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.
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.
Vermeulen
Katrien
K
Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Edegem, Belgium.
Strnad
Miroslav
M
Havlícek
Libor
L
Van Onckelen
Harry
H
Lenjou
Marc
M
Nijs
Griet
G
Van Bockstaele
Dirk R
DR
Berneman
Zwi N
ZN
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
D017304:Annexin A5; D003583:Cytokinins; D010936:Plant Extracts; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases