title: Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: determination by a multiparametric flow cytometric analysis.
abstract: OBJECTIVE
  Regulation of the cell cycle by cyclin-dependent kinase (CDK) activity occurs at multiple levels and is often altered in human cancers. Therefore, CDK activity has been targeted for drug discovery, and a number of small molecules have now been identified as CDK inhibitors. Plant cytokinin analogues with CDK inhibitory activity and antiproliferative effects were studied to characterize the cellular basis of the cytotoxic effect.
  
  
  METHODS
  The IC(50) value (concentration at which 50% of the cell proliferation is inhibited) and AC(50) value (concentration at which 50% of the cell population is apoptotic) were determined by flow cytometry and microscopy, respectively. A new multiparametric flow cytometric analysis was used to study the sequence of different apoptotic events. In this assay, analysis of phosphatidylserine exposure, mitochondrial membrane depolarization, activation of caspases and DNA condensation were combined.
  
  
  RESULTS
  Treatment of Jurkat and KG1 cells with the CDK inhibitors results in a decrease of viable cells and a parallel increase in percentage of apoptotic cells. Apoptosis was accompanied by a rapid decrease of mitochondrial membrane potential, which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases.
  
  
  CONCLUSIONS
  The main cellular mechanism of the antiproliferative effect of plant cytokinin analogues with CDK inhibitory activity is the induction of apoptosis. The multiparametric flow cytometric technique allowed to follow the kinetics of various aspects of apoptotic cell changes and demonstrated that cytokinin analogue-induced apoptosis starts through the mitochondrial pathway. This technique could also become of value for the rapid screening of pro-apoptotic properties of chemotherapeutic compounds.
authors.affiliation: Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Edegem, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Vermeulen
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.forename: Libor
authors.initials: L
authors.lastname: Havlícek
authors.forename: Harry
authors.initials: H
authors.lastname: Van Onckelen
authors.forename: Marc
authors.initials: M
authors.lastname: Lenjou
authors.forename: Griet
authors.initials: G
authors.lastname: Nijs
authors.forename: Dirk R
authors.initials: DR
authors.lastname: Van Bockstaele
authors.forename: Zwi N
authors.initials: ZN
authors.lastname: Berneman
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D003583:Cytokinins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D019169:Jurkat Cells; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D010936:Plant Extracts / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D017304:Annexin A5; D003583:Cytokinins; D010936:Plant Extracts; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases