Bortezomib is a proteasome inhibitor for the treatment of relapsed/refractory multiple myeloma (MM). Mechanisms of resistance to Bortezomib are undefined. Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic protein, which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In MM, specific downregulation of Mcl-1 induces apoptosis. Here, we examined the role of Mcl-1 in Bortezomib- and doxorubicin-induced apoptosis. We demonstrate that Bortezomib, but not doxorubicin, triggers caspase-dependent generation of a 28 kDa Mcl-1-fragment, in several MM cell lines, including MM.1S cells. Conversely, transient transfection of MM.1S cells with a previously reported 28 kDa Mcl-1128–350 fragment, but not with the Mcl-11–127 fragment, induces apoptosis. Therefore, both downregulation of full-length antiapoptotic Mcl-1, as well as Bortezomib-induced generation of Mcl-1128–350 cleaved protein, contribute to MM cell apoptosis. To verify further these findings, we next compared effects triggered by Bortezomib, doxorubicin and melphalan in Mcl-1wt/wt and Mcl-1Δ/null murine embryonic fibroblasts (MEFs). Our results show that Bortezomib, but not doxorubicin or melphalan, triggers Mcl-1 cleavage in Mcl-1wt/wt, but not Mcl-1Δ/null MEFs and induces sub-G1 phase cells; caspase-3 and -9, and PARP cleavage as well as morphological signs of apoptosis. Taken together, these results support an important role of Mcl-1 and a Mcl-1 fragment in Bortezomib-induced cell death in general, and in MM in particular. To prevent relapse of MM in patients treated with Bortezomib, we therefore recommend the combination of Bortezomib with agents that induce MM cell death independent of Mcl-1.
Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic protein in the Bcl-2 family. Among Bcl-2 family members, Mcl-1 distinguishes itself by its ability to oppose several apoptotic stimuli, as well as the presence of polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) domains (PEST), which could be responsible for its short half-life (3–4 h). Thus, Mcl-1 is involved in both cell homeostasis and differentiation (Craig, 2002; Opferman, 2006). Deletion of Mcl-1 prevents embryonic development, since Mcl-1 null embryos fail to survive pre-implantation development and implantation (Rinkenberger et al., 2000). Mcl-1 is also required for development and maintenance of B and T cells (Opferman et al., 2003) and plays an obligate role in the survival of hematopoietic stem cells (Opferman et al., 2005). Overexpression of Mcl-1 promotes cell immortalization whereas downregulation induces apoptosis (Zhou et al., 1998; Nijhawan et al., 2003). This prosurvival function is conferred predominantly by Mcl-1 binding to proapoptotic proteins such as Bak, to form heterodimers and thereby prevent cell death (Leu et al., 2004). Conversely, release of Bak from its interaction with Mcl-1 induces apoptosis (Willis et al., 2005). Interestingly, Mcl-1 is regulated at transcriptional, post-transcriptional and post-translational levels (Craig, 2002; Le Gouill et al., 2004b; Opferman, 2006). Mcl-1 transcription is upregulated via several cytokines and growth factors via various signaling pathways. At the post-transcriptional level, Mcl-1 (located on chromosome 1q21) undergoes alternative splicing: the junction of the first and third exons, without exon 2, encodes for a 271-amino-acid Mcl-1 short protein, which has a proapoptotic function (Bae et al., 2000). At the post-translational level, caspase-3 triggers Mcl-1 cleavage with resultant Mcl-1 cleaved forms, some of which have potent proapoptotic functions (Michels et al., 2004; Weng et al., 2005). Moreover, Mcl-1 is regulated by phosphorylation. For example, oxidative stress triggers Mcl-1 phosphorylation via JNK at serine-121 and threonine-163 resulting in the loss of its antiapoptotic function (Inoshita et al., 2002). In addition, Mcl-1 is phosphorylated at serine-159 by glycogen synthase kinase-3 and thereby destabilizes Mcl-1 protein and induces apoptosis (Maurer et al., 2006).
Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow (BM) (Bataille and Harousseau, 1997). Despite intensive therapy including autologous hematopoietic stem cell support, MM remains incurable. Specific inhibition of Mcl-1, but not of Bcl-2 or Bcl-XL, using antisense oligonucleotide or Mcl-1 small interfering RNA (siRNA) induces apoptosis of MM cells, confirming the crucial antiapoptotic role of Mcl-1 in malignant plasma cells (Derenne et al., 2002; Zhang et al., 2002; Le Gouill et al., 2004b). Moreover, interleukin-6 (IL-6) and vascular endothelial growth factor upregulate Mcl-1 expression in MM cells and thereby protect against apoptosis (Puthier et al., 1999; Le Gouill et al., 2004a). Mcl-1 is therefore a potential novel therapeutic target in MM.
