Received for publication March 16, 2006.
Revised May 3, 2006.
Accepted for publication May 3, 2006.

The 3-substitute indolinone CDK2 inhibitor SU-9516 kills human leukemia cells via downregulation of Mcl-1 through a transcriptional mechanism

Abstract

Mechanisms of lethality of the 3-substituted indolinone and putatively selective CDK2 inhibitor SU9516 were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations 5 µM rapidly (i.e., within 4 hr) induced cytochrome c release, Bax mitochondrial translocation, and apoptosis in association with pronounced down-regulation of the anti-apoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II on serine 2 but not serine 5. RT-PCR analysis revealed pronounced down-regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore, co-treatement with the proteasome inhibitor MG-132 blocked SU9516-mediated Mcl-1 down-regulation, implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome c release, Bax translocation, and apoptosis, whereas knockdown of Mcl-1 by siRNA potentiated SU9516 lethality, confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. Notably, SU9516 treatment resulted in a marked increase in ROS production, which was diminished, along with cell death, by the free radical scavenger NAC. Unexpectedly, NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2, reductions in Mcl-1 mRNA levels, and Mcl-1 down-regulation. Collectively, these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level, culminating in mitochondrial injury and cell death.

Key words: Oxidative stress/antioxidants, Mechanisms of cell killing/apoptosis

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