Abstract
Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 micromol/L (95% confidence interval [CI] +/-0.31), 0.18 micromol/L (95% CI +/-0.04), and 0.16 micromol/L (95% CI +/-0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 micromol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol's dependence on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.
MeSH terms
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Animals
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Antineoplastic Agents / administration & dosage
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Antineoplastic Agents / pharmacology*
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Apoptosis / drug effects*
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Caspase 3
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Caspases / physiology*
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Cell Cycle Proteins*
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Chromosomes, Human, Pair 17 / genetics
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Cyclin-Dependent Kinase Inhibitor p27
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Drug Administration Schedule
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Drug Resistance, Neoplasm
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Drug Screening Assays, Antitumor
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Enzyme Activation / drug effects
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Enzyme Precursors / metabolism*
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Flavonoids / administration & dosage
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Flavonoids / pharmacology*
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Gene Deletion
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Genes, p53
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Growth Inhibitors / administration & dosage
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Growth Inhibitors / pharmacology*
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Humans
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In Situ Hybridization, Fluorescence
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Interleukin-4 / pharmacology
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Leukemia, Lymphocytic, Chronic, B-Cell / drug therapy
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Leukemia, Lymphocytic, Chronic, B-Cell / enzymology
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Leukemia, Lymphocytic, Chronic, B-Cell / pathology*
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Microtubule-Associated Proteins / biosynthesis
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Microtubule-Associated Proteins / genetics
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Neoplasm Proteins / biosynthesis
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Neoplasm Proteins / genetics
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Neoplasm Proteins / physiology*
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Piperidines / administration & dosage
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Piperidines / pharmacology*
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Proto-Oncogene Proteins / biosynthesis
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins c-bcl-2 / biosynthesis
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / physiology*
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Tumor Cells, Cultured
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Tumor Suppressor Protein p53 / physiology*
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Tumor Suppressor Proteins*
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Vidarabine / analogs & derivatives
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Vidarabine / pharmacology
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bcl-2-Associated X Protein
Substances
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Antineoplastic Agents
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Cdkn1b protein, mouse
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Cell Cycle Proteins
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Enzyme Precursors
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Flavonoids
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Growth Inhibitors
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Microtubule-Associated Proteins
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Neoplasm Proteins
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Piperidines
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Tumor Suppressor Protein p53
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Tumor Suppressor Proteins
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bcl-2-Associated X Protein
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Cyclin-Dependent Kinase Inhibitor p27
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Interleukin-4
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alvocidib
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CASP3 protein, human
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Casp3 protein, mouse
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Caspase 3
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Caspases
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Vidarabine
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fludarabine