The proteasome inhibitor Bortezomib (formerly PS-341; Velcade, Millenium Pharmaceuticals Inc., Cambridge, MA, USA), a novel dipeptide boromic acid, has been Food and Drug Administration (FDA) approved in May 2003 for treatment of relapsed or refractory MM and is currently under clinical investigation as front-line therapy (Richardson et al., 2003). Bortezomib has various mechanisms of action against MM cells including induction of apoptosis, inhibition of nuclear factor-κB activation, triggering of caspase-dependent cleavage of gp130, downregulation of adhesion molecule expression on MM cells and BM stromal cells, inhibition of angiogenesis and decreasing secretion of survival and proliferative cytokines (Hideshima et al., 2001, 2003a, 2003b; LeBlanc et al., 2002; Podar et al., 2004b; Roccaro et al., 2006). However, mechanisms mediating Bortezomib resistance are not fully understood. Here, we add another facet to the anti-MM functions of Bortezomib by characterizing the role of Mcl-1 and Mcl-1128–350-cleaved protein in Bortezomib-induced cell apoptosis using both MM cells as well as Mcl-1-lacking murine embryonic fibroblasts (MEFs). On the basis of our data, we recommend the combination of Bortezomib with agents that induce MM cell death independent of Mcl-1.
Bortezomib reduces cell viability and induces Mcl-1 cleavage associated with cleavage of caspase -3, -8, -9 and PARP
Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we first determined the affects of both Bortezomib and doxorubicin on MM.1S cell viability. Cells were cultured with or without various doses of Bortezomib or doxorubicin for 48 h. Treatment with Bortezomib (Figure 1a) and doxorubicin (Figure 1b) decreases MM.1S viability in a dose-dependent manner. The median inhibitory concentration (IC50) was 4 and 45 nM for Bortezomib and doxorubicin, respectively.
Next, we used western blot analysis to determine whether Bortezomib and doxorubicin treatment modifies Mcl-1, caspase-3, -8, -9 and poly (ADP) ribose polymerase (PARP), as well as Bcl-2 expression in MM.1S cells in a dose- and/or time-dependent fashion. Specifically, our results show that high-dose Bortezomib (10 nM) induces Mcl-1 cleavage only after 6 h, resulting in the accumulation of a 28 kDa Mcl-1-cleaved product and cleavage of caspase-3, -8, -9 and PARP. Importantly, Mcl-1 downregulation and cleavage was also observed at concentrations as low as 2.5 nM after longer periods of Bortezomib treatment (24 h). In contrast to Mcl-1, no modification of Bcl-2 was observed at any time point (Figure 2a). Moreover, although high-dose treatment with doxorubicin induced Mcl-1 downregulation in MM.1S cells, we did not observe any accumulation of the Mcl-1-cleaved protein (Figure 2b). We hypothesize that this late effect reflects an apoptotic shutdown of the MM cell and that doxorubicin-induced decrease of Mcl-1 may be either due to the short half-life of Mcl-1 mRNA or reduced Mcl-1 promoter activity. However, PARP cleavage, indicative for doxorubicin-induced cell death, was observed also at lower doses of doxorubicin. Taken together, these results demonstrate that low-dose Bortezomib, but not doxorubicin, induces early generation of a 28 kDa Mcl-1 fragment associated with cleavage of caspase-3, -8, -9 and PARP.
Bortezomib-induced fragmentation of Mcl-1 is caspase dependent
To investigate the potential role of caspases in Bortezomib-induced Mcl-1 cleavage, MM.1S cells were preincubated with pan-caspase inhibitor (z-VAD-fmk, z-Val-Ala-Asp(OCH3)-Fluoromethylketone), caspase-9 inhibitor (z-LEHD-fmk, z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethylketone) and caspase-8 inhibitor (z-IETD-fmk, z-lle-Glu-Thr-Asp-Fluoromethylketone) (3 h) and then treated with Bortezomib (6 h). Pretreatment with the pan-caspase inhibitor as well as with the caspase-9 inhibitor, and to a lesser extent with the caspase-8 inhibitor, blocked Bortezomib-induced Mcl-1 cleavage and thus the appearance of the 28 kDa Mcl-1-cleaved protein (Figure 3a).
Bortezomib generates a previously reported 28 kDa Mcl-1 fragment
The generation of a 28 kDa cleaved Mcl-1 product was previously reported in several cisplatin-treated malignant B cells. Importantly, this fragment was identified as Mcl-1128–350 and functionally linked to proapoptosis (Michels et al., 2004). We therefore hypothesized that the 28 kDa Mcl-1-fragment generated in Bortezomib-treated MM cells is identical with Mcl-1128–350 and also contributes to Bortezomib-induced MM cell death. Indeed, the Bortezomib-induced Mcl-1 fragment migrates at the same molecular weight as the Mcl-1 fragment pcDNA3-Mcl-1128–350 transiently transfected into MM.1S cells. Moreover, MM cells that overexpress the 28 kDa Mcl-1 fragment show both caspase-3 and PARP cleavage indicative of Mcl-1128–350-induced apoptosis (Figure 3b).
Exogenous Mcl-1 fragment Mcl-1128–350, but not Mcl-1 fragment Mcl-11–127 or Mcl-1wt, triggers increased apoptosis and G2 cell-cycle arrest
To verify whether Mcl-1128–350 is able to induce MM cell apoptosis, MM.1S cells were transiently transfected with empty vector (pcDNA3), Mcl-1wt, or either of two fragments, pcDNA3-Mcl-11–127 or pcDNA3-Mcl-1128–350, respectively. Our results show that pcDNA3-Mcl-1128–350, but not the empty vector, Mcl-1wt, or pcDNA3-Mcl-11–127 increases cell-cycle phase sub-G1 indicative of apoptosis (Figure 4a), and G2 indicative of G2 arrest (Figure 4b) in MM cells; effects were seen at 24 h (Figures 4a and b, left panels) and more pronounced at 48 h (Figures 4a and b, right panels) after transfection. Similar results were previously observed in NIH3T3 cells (Michels et al., 2004). Taken together, these data indicate that Bortezomib-triggered apoptosis is, at least in part, mediated by Mcl-1128–350 fragment, but not Mcl-1wt or Mcl-11–127 fragment via G2 cell-cycle arrest.
Depletion of Mcl-1 and Mcl-1128–350-cleaved protein alleviates Bortezomib-induced MM cell death
To verify further the role of Mcl-1128–350-cleaved protein in Bortezomib-induced MM cell apoptosis, MM.1S cells were transiently transfected with Mcl-1 siRNA before treatment with Bortezomib or doxorubicin, respectively. As shown in Figure 5a, transfection efficiency of siRNA in MM.1S cells, using siGLO Green as a control, is >87%. Transient transfection with specific Mcl-1 siRNA markedly downregulated protein expression of both uncleaved and cleaved Mcl-1 compared to cells transfected with siGLO Green or mock siRNA (Figure 5b). Downregulation of expression of Mcl-1, similar to Bortezomib 2.5 nM induced cell apoptosis in 30–40% of MM cells. These results are consistent with our own and other previous data (Derenne et al., 2002; Le Gouill et al., 2004a). However, Bortezomib-induced cell death was significantly alleviated in cells treated with Mcl-1 siRNA versus cells treated with mock siRNA at Bortezomib concentrations of 5 nM (Figure 5c). In contrast doxorubicin-induced cell death was similar in cells treated with either mock siRNA or Mcl-1 siRNA at concentrations of 25 or 50 nM (Figure 5d). Taken together, these data indicate that MM cell apoptosis can be induced both by depletion of Mcl-1 full-length protein, as well as Bortezomib-triggered generation of a proapoptotic Mcl-1128–350 fragment.
Bortezomib induces NOXA upregulation and its association with Mcl-1
Similar to MM.1S cells, Bortezomib, but not doxorubicin, triggered the generation of Mcl-1128–350 fragment in U266, KMS18, NCI (Figure 6a) and RPMI cells (data not shown). In contrast, no fragment was generated in the INA-6 MM cell line treated with Bortezomib (data not shown). Previous data show that proteasome inhibitors such as Bortezomib induce increased expression of BH3-only protein NOXA in several tumor cells including melanoma, mantle-cell lymphoma and MM. Importantly, NOXA upregulation is associated with tumor cell apoptosis (Qin et al., 2005; Perez-Galan et al., 2006). Indeed, in contrast to other BH3-only proteins, which exhibit promiscuous binding, NOXA only binds Mcl-1 and A1 (Chen et al., 2005). Mcl-1 binding to NOXA induces apoptosis both by releasing Bak from its interaction with Mcl-1 and by promoting Mcl-1 degradation (Willis et al., 2005). Consistent with these data, we observed that Bortezomib-induced cleavage of Mcl-1 was accompanied by expressional upregulation of NOXA in all MM cell lines (Figure 6a). In contrast, Bortezomib did not change either expression of Bim or its cell line-dependent association with Mcl-1 (Figures 6a and b). Moreover, Bortezomib, but not doxorubicin, specifically triggered increased binding of Mcl-1 with NOXA (Figure 6b). Ongoing studies are now evaluating the impact of this complex formation on Bak release and Mcl-1 fragmentation in MM cells. Taken together, these data demonstrate that Mcl-1 fragmentation in the majority of MM cell lines is associated with upregulation of the proapoptotic BH3-only protein NOXA and its increased binding to Mcl-1.
Lack of Mcl-1 protects against Bortezomib- but not doxorubicin-induced apoptosis
We next compared effects triggered by Bortezomib, doxorubicin and melphalan in Mcl-1Δ/null and Mcl-1wt/wt MEFs. Changes in sub-G1 were observed at 48 h (Figure 7c), but not at 12 h (Figure 7a) or 24 h (Figure 7b). Both Mcl-1wt/wt and Mcl-1Δ/null MEFs were sensitive to doxorubicin: 46 versus 40% apoptosis at 50 nM; and 85 versus 80% apoptosis at 100 nM in Mcl-1wt/wt and Mcl-1Δ/null MEFs, respectively (Figure 7c, right panel). In contrast, Bortezomib failed to induce apoptosis in Mcl-1Δ/null versus Mcl-1wt/wt MEFs at concentrations up to 10 nM (0.7 versus 27.6%, respectively) (Figure 7c, left panel). At higher concentrations of Bortezomib (20 nM), 73 versus 18% apoptotic cells were observed (Figure 7c, left panel). Lack of Bortezomib-triggered induction of sub-G1 in Mcl-1Δ/null cells correlated with the absence of morphologic changes (Figure 7d). We therefore conclude that Mcl-1 and Mcl-1 fragmentation is pivotal for Bortezomib-, but not doxorubicin-induced apoptosis.
Lack of Mcl-1 protects against Bortezomib- but not doxorubicin- and melphalan-induced PARP and caspase-3 cleavage
Finally, the mechanism whereby Mcl-1 mediates Bortezomib-induced apoptosis was investigated. We therefore compared caspase-3, -9 and PARP cleavage triggered by doxorubicin, melphalan and Bortezomib in Mcl-1Δ/null versus Mcl-1wt/wt MEFs. Mcl-1Δ/null and Mcl-1wt/wt MEFs were cultured with or without compounds for 24–36 h. As in MM.1S cells, Bortezomib, but not doxorubicin and melphalan, triggered Mcl-1 cleavage and appearance of Mcl-1-cleaved product in Mcl-1wt/wt MEFs. However, like Bortezomib, doxorubicin and melphalan-induced PARP cleavage. In contrast, PARP cleavage triggered by Bortezomib, but not doxorubicin or melphalan is significantly inhibited in Mcl-1Δ/null MEFs. Moreover, cleavage of caspase-9 and -3 was also significantly diminished in Mcl-1Δ/null MEFs (Figure 8). Taken together, these experiments demonstrate that Bortezomib induces Mcl-1-dependent apoptotic signaling events, whereas doxorubicin and melphalan trigger Mcl-1-independent apoptotic cascades.
Bortezomib is a proteasome inhibitor for the treatment of relapsed/refractory MM (Chauhan et al., 2005). However, the majority of patients with relapsed or refractory MM does not respond, and occurrence of resistance is common. Mechanisms conferring Bortezomib resistance are undefined. Mcl-1 is an antiapoptotic protein, originally identified as an ‘early induction’ gene during myeloblastic leukemic cell differentiation (Kozopas et al., 1993), which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. Mcl-1 forms heterodimers with proapoptotic BH3-only-proteins Bim and NOXA, or with Bak, thereby preventing cytochrome c release and caspase activation. Owing to its binding with BH3-only protein and short half-life, Mcl-1 is therefore an early checkpoint regulating the apoptotic/survival balance (Opferman et al., 2003; Han et al., 2004; Willis et al., 2005). In MM, specific downregulation of Mcl-1 in tumor cells induces apoptosis (Derenne et al., 2002; Zhang et al., 2002; Le Gouill et al., 2004a). Moreover, apoptotic stimuli in MM cells, such as IL-6 withdrawal or inhibition of JAK/Stat- pathways by AG490, lead to upregulation of Bim associated with Mcl-1 downregulation, Bim release from the Mcl-1 complex, and MM cell apoptosis (Gomez-Bougie et al., 2004).
