause phenotypic conversion. The down-regulation and dephosphorylation of RB by radicicol may thus facilitate cell differentiation.