GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Inhibitors of histone deacetylase (HDAC) block cell cycle progression at Gl in many cell types. Weinvestigated the mechanism by which trichostatin A (TSA), a specific inhibitor of HDAC, induces Gl arrest in human cervix carcinoma HeLa cells. TSAtreatment induced histone hyperacetylation followed by growth arrest in Gl as well as hypophosphorylation of pRb. The Cdk4 kinase activity was essentially unchanged during the TSA-induced Gl arrest. On the other hand, the arrest was accompanied by down-regulation of kinase activity of Cdk2, although the total protein levels of Cdk2 and its activator Cdc25A were unaffected. Upon TSA treatment, amounts of cyclin E and the CDKinhibitor p2lWAF1/QP1were markedly increased, while that of cyclin A was reduced. The induction of p21 and down-regulation of cyclin A correlated well with the decreased Cdk2 activity and cell cycle arrest. Furthermore, gel filtration chromatography showed the association of p21 with the cyclin E-Cdk2 complex, suggesting that the activation of Cdk2 by the enhanced expression of cyclin E is blocked by the increased p21. The elevated expression of p21 is also observed in cells treated with trapoxin and FR901228, structurally unrelated histone deacetylase inhibitors.A human colorectal carcinoma cell line lacking both alleles ofthep21 gene (p21 -/-) was resistant to TSAseveral times more than the parental line (p21 +/+). These results suggest that the suppression of Cdk2 kinase activity due to p21 overexpression play a critical role in HDACinhibitor-induced growth inhibition.

Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a noncompetitive inhibitor of ATP citrate lyase with K i values for citrate and ATP of 13 and 7 M, respectively.Radicicol (also known as monorden), a 14-membered macrolide originally isolated from Monosporium bonorden as an antifungal antibiotic in 1953 (1), is a compound showing a variety of biological activities. It was reported again as a potent tranquilizer with low toxicity in 1964 (2). We rediscovered radicicol as a potent inducer of reversal of the transformed phenotype in v-src-transformed fibroblasts to the normal one (3, 4). We also showed that radicicol caused cell cycle arrest in G 1 and G 2 phases, and Oikawa et al. (5) demonstrated that it inhibited in vivo angiogenesis. Furthermore, radicicol was reported to induce morphological reversion of not only src but also ras, mos, raf, fos, and SV40-transformed cell lines and inhibited the expression of mitogen-inducible cyclooxygenase in macrophages (6 -8). Some of leukemia cell lines were differentiated in response to radicicol (4, 9). Recently, KF25706, a novel oxime derivative of radicicol, was reported to show potent antitumor activity and is currently under consideration as an anticancer drug (10). The in vivo inhibition of tyrosine kinases and MAP kinases has been suggested to be involved in these characteristic phenotypes elicited by radicicol (6, 7). Increased expression of gelsolin, an actin regulatory protein, has also been observed during the induction of morphological changes in various transformed cells (11). Recent studies showed that radicicol disrupted the Ras-activated signaling pathway by selectively depleting Raf kinase or reducing Ras/Raf molecular interaction (12, 13). The target molecule of radicicol was proposed to be Hsp90, because it strongly binds Hsp90 in a manner competitive with ATP and...

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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-rasbinding 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.The ras proto-oncogene is a central component of mitogenic signal-transduction pathways, and is essential for cells to progress the cell cycle through a quiescent kinase, is part of a highly conserved kinase cascade that mediates signaling from extracellular growth factors to mitogen-activated protein kinases. Raf-1 functions downstream of Ras, which in its active, GTP-bound state binds directly to the N-terminal regulatory domain of Raf-15). This interaction serves to recruit Raf-1 to the cell membrane, which is necessary for Raf-1 activation6,7). Thus, activation of Raf-1 requires direct interaction with Ras. It is therefore likely that a specific inhibitor of Ras/Raf binding would be a promising chemotherapeutic agent against Ras-activated cancers.To identify such inhibitors, we employed the yeast two-hybrid system8), since it seems to be a powerful strategy for identifying not only a protein that is physically associated with a certain target protein but also an agent that blocks protein-protein interaction. In this system, the interaction of Ras and Raf-1 can be monitored by transcriptional activation of two reporter genes, HIS3 and lacZ9). We screened a variety of microbial metabolites for inhibitors blocking Ras/Raf interaction by this system, and identified radicicol (Fig.