Mcl-1 is highly and tightly regulated. Mcl-1, like other Bcl-2 family members, is subjected to post-translational regulation, including phosphorylation and cleavage. Specifically, extracellular signal-regulated kinase (ERK)-dependent or -independent pathways mediate phosphorylation of Mcl-1. Taxol-triggered ERK-independent Mcl-1 phosphorylation promotes cell death, whereas 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-triggered ERK-dependent Mcl-1 phosphorylation promotes cell viability via slowing the cell cycle and reducing Mcl-1 turnover (Domina et al., 2000, 2004). At the post-translational level, Mcl-1 is cleaved (Michels et al., 2004). Two distinct cleavage sites have been identified and are conserved from one species to another. Both sites (Asp127 and Asp157) are located in the PEST domain of the protein: Mcl-1 cleavage at Asp127 results in 28 kDa Mcl-1128–350 and 17 kDa Mcl-11–127 cleaved products, whereas cleavage at Asp157 results in 23 kDa Mcl-1158–350 and 21 kDa Mcl-11–157-cleaved protein products. The two C-terminal Mcl-1-cleaved products (Mcl-1158–350 and Mcl-1128–350) have lost the BH4 domain and are structurally similar to Bax, a proapoptotic protein of the Bcl-2 family. Indeed, Michels et al. (2004) have demonstrated that Mcl-1128–350-cleaved protein triggered by cisplatin accumulates in Akata6 B-lymphoma cells, and that its overexpression induces apoptosis. In Jurkat T cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers Mcl-1 cleavage through caspase-3 activation and conversely, mutations in Mcl-1-cleaved sites protect against TRAIL-induced apoptosis (Weng et al., 2005). Moreover, the C-terminal Mcl-1128–350-cleaved product mediates TRAIL-induced apoptosis, by acting as a proapoptotic protein cooperating with tBid and Bak to regulate mitochondrial apoptotic events.
Here, we demonstrate that Bortezomib, but not doxorubicin, triggers caspase-3, -8 and -9 dependent generation of a previously reported 28 kDa Mcl-1 fragment, Mcl-1128–350, in several MM cell lines including MM.1S cells. However, whether caspase-8 processing results in active caspase-8 is controversial to date. We were unable to detect other Mcl-1-cleaved products, since the epitope targeted by S-19 antibody is not present in the Mcl-1158–350-cleaved product (Michels et al., 2004; Weng et al., 2005). Furthermore, we do not exclude additional modifications, which can switch Mcl-1 to a proapoptotic form. Importantly, we demonstrate that downregulation of full-length antiapoptotic Mcl-1and Bortezomib-induced generation of Mcl-1128–350-cleaved protein contribute to MM cell apoptosis. Interestingly, Bortezomib-induced generation of the Mcl-1128–350 fragment was accompanied by the induction of the BH3-only protein NOXA and its increased binding to Mcl-1. Indeed, NOXA tightly binds Mcl-1 but not Bcl-2, Bcl-XL, or Bcl-w, thereby displacing Bak. In turn, free Bak induces apoptosis via permeabilization of organellar membranes and induction of caspase activation. Besides Mcl-1, Bcl-XL is also a guardian of Bak (Willis et al., 2005). Consistent with these data, our results show that Bortezomib induces apoptosis dependent on Mcl-1. We hypothesize that combined neutralization of both Mcl-1 and Bcl-XL drive efficient Bak-mediated apoptosis (Willis et al., 2005). Ongoing experiments in MM are evaluating this hypothesis.
