phosphothreonine and alvocidib

Survivin is a member of the inhibitor of apoptosis gene family that is expressed in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Here, exposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells to anticancer agents, including Adriamycin, Taxol, or UVB resulted in a 4-5-fold increased survivin expression. Changes in survivin levels after anticancer treatment did not involve modulation of survivin mRNA expression and were independent of de novo gene transcription. Conversely, inhibition of survivin phosphorylation on Thr(34) by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, and nonphosphorylatable survivin Thr(34)-->Ala exhibited accelerated clearance as compared with wild-type survivin. Sequential ablation of survivin phosphorylation on Thr(34) enhanced tumor cell apoptosis induced by anticancer agents independently of p53 and suppressed tumor growth without toxicity in a breast cancer xenograft model in vivo. These data suggest that Thr(34) phosphorylation critically regulates survivin levels in tumor cells and that sequential ablation of p34(cdc2) kinase activity may remove the survivin viability checkpoint and enhance apoptosis in tumor cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Doxorubicin; Female; Flavonoids; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Paclitaxel; Phosphorylation; Phosphothreonine; Piperidines; RNA, Messenger; Survivin; Tumor Cells, Cultured; Ultraviolet Rays

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.

Topics: Adenosine Triphosphate; Alkaloids; Alzheimer Disease; Animals; Antibiotics, Antineoplastic; Calcium-Calmodulin-Dependent Protein Kinases; CDC2 Protein Kinase; Cyclin B; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Dopamine and cAMP-Regulated Phosphoprotein 32; Drugs, Chinese Herbal; Enzyme Inhibitors; Flavonoids; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Indoles; Inhibitory Concentration 50; Mice; Molecular Structure; Neostriatum; Nerve Tissue Proteins; Phosphoproteins; Phosphorylation; Phosphothreonine; Piperidines; Staurosporine; tau Proteins