Mechanistic Insight Into Taxol-Induced Cell Death

F Impens1,2, P Van Damme1,2, H Demol1,2, J Van Damme1,2, J Vandekerckhove1,2 and K Gevaert1,2

1Department of Medical Protein Research, VIB, Ghent, Belgium and 2Department of Biochemistry, Ghent University, Ghent, Belgium

We analysed the involvement of proteases during taxol- One class of chemotherapeutic drugs that induce such mediated cell death of human A549 non-small-cell lung alternative forms of PCD is microtubule-stabilizing carcinoma cells using a proteomics approach that specifi- agents with their prototypical representative taxol cally targets protein N termini and further detects newly (paclitaxel). Taxol and derivatives are used as potent formed N termini that are the result of protein processing. drugs against several solid tumors. Although their Our analysis revealed 27 protease-mediated cleavages, cytotoxic mechanism depends on cell type, concentra- which we divided in sites C-terminal to aspartic acid (Asp) tion and exposure duration, in most studies with and sites C-terminal to non-Aspresidues, as the result of clinically relevant taxol concentrations (10–200 nM), caspase and non-caspase protease activities, respectively. apoptosis is induced by blocking the mitotic spindle Remarkably, some of the former were insensitive to potent and a G2/M arrest (Schiff and Horwitz, 1980; Torres pancaspase inhibitors, and we therefore suggest that and Horwitz, 1998; Blagosklonny and Fojo, 1999; Zhao previous inhibitor-based studies that report on the caspase- et al., 2005). The signaling pathways leading to cell independent nature of taxol-induced cell death should be death have been extensively studied (Blagosklonny and judged with care. Furthermore, many of the sites C-terminal Fojo, 1999; Zhao et al., 2005) and recently several to non-Aspresidues were also uniquely observed in a model papers on the proteases involved were published. It is of cytotoxic granule-mediated cell death and/or found by now clear that taxol triggers apoptosis by both caspase- in vitro cataloging human l-calpain substrates using a dependent (Park et al., 2004; Ehrlichova et al., 2005; Li similar proteomics technique. This thus raises the hypothesis et al., 2005; Lu et al., 2005; Day et al., 2006; Janssen that killing tumor cells by chemotherapy or by immune cells et al., 2007; Pineiro et al., 2007) and caspase-indepen- holds similar non-Asp-specific proteolytic components with dent pathways (Broker et al., 2002, 2004; Huisman et al., strong indications to calpain activity. 2002; Ofir et al., 2002). One of the main supporting Oncogene (2008) 27, 4580–4591; doi:10.1038/onc.2008.96; observations for the latter is the failure of the published online 14April 2008 pancaspase inhibitor zVADfmk (N-benzyloxycarbonyl- Val-Ala-Asp(O-Me) fluoromethyl ketone) to protect Keywords: protease substrates; caspase-like activity; against taxol-induced apoptosis (Broker et al., 2002; taxol; cytotoxic granule Huisman et al., 2002). Here, we identified protease substrates targeted during taxol treatment of human A549 non-small-cell Introduction lung carcinoma cells using the peptide-centric proteomics technique known as N-terminal COFRADIC Chemotherapeutic agents kill tumor cells by different (COmbined FRActional DIagonal Chromatography). forms of programmed cell death (PCD). Next to This method specifically isolates (neo-)N-terminal pep- apoptosis with activation of the caspase cascade as the tides from digested proteomes before identification by most studied form, alternatives such as ‘apoptosis-like tandem mass spectrometry (Gevaert et al., 2003) and has PCD’ and ‘necrosis-like PCD’ have been described proven to be a powerful tool for substrate degradome (Leist and Jaattela, 2001). These modes of PCD are analysis (Lopez-Otin and Overall, 2002; Van Damme characterized by an essential activity of proteases other et al., 2005; Vande Walle et al., 2007): not only the than caspases during the onset or execution phase of cell protein substrates but also the exact cleavage sites death. For example, cathepsins (Foghsgaard et al., 2001; are simultaneously assigned, providing links to the Bidere et al., 2003; Broker et al., 2004), calpains functional implication of the cleavage events and to (Karmakar et al., 2007; Pineiro et al., 2007) and yet the type(s) of protease(s) creating the cleavage patterns. uncharacterized proteases (de Bruin et al., 2003) were reported to play key roles in different cell death models. Results Correspondence: Professor Dr K Gevaert, Department of Biochem- istry, Faculty of Medicine and Health Sciences, Ghent University, Treatment with taxol results in detachment and apoptosis A Baertsoenkaai 3, B-9000 Ghent, Belgium. E-mail: [email protected] of G2/M-arrested A549 cells Received 12 September 2007; revised 18 February 2008; accepted 5 Twenty-four hours after induction with 200 nM taxol, a March 2008; published online 14April 2008 fraction of A549 cells had detached from the culture Substrate degradomics of taxol-treated A549 cells F Impens et al 4581 dish and died during the next 24h, whereas a significant all parameters; however, the remarkable changes fraction of cells remained attached and did not show any between 24and 48h observed for detached cells as a apoptotic alterations, even after 48 h (Figure 1). Cell result of the execution phase of cell death are not cycle analysis by flow cytometry revealed that detach- present, pointing to stress conditions without triggering ment and associated cell death was the result of a mitotic cell death. Together, these observations clearly indicate arrest in the G2/M phase. This was not the case for the need to distinguish between detached and adherent adherent cells, where next to a limited increase in G2/M- A549 cell populations for further substrate degradomics. phase cells, a significant fraction of the cell population resided in other phases of the cell cycle (Figure 1). Besides the fact that the latter stopped dividing and that Lysosomal membrane permeabilization and activation stress granules were formed, no signs of PCD were of caspase-3 and calpains occur only in detached cells present. Next to caspases (Park et al., 2004; Ehrlichova et al., Time course flow cytometry experiments (Figure 2) 2005; Li et al., 2005; Lu et al., 2005; Day et al., 2006; further confirmed that detached cells were dying by Janssen et al., 2007; Pineiro et al., 2007), involvement of apoptosis, as a high proportion of these cells stained calpains (Pineiro et al., 2007) and cathepsin-B (Broker positive for annexin V and showed a hypoploid (sub- et al., 2004) was described in taxol-induced apoptosis. G1) DNA content at early time points (p24h). A large We compared taxol-treated detached and adherent fraction of these cells became positive for propidium A549 cells for activation of caspase-3 and calpains iodide (PI) only at late time points (X48 h). In adherent on immunoblots (Figure 3a). Lysosomal membrane cells, we measured a rather slow increase over time for permeabilization (LMP) and intracellular calcium