Our findings in MM cells were subsequently verified in Mcl-1wt/wt and Mcl-1Δ/null MEFs. Specifically, Bortezomib, but not doxorubicin or melphalan, triggered Mcl-1 cleavage in Mcl-1wt/wt but not Mcl-1Δ/null MEFs, as well as induced sub-G1 phase cells, with caspase-3, -9 and PARP cleavage. In contrast, doxorubicin-induced apoptosis was similar in Mcl-1wt/wt and Mcl-1Δ/null MEFs. Thus, cleavage of caspase-3 and Mcl-1 are simultaneous events, which together enhance Bortezomib-induced apoptosis. As reported by Hideshima et al. (2001), Chauhan et al. (2004) Bortezomib induces apoptosis by triggering mitochondrial events leading to caspase activation. Indeed, DHL-4 cell line expresses low levels of caspase-3 and -8, which confers resistance to Bortezomib. In contrast, doxorubicin induces apoptosis through activation of several apoptotic pathways, including DNA damage through topoisomerase II inhibition and free radical production (Gewirtz, 1999; Hurley, 2002). Indeed, doxorubicin-triggered caspase-3 activity is due to stress response (SR)-mediated stress (Jang et al., 2004). The comparison therefore shows that Bortezomib and doxorubicin induce distinct Mcl-1-dependent and -independent apoptotic pathways.
In conclusion, our study demonstrates that lack of Mcl-1 and Mcl-1128–350 fragment confers resistance to Bortezomib and protects against Bortezomib-induced caspase-3 and PARP cleavage, highlighting the complexity of Mcl-1 post-translational regulation and its role in mitochondrial and caspase-3-mediated drug-induced apoptosis. Furthermore, our results identify a new mechanism of drug resistance, implicating a role for Mcl-1 not only as an antiapoptotic protein that opposes drug-induced apoptosis, but also as a proapoptotic cleaved protein enhancing mitochondrial/caspase activation and thereby leading to apoptosis. From a clinical point of view, our results suggest the potential utility of combining therapies that trigger Mcl-1-dependent and -independent pathways, that is Bortezomib and doxorubicin or Bortezomib with histone deacetylase inhibitors (Pei et al., 2004) and Bortezomib and doxorubicin with seliciclib, a small molecule cyclin-dependent kinase inhibitor (Raje et al., 2005). Recently, Orlowski et al. (2005) have combined these two drugs and demonstrated remarkably activity in patients with relapsed or refractory hematologic malignancies, supporting the potential utility of this therapeutic strategy.
Materials and methods
Cells and cell culture
MEF cell lines Mcl-1wt/wt and Mcl-1Δ/null were generated by SV40 large T transformation followed by Tat-Cre-mediated deletion. Single cell clones were selected and then grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 10 μg streptomycin and 2 mM L-glutamine, 2-mercapto-ethanol (Sigma, St Louis, MO, USA) and minimum essential meduium (MEM) non-essential amino acid (Gibco, Grand Island, NY, USA) to be used from early passages. Both Mcl-1wt/wt and Mcl-1Δ/null were extensively characterized as being hypersensitive to various death stimuli with restorable resistance upon re-expression of Mcl-1 (Opferman et al., 2003). The human MM cell line MM.1S was maintained in RPMI-1640 medium with 2 mM L-glutamine (Mediatech, Cellgro, AK, USA) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 10 μg streptomycin (Mediatech).
Cell viability assays
Cell viability was assessed by MTT (Chemicon International, Temecula, CA, USA) assay, according to manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA). Cells were seeded in 96-well plates. Cell viability was evaluated as described previously and survival estimated relative to untreated controls (Chauhan et al., 1996).
For cell-cycle analysis, DNA was stained with propidium iodide as described previously (Podar et al., 2004a). Apoptotic cells were detected as a subdiploid peak, as described by Zamai et al. (1993). Flow cytometry was analysed using Cytomics RXP program (Beckman Coulter, Inc., Fullerton, CA, USA).
Cell lysis and western blot
Cells were treated with various concentrations of Bortezomib (Millenium Pharmaceuticals), doxorubicin or melphalan, washed two times with 1 × phosphate-buffered saline, and suspended in lysis buffer (10 mM Tris, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium vanadate, 1 mM N-phenylmethyl sulfonyl fluoride and 2 mg/ml aprotinin). After 10 min on ice, lysates were cleared by centrifugation at 13 000 g/min for 30 min at 4°C; and were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, before electrophoretic transfer onto Hybond C supermembrane (Amersham, Arlington Heights, IL, USA). The blots were probed overnight with anti-human Mcl-1, Bcl-2, actin, caspase-3, -8, Bim and NOXA (Santa Cruz Biotechnology; Santa Cruz, CA, USA); anti-murine Mcl-1 (Rockland, Gilbertsville, PA, USA); anti-actin (Santa Cruz Biotechnology); and anti-PARP (Cell Signaling Technology, Beverly, MA, USA) antibodies. After washing, blots were incubated with secondary antibodies, followed by exposure to enhanced chemiluminescence substrate.
