Involvement of Asp-Glu-Val-Asp-directed, caspase-mediated mitogen-activated protein kinase kinase 1 cleavage, c-Jun N-terminal kinase activation, andsubsequent Bcl-2 phosphorylation for paclitaxel-induced apoptosis in HL-60cells

Paclitaxel is a novel anticancer drug that has demonstrated efficacy toward treating several malignant tumor types. Here, we demonstrate that c-Jun NH 2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase or ex tracellular signal-regulated kinase 1/2, was persistently activated by pacl itaxel or other microtubule-damaging agents within human leukemia HL-60 cel ls. Overexpression of a dominant-negative mutant, mitogen-activated protein kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligon ucleotide prevented paclitaxel-induced JNK activation, Bcl-2 phosphorylatio n and apoptosis. Furthermore, we found that the full-length MEKK1 was cleav ed to a 91-kDa carboxyl-terminal fragment at the earlier time of apoptosis induced by microtubule-damaging agents. This cleavage, however, occurred co nsistently with JNK activation and Bcl-2 phosphorylation, but preceded DNA fragmentation in cells in response to paclitaxel activity. The caspase inhi bitor Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO), but not Ac-Tyr-Val-Ala-Asp-CHO (Ac -YVAD-CHO), effectively blocked MEKK1 cleavage, JNK activation, Bcl-2 phosp horylation, and subsequent apoptosis. Subcellular fractionation revealed th at the 91-kDa C-terminal MEKK1 fragment was translocated to cytosol. Notabl y, the MEKK1 fragment could be coimmunoprecipitated with anti-JNK antibodie s, suggesting that a signaling complex of C-terminal MEKK1/stress-activated protein kinase/extracellular-signal regulated kinase 1/JNK formed during a poptosis induced by microtubule-damaging agents. Taken together, our result s suggest that disruption of cytoarchitecture by paclitaxel triggers a nove l apoptosis-signaling pathway, wherein an active DEVD-directed caspase (DEV Dase) initially cleaves MEKK1to generate a proapoptotic kinase fragment tha t is able to activate JNK and subsequent Bcl-2 phosphorylation, finally eli citing cell death.