A novel mechanism for imatinib mesylate–induced cell death of BCR-ABL–positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity

Abstract

Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL-positive cells in a caspasedependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate-induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate-treated BCR-ABL-positive human leukemic cells. Moreover, zVAD-fmk-preincubated, imatinib mesylate-treated cells exhibited a necrosislike morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2. IntroductionImatinib mesylate (imatinib, Gleevec) was developed as a potent and specific inhibitor of ABL tyrosine kinase. 1 Preclinical studies and clinical trials showed that imatinib exhibited a remarkable single-agent activity against BCR-ABL-expressing cells with acceptable toxicity in vitro and in vivo. 1,2 BCR-ABL tyrosine kinase activates several signaling pathways such as the Ras/mitogenactivated protein kinase, 2-4 signal transducer and activator of transcription 5, 2,4 and phosphatidylinositol 3 kinase/Akt pathways 2,4 ; enhances nuclear factor B (NF-B) activity 5 ; upregulates the level of 6 ; and suppresses the mitochondrial apoptotic pathway. 5,7 Imatinib counteracts BCR-ABL tyrosine kinase and induces apoptosis in BCR-ABL-positive cells [8][9][10] in a caspase-dependent fashion. 11,12 Recently, however, it has been revealed that responses to imatinib are not necessarily remarkable or durable in some patients with BCR-ABL-positive leukemia, [13][14][15][16][17][18][19][20][21][22] and thus an increasing number of studies have searched for a novel therapy targeting the BCR-ABL-induced signaling pathways. 5 Cell death is generally classified into 2 categories, apoptosis 23,24 and necrosis. Apoptosis is a well-documented active programmed cell death (PCD) in which the activation of caspases plays a central role. 25 In contrast, necrosis has been conceived as a passive cell death without established regulatory mechanisms. However, it has recently been reported that necrosis-like cell death may be re...

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition

“…This finding is also reinforced by the observation of a diminished cell rate proliferation in K562 clones, nevertheless resistant in term of apoptosis to Bcr-Abl inhibitors. The caspaseindependent counterpart of the imatinib-induced cell death could be likely owing to activation of HtrA serine peptidase 2 (HTRA2) as described recently (Okada et al, 2004) or to other currently unrevealed mechanisms. However, in our hands, neither tosyl-L-lysine chloromethylketone, a general trypsin-like inhibitor, nor the more specific OMI/HTRA2 inhibitor, UFC101, were able to further increase cell metabolism in the presence of Bcr-Abl inhibitors.…”

“…Finally, imatinib-induced caspase activation and differentiation were unaffected by antioxidants, suggesting that reactive oxygen species production is not involved in these processes (not shown). A recent study by Okada et al (2004) reported that imatinib-induced cell death in CML cells occurs by both caspase-dependent and -independent mechanisms. The partial caspase independence of Bcr-Abl inhibitors is confirmed in our study by the observation that even in the presence of ZVAD-fmk, a significant loss of cell metabolism (around 50%) could be still detected upon imatinib and PD166326 treatment of CML cell lines.…”

“…Subsequently, the cell cycle progression is impaired and cells undergo an apoptotic programme characterized by activation of caspases 9, 8 and 3 (Jacquel et al, 2003). Furthermore, it has been recently shown that inhibition of caspases in imatinib-treated K562 cells shifted the cells from a caspase-dependent to a necrosis-like cell death process, suggesting that imatinib also triggered caspase-independent cell death (CID) (Okada et al, 2004).…”

Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomerase-expressing cell lines

“…This finding is in accordance with a recent study conducted on K-562, a BCR-ABL-positive cell line, exposed to IM. The authors demonstrated that IM induced a caspase-independent, necrosis-like cell death mediated by the serine protease activity of Omi/HtrA2 (Okada et al, 2004). Other studies demonstrated an inhibitory effect of IM on PDGF-R-expressing cell lines, suggesting that this effect was mediated mainly through promoting growth arrest rather than apoptosis (Kilic et al, 2000).…”

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K+ ions

“…Specifically, TLCK and TPCK have been shown to block activation of NFκB [35], prevent the activation of pp70(s6k), a mitogenrelated kinase [36], and to suppress the processing of caspases in some model systems [37,38]. In contrast, these two proteases have been shown to both induce and enhance cell death upon treatment with various cytotoxic agents [39][40][41].…”