Transfection of empty vector (pcDNA3), Mcl-1wt and Mcl-1-cleaved products
pcDNA3, pcDNA3-Mcl-1wt and pcDNA3-Mcl-1128–350, pcDNA3-Mcl-11–127 were kindly provided by Dr G Packham and transfected using Nucleofactor Kit V according to the manufacturer's instructions (Amaxa Biosystems, Cologne, Germany) (Michels et al., 2004). For Mcl-1-specific knockdown experiments, MM.1S cells were transiently transfected with 200 nM of siRNA SMARTpool for Mcl-1, siGLO Green or non-specific control duplexes (pool of four; Upstate Cell Signalling Solutions, Charlottesville, VA, USA/Dharmacon RNA Technologies, Lafayette, CO, USA) as described previously (Le Gouill et al., 2004a).
Bae J, Leo CP, Hsu SY, Hsueh AJ . (2000). MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain. J Biol Chem 275: 25255–25261.
Bataille R, Harousseau JL . (1997). Multiple myeloma. N Engl J Med 336: 1657–1664.
Chauhan D, Hideshima T, Mitsiades C, Richardson P, Anderson KC . (2005). Proteasome inhibitor therapy in multiple myeloma. Mol Cancer Ther 4: 686–692.
Chauhan D, Li G, Podar K, Hideshima T, Mitsiades C, Schlossman R et al. (2004). Targeting mitochondria to overcome conventional and Bortezomib/proteasome inhibitor PS-341 resistance in multiple myeloma (MM) cells. Blood 104: 2458–2466.
Chauhan D, Uchiyama H, Akbarali Y, Urashima M, Yamamoto K, Libermann TA et al. (1996). Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood 87: 1104–1112.
Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG et al. (2005). Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17: 393–403.
Craig RW . (2002). MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis. Leukemia 16: 444–454.
Derenne S, Monia B, Dean NM, Taylor JK, Rapp MJ, Harousseau JL et al. (2002). Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. Blood 100: 194–199.
Domina AM, Smith JH, Craig RW . (2000). Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition. J Biol Chem 275: 21688–21694.
Domina AM, Vrana JA, Gregory MA, Hann SR, Craig RW . (2004). MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol. Oncogene 23: 5301–5315.
Gewirtz DA . (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol 57: 727–741.
Gomez-Bougie P, Bataille R, Amiot M . (2004). The imbalance between Bim and Mcl-1 expression controls the survival of human myeloma cells. Eur J Immunol 34: 3156–3164.
Han J, Goldstein LA, Gastman BR, Froelich CJ, Yin XM, Rabinowich H . (2004). Degradation of Mcl-1 by granzyme B: implications for Bim-mediated mitochondrial apoptotic events. J Biol Chem 279: 22020–22029.
Hideshima T, Chauhan D, Hayashi T, Akiyama M, Mitsiades N, Mitsiades C et al. (2003a). Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades via caspase-dependent downregulation of gp130 in multiple myeloma. Oncogene 22: 8386–8393.
Hideshima T, Mitsiades C, Akiyama M, Hayashi T, Chauhan D, Richardson P et al. (2003b). Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341. Blood 101: 1530–1534.
Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J et al. (2001). The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 61: 3071–3076.
Hurley LH . (2002). DNA and its associated processes as targets for cancer therapy. Nat Rev Cancer 2: 188–200.
Inoshita S, Takeda K, Hatai T, Terada Y, Sano M, Hata J et al. (2002). Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress. J Biol Chem 277: 43730–43734.
Jang YM, Kendaiah S, Drew B, Phillips T, Selman C, Julian D et al. (2004). Doxorubicin treatment in vivo activates caspase-12 mediated cardiac apoptosis in both male and female rats. FEBS Lett 577: 483–490.
Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW . (1993). MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci USA 90: 3516–3520.
LeBlanc R, Catley LP, Hideshima T, Lentzsch S, Mitsiades CS, Mitsiades N et al. (2002). Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model. Cancer Res 62: 4996–5000.
Le Gouill S, Podar K, Amiot M, Hideshima T, Chauhan D, Ishitsuka K et al. (2004a). VEGF induces Mcl-1 up-regulation and protects multiple myeloma cells against apoptosis. Blood 104: 2886–2892.
Le Gouill S, Podar K, Harousseau JL, Anderson KC . (2004b). Mcl-1 regulation and its role in multiple myeloma. Cell Cycle 3: 1259–1262.
