Multiple Pathways of Apoptosis Induced by Roscovitine in Leukemic Cell Lines in vitro



Hairong Song1, Åke Sidén 2, Zuzana Hassan 1, 3, *
1 Experimental Cancer Medicine, Clinical Research Center, Novum, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden
2 >Department of Neurology, Karolinska University Hospital, Huddinge, 14186, Stockholm, Sweden
3 >Center for allogeneic stem cell transplantation, Karolinska University Hospital, Huddinge, 14186, Stockholm, Sweden


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© 2008 Song et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Experimental Cancer Medicine, Clinical Research Center, Novum, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; Tel: +46-8-58583856; Fax: +46-8-58583850; E-mail: Zuzana.Hassan@ki.se


Abstract

Roscovitine is a potent inhibitor of cyclin-dependent kinases (CDKs) that competes with the ATP binding pocket of kinases. Roscovitine has been shown to have cytotoxic effect on cancer cell lines in vitro and also in tumor xenografts in vivo. A strong synergistic effect in combination with conventional cytostatics has been reported in cancer cell lines in vitro. In this study, the mechanisms of roscovitine- induced cell death were investigated in human leukemic cell lines HL-60, Jurkat and K562. Cells were incubated with roscovitine (0.5-200 􀀁mol/L) up to 24 hours and cell viability and proliferation were studied using resazurin and 3H-thymidine incorporation assays, respectively. Cell cycle and mitochondrial membrane potential were analyzed using flow cytometry, apoptosis was assessed using morphological criteria in Giemsa staining and apoptotic pathways using Western blot analysis. Both viability and proliferation were inhibited in a concentration-dependent manner in all cell lines. Estimated IC50 was 17, 24 and 47 􀀁mol/L for HL-60, Jurkat and K562, respectively. Loss of mitochondrial membrane potential, release of cytochrome c, active fragment of caspase-3 and cleaved PARP were observed in all three cell lines. The cleaved fragments of caspase-2 and -8 were observed in HL-60 and Jurkat cells and the order of appearance differed between these two cell lines, while none of these fragments was observed in K562 cells. Thus, roscovitine is a potent inducer of apoptosis in leukemic cells and apoptosis has been mediated through different pathways depending on the cell line.

Keywords: Roscovitine, CDKI, apoptosis, cell cycle, leukemia.