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Departments of ¹ Medicine, ² Biochemistry, and ³ Pharmacology, Virginia Commonwealth University/Medical College of Virginia, Richmond, Virginia

Requests for reprints: Steven Grant, Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298. Phone: 804-828-5211; Fax: 804-828-8079. E-mail: stgrant{at}hsc.vcu.edu

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 were examined in human multiple myeloma cells. Whereas individual treatment of U266 myeloma cells with 10 µmol/L HA14-1 or 100 nmol/L flavopiridol had little effect, exposure of cells to flavopiridol (6 hours) followed by HA14-1 (18 hours) resulted in a striking increase in mitochondrial dysfunction (cytochrome c and Smac/DIABLO release; loss of mitochondrial membrane potential), activation of the caspase cascade, apoptosis, and diminished clonogenic survival. Similar findings were noted in other myeloma cell lines (e.g., MM.1S, RPMI8226, and NCI-H929) as well as in those resistant to dexamethasone and cytotoxic agents (e.g., MM.1R, 8226/Dox40, and 8226/LR5). Combined exposure to flavopiridol and HA14-1 was associated with down-regulation of Mcl-1 and Bcl-x_L, Bid cleavage, and mitochondrial translocation of Bax. Flavopiridol/HA14-1-treated cells also exhibited a pronounced activation of Jun NH₂-terminal kinase, a modest activation of p38 mitogen-activated protein kinase, and down-regulation of cyclin D1. Flavopiridol/HA14-1-induced apoptosis was associated with a marked increase in reactive oxygen species generation; moreover,both events were attenuated by the antioxidant N-acetyl-L-cysteine. Finally, in contrast to dexamethasone, flavopiridol/HA14-1-induced lethality was unaffected by exogenous interleukin-6 or insulin-like growth factor-I. Together, these findings indicate that flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 cooperate to trigger oxidant injury, mitochondrial dysfunction, caspase activation, and apoptosis in human multiple myeloma cells and suggest that this approach may warrant further evaluation as an antimyeloma strategy.

Key Words: HA14-1 • Bcl-2 • flavopiridol • apoptosis • myeloma

Grant support: National Cancer Institute awards CA63753, CA 93738,and CA 100866 and Leukemia and Lymphoma Society of America award 6045-03.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/24/04; revised 8/13/04; accepted 9/17/04.