cytochrome-c-t and Blast-Crisis

The Bcr-Abl fusion gene encodes for the p210(Bcr-Abl) or p185(Bcr-Abl) tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia, respectively. Because Bcr-Abl TK is chaperoned by Hsp90 (90 kDa heat-shock protein), we investigated the effects of novobiocin (NB), an Hsp90 C-terminal inhibitor, on the viability of the Bcr-Abl-positive human leukemia cells HL-60/Bcr-Abl and K562, the expression of Bcr-Abl protein and the interaction between Hsp90 and Bcr-Abl TK. Present studies demonstrate that NB is a potent inhibitor of the growth of Bcr-Abl-positive human leukemia cells. NB induces cytosolic accumulation of cytochrome c and activation of caspase-9 and caspase-3, triggering apoptosis of HL-60/Bcr-Abl and K562 cells. Treatment of cell lines with NB disrupts Bcr-Abl /Hsp90 and Bcr-Abl /Hsp70 interactions, resulting in a decreased amount of intracellular Bcr-Abl protein levels. Co-treatment with the proteasome inhibitor N-acetyl leucyl-leucyl norlucinal increases NB-mediated accumulation of Bcr-Abl in the detergent-insoluble cellular fraction, which demonstrates that NB promotes proteasomal degradation of Bcr-Abl. Moreover, both imatinib-resistant K562/G01 and primary CML CD34(+) cells are sensitive to NB.

Topics: Apoptosis; Benzamides; Benzoquinones; Blast Crisis; Caspase 3; Caspase 9; Cell Proliferation; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Fusion Proteins, bcr-abl; HL-60 Cells; HSP90 Heat-Shock Proteins; Humans; Imatinib Mesylate; Immunoprecipitation; K562 Cells; Lactams, Macrocyclic; Leukemia; Novobiocin; Piperazines; Proteasome Endopeptidase Complex; Pyrimidines

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blast Crisis; Blotting, Western; Butyrates; Caspases; Chromatin Immunoprecipitation; Cyclin-Dependent Kinases; Cytochromes c; Down-Regulation; Drug Interactions; Flavonoids; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Membrane Potential, Mitochondrial; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Piperidines; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Stem Cell Assay; U937 Cells; Vorinostat; X-Linked Inhibitor of Apoptosis Protein