Leu JI, Dumont P, Hafey M, Murphy ME, George DL . (2004). Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol 6: 443–450.
Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR . (2006). Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell 21: 749–760.
Michels J, O'Neill JW, Dallman CL, Mouzakiti A, Habens F, Brimmell M et al. (2004). Mcl-1 is required for Akata6 B-lymphoma cell survival and is converted to a cell death molecule by efficient caspase-mediated cleavage. Oncogene 23: 4818–4827.
Nijhawan D, Fang M, Traer E, Zhong Q, Gao W, Du F et al. (2003). Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev 17: 1475–1486.
Opferman JT . (2006). Unraveling MCL-1 degradation. Cell Death Differ 13: 1260–1262.
Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K et al. (2005). Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 307: 1101–1104.
Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, Korsmeyer SJ . (2003). Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 426: 671–676.
Orlowski RZ, Voorhees PM, Garcia RA, Hall MD, Kudrik FJ, Allred T et al. (2005). Phase 1 trial of the proteasome inhibitor Bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies. Blood 105: 3058–3065.
Pei XY, Dai Y, Grant S . (2004). Synergistic induction of oxidative injury and apoptosis in human multiple myeloma cells by the proteasome inhibitor Bortezomib and histone deacetylase inhibitors. Clin Cancer Res 10: 3839–3852.
Perez-Galan P, Roue G, Villamor N, Montserrat E, Campo E, Colomer D . (2006). The proteasome inhibitor Bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and NOXA activation independent of p53 status. Blood 107: 257–264.
Podar K, Catley LP, Tai YT, Shringarpure R, Carvalho P, Hayashi T et al. (2004a). GW654652, the pan-inhibitor of VEGF receptors, blocks the growth and migration of multiple myeloma cells in the bone marrow microenvironment. Blood 103: 3474–3479.
Podar K, Shringarpure R, Tai YT, Simoncini M, Sattler M, Ishitsuka K et al. (2004b). Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by Bortezomib. Cancer Res 64: 7500–7506.
Puthier D, Bataille R, Amiot M . (1999). IL-6 up-regulates Mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway. Eur J Immunol 29: 3945–3950.
Qin JZ, Ziffra J, Stennett L, Bodner B, Bonish BK, Chaturvedi V et al. (2005). Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. Cancer Res 65: 6282–6293.
Raje N, Kumar S, Hideshima T, Roccaro A, Ishitsuka K, Yasui H et al. (2005). Seliciclib (CYC202 or R-roscovitine), a small-molecule cyclin-dependent kinase inhibitor, mediates activity via down-regulation of Mcl-1 in multiple myeloma. Blood 106: 1042–1047.
Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D et al. (2003). A phase 2 study of Bortezomib in relapsed, refractory myeloma. N Engl J Med 348: 2609–2617.
Rinkenberger JL, Horning S, Klocke B, Roth K, Korsmeyer SJ . (2000). Mcl-1 deficiency results in peri-implantation embryonic lethality. Genes Dev 14: 23–27.
Roccaro AM, Hideshima T, Raje N, Kumar S, Ishitsuka K, Yasui H et al. (2006). Bortezomib mediates antiangiogenesis in multiple myeloma via direct and indirect effects on endothelial cells. Cancer Res 66: 184–191.
Weng C, Li Y, Xu D, Shi Y, Tang H . (2005). Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem 280: 10491–10500.
Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI et al. (2005). Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19: 1294–1305.
Zamai L, Falcieri E, Zauli G, Cataldi A, Vitale M . (1993). Optimal detection of apoptosis by flow cytometry depends on cell morphology. Cytometry 14: 891–897.
Zhang B, Gojo I, Fenton RG . (2002). Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood 99: 1885–1893.
Zhou P, Qian L, Bieszczad CK, Noelle R, Binder M, Levy NB et al. (1998). Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 92: 3226–3239.
We are grateful for reagents provided by G Packham (University of Southampton School of Medicine, Southampton General Hospital, Southampton, UK). K Podar is the recipient of the Dunkin Donut's Rising Star Award 2007. This work was supported by a Multiple Myeloma Foundation (MMRF) Senior Research Grant Award (KP), National Institute of Health Grants RO CA 50947, PO-1 CA 78378, and P50 CA 10070; and the Doris Duke Distinguished Clinical Research Scientist Award (KCA).
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Podar, K., Gouill, S., Zhang, J. et al. A pivotal role for Mcl-1 in Bortezomib-induced apoptosis. Oncogene 27, 721–731 (2008) doi:10.1038/sj.onc.1210679
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