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Departments of ¹ Medicine, ² Biochemistry, ³ Pharmacology, and ⁴ Radiation Oncology, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia

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

Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH₂-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-x_L and diminished the expression of cyclin D₁ and p34^cdc2. Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin–mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.

Key Words: leukemia • Rapamycin • UCN-01 • apoptosis • Raf-1 • MEK • ERK • JNK • Akt

Grant support: NIH grants CA63753, CA 100866, and CA 93738; Leukemia and Lymphoma Society of America award 6045-03; Department of Defense award DAMD-17-03-1-0209; and an award from V Foundation for Cancer Research.

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 5/31/04; revised 11/30/04; accepted 1/25/05.