Effects of plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases on the proliferation of human hematopoietic cells

The frequent dysregulation of cell cycle and/or apoptosis in cancer has stimulated the search for new chemical molecules targeting events in these biochemical pathways. Therefore, CDK activity has been targeted for drug discovery and a number of small molecules have now been identified as CDK inhibitors. The first compound identified as a (nonspecific) CDK1 inhibitor was N 6-isopentenyladenine, a natural plant cytokinin or phytohormone. Structural analogy searches led to the development of olomoucine and roscovitine; both are 2,6,9-trisubstituted purines with potent, specific and selective inhibition of CDK1/CDK2 activity. Since then, cytokinin analogues have been synthesized and are the subject of this study.; More than 20 cytokinin analogues were synthesized and a majority of them has inhibitory activity on CDK1 and CDK2. In addition, the biological effects on proliferation of hematopoietic cells was studied. It was shown that a significant correlation exists between the inhibition of CDK activity and the anti proliferative effect on myeloid and lymphoblastic cell lines and on primary human lymphocytes. Furthermore, IC₅₀ value (concentration where 50% of the cell proliferation is inhibited) and AC₅₀ value (concentration where 50% of the cell population is apoptotic) were in the same order of magnitude, indicating that the main mechanism of the anti proliferative effect of these cytokinin analogues is the induction of apoptosis. Cell cycle distribution studies showed preferential, but not exclusive, induction of apoptosis in specific phases of the cell cycle, rather than a cell cycle phase arrest. In a further study, the mechanisms of cytokinin analogue-induced apoptosis were characterized using a new multiparametric flow cytometric analysis. The data indicate that treatment with cytokinin analogues results in a rapid decrease of mitochondrial transmembrane potential which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases. This indicates that cytokinin analogues, with inhibitory activity on CDK, induce apoptosis through the mitochondrial pathway. The expression of Bcl-2 family members (Bcl-2 and Bax) was examined in malignant hematopoietic cell lines after treatment with cytokinin analogues. Intracellular flow cytometric detection of both proteins revealed that CDK inhibitors do not influence the expression level of these Bcl-2 family members. We also investigated the influence of cytokinin analogues on the progression of cells through the cell cycle. When these cells were synchronized either at the G₁/S (by hydroxyurea) or G₂/M (by nocodazole) boundaries of the cell cycle, addition of a cytokinin analogue markedly, delayed but did not block progression through the cell cycle. (Abstract shortened by UMI.)...