Apaf-1 and caspase-9 deficiency prevents apoptosis in a Bax-controlled pathway and promotes clonogenic survival during paclitaxel treatment

Abstract

Taxane derivatives such as paclitaxel elicit their antitumor effects at least in part by induction of apoptosis, but the underlying mechanisms are incompletely understood. Here, we used different cellular models with deficiencies in key regulators of apoptosis to elucidate the mechanism of paclitaxel-induced cell death. Apoptosis by paclitaxel was reported to depend on the activation of the initiator caspase-10; however, we clearly demonstrate that paclitaxel kills murine embryonic fibroblasts (MEFs) devoid of caspase-10 as well as human tumor cell lines deficient in caspase-10, caspase-8, or Fas-associating protein with death domain. In contrast, the lack of Apaf-1 or caspase-9, key regulators of the mitochondrial pathway, not only entirely protected against paclitaxel-induced apoptosis but could even confer clonogenic survival, depending on the cell type and drug concentration. Thus, paclitaxel triggers apoptosis not through caspase-10, but via caspase-9 activation at the apoptosome. This conclusion is supported by the fact that Bcl-2-overexpressing cells and Bax/Bak doubly-deficient MEFs were entirely resistant to paclitaxel-induced apoptosis. Interestingly, also the single knockout of Bim or Bax, but not that of Bak or Bid, conferred partial resistance, suggesting a particular role of these mediators in the cell-death pathway acti- IntroductionPaclitaxel (Taxol; Calbiochem, Darmstadt, Germany) is one of the most effective antitumor drugs and is approved for chemotherapy of a variety of human malignancies, including breast, ovarian and non-small cell lung cancer. 1 Like other taxane derivatives, paclitaxel binds to the ␤-subunit of tubulin, thus preventing microtubule depolymerization that is required for proper mitosis and cell division. During mitosis, the spindle checkpoint ensures proper chromosome segregation by blocking the anaphase onset until all chromosomes are attached to microtubules and tension across the kinetochores is generated. Paclitaxel inhibits the dynamic instability of the mitotic spindle, leading to impaired chromosome alignment. Consequently, the cells become arrested by the spindle checkpoint at the G 2 /M phase and then die eventually by an apoptotic pathway, called mitotic catastrophe. 2 Cells that escape this pathway can be also arrested by a second, postmitotic checkpoint, which is activated by aberrant division of cells with multipolar spindles, and leads to cell death in polyploid cells following paclitaxel treatment. 3 Although both p53-dependent and -independent mechanisms have been implicated in cell death during aberrant mitosis, the exact mechanisms of paclitaxelinduced apoptosis are currently unknown.Apoptosis is largely controlled by a family of aspartate-specific cysteine proteases, called caspases, that function as initiators and executioners of the apoptotic process. 4,5 Caspases are activated by two major signaling routes, the extrinsic death receptor and the intrinsic mitochondrial pathway, that both depend on the formation of large multiprotein complexes. 4,6 Initia...

The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program

“…Both cell lines were engineered to express control or TACC3-specific shRNAs upon doxycycline (DOX) treatment (Supplementary Material & Methods). Consistent with earlier studies (Janssen et al, 2007), prolonged treatment with increasing doses of PTX (10 and 100 nM) elicited a strong cell-death response in both MCF-7 lines that was comparable to that induced by the topoisomerase inhibitor etoposide (Figures 1a and b). As demonstrated previously (Essmann et al, 2004), exposure of the cells to gIR, on the other hand, led to a reduced apoptotic response when DNA fragmentation was determined ( Figure 1a).…”

“…Thus, these data imply that the reduction of Bim expression might be the reason for the apoptosis-resistant phenotype of TACC3-depleted cells. In agreement with this hypothesis are recent reports demonstrating that Bim is an important mediator of apoptosis induced by microtubule-interfering agents such as PTX (Janssen et al, 2007;Li et al, 2007), although contrary reports also exist (Czernick et al, 2009). …”

Mechanistic insight into taxol-induced cell death

“…Lysosomal membrane permeabilization and activation of caspase-3 and calpains occur only in detached cells Next to caspases (Park et al, 2004;Ehrlichova et al, 2005;Li et al, 2005;Lu et al, 2005;Day et al, 2006;Janssen et al, 2007;Pineiro et al, 2007), involvement of calpains (Pineiro et al, 2007) and cathepsin-B (Broker et al, 2004) was described in taxol-induced apoptosis. We compared taxol-treated detached and adherent A549 cells for activation of caspase-3 and calpains on immunoblots (Figure 3a).…”

“…The signaling pathways leading to cell death have been extensively studied (Blagosklonny and Fojo, 1999;Zhao et al, 2005) and recently several papers on the proteases involved were published. It is now clear that taxol triggers apoptosis by both caspasedependent (Park et al, 2004;Ehrlichova et al, 2005;Li et al, 2005;Lu et al, 2005;Day et al, 2006;Janssen et al, 2007;Pineiro et al, 2007) and caspase-independent pathways (Broker et al, , 2004Huisman et al, 2002;Ofir et al, 2002). One of the main supporting observations for the latter is the failure of the pancaspase inhibitor zVADfmk (N-benzyloxycarbonylVal-Ala-Asp(O-Me) fluoromethyl ketone) to protect against taxol-induced apoptosis Huisman et al, 2002).…”

Caspases and cancer

“…It was shown that the apoptosome is critical for tumor suppression in a cell-specific manner and that failure of apoptosome formation may be important for tumor cell survival only in cell types unable to switch their death program from apoptosis to caspase-independent cell death. 2,66 Mitochondrial outer membrane potential (MOMP)-dependent, caspase-independent cell death may be induced by damaged mitochondrial function or by the release of mitochondrial proteins, such as AIF, Omi/HtrA2 and endonuclease G. Cell survival in spite of MOMP, in contrast, requires recovery of mitochondrial function. Live cell imaging revealed that during apoptosis some mitochondria escape MOMP.…”