The three-substituted indolinone cyclin-dependent kinase 2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) kills human leukemia cells via down-regulation of Mcl-1 through a transcriptional mechanism.

Abstract

Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations > or =5 microM rapidly (i.e., within 4 h) induced cytochrome c release, Bax mitochondrial translocation, and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction 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, cotreatment with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) 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 small interfering RNA potentiated SU9516 lethality, confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production, which was diminished, along with cell death, by the free radical scavenger N-acetylcysteine (NAC). We were surprised to find that 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. Together, 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.

• Authors: Ning Gao, Lora Kramer, Mohamed Rahmani, Paul Dent, Steven Grant
• Journal: Molecular pharmacology (Mol Pharmacol) Vol. 70 Issue 2 Pg. 645-55 (Aug 2006) ISSN: 0026-895X [Print] United States
• PMID: 16672643 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Imidazoles
• Indoles
• Myeloid Cell Leukemia Sequence 1 Protein
• Neoplasm Proteins
• Protein Kinase Inhibitors
• Proto-Oncogene Proteins c-bcl-2
• Reactive Oxygen Species
• SU 9516
• Cyclin-Dependent Kinase 2
• RNA Polymerase II
• Caspases

Topics

• Apoptosis (drug effects)
• Caspases (physiology)
• Cyclin-Dependent Kinase 2 (antagonists & inhibitors)
• Down-Regulation
• Gene Expression Regulation (drug effects)
• Humans
• Imidazoles (pharmacology)
• Indoles (pharmacology)
• Mitochondria (drug effects)
• Myeloid Cell Leukemia Sequence 1 Protein
• Neoplasm Proteins (antagonists & inhibitors, genetics)
• Phosphorylation
• Protein Kinase Inhibitors (pharmacology)
• Protein Processing, Post-Translational
• Proto-Oncogene Proteins c-bcl-2 (antagonists & inhibitors, genetics)
• RNA Polymerase II (metabolism)
• Reactive Oxygen Species (metabolism)
• Transcription, Genetic (drug effects)
• U937 Cells