control 24h 200 nM taxol 48h 200 nM taxol

adherent cells adherent cells 1400 1400 1400

1120 1120 1120

840 G1 840 840 counts 560 S counts 560 counts 560 G2/M 280 280 sub-G1 280

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 red fluorescence red fluorescence red fluorescence

detached cells detached cells 1400 1400

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840 840

counts 560 counts 560

280 280

0 0 100 101 102 103 104 100 101 102 103 104 red fluorescence red fluorescence Figure 1 Taxol induces detachment and cell death of A549 cells suffering from mitotic arrest. Results of the analysis of the cell cycle by ﬂow cytometry are shown as overlay histograms (open line) with control cells (ﬁlled line). Microscopic analysis revealed that detached cells are present after 24h taxol treatment and further die during the next 24h. In contrast to cells that remain adherent, detached cells are exclusively present in the G2/M phase of the cell cycle after 24h and show a high sub-G1 population after 48h, indicating cell death by apoptosis.

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4582 detached cells adherent cells not shown). Activation of the classical calpains (m- and m-calpain) was initiated 24h after taxol treatment and 100 was clearly visible after 48 h as shown by the formation of smaller autolytic fragments of the common small 75 calpain subunit (Wood and Newcomb, 1999). Flow cytometry revealed that the activation of calpains was accompanied by an increase of intracellular calcium 50 concentration. Finally, by measuring AO ﬂuorescence, we observed LMP between 24and 48h in detached cells. 25 No evidence for the activation of proteases involved in cell death or LMP in adherent cells was obtained, in line with the fact that no PCD was observed in these 0 control 8h 16h24h 48h 72h cells (Figure 2). Based on these results, we screened for protease substrates in 48 h taxol-treated detached 100 A549 cells.

75 Screening by N-terminal COFRADIC in taxol-treated detached A549 cells identiﬁed 27 processed sites in 50 22 proteins Two proteomic screens were performed using N-terminal COFRADIC (Gevaert et al., 2003). In the % PI positive cells25 % annexin V positive cells ﬁrst experiment, we post-metabolically labeled the 16 samples at the peptide level with light ( O2, control) 18 0 or heavy ( O2, taxol-treated) isotopes, introducing a control 8h 16h24h 48h 72h mass difference of 4Da (Staes et al., 2004). In the second 100 screen, cells were metabolically labeled during cell culture by SILAC (stable isotope labeling by amino 12 acids in cell culture) (Ong et al., 2002) with light ( C6, 75 13 taxol-treated) or heavy ( C6, control) arginine, introducing a mass difference of 6 Da. In both setups, equal 50 amounts of control and taxol-treated samples were mixed before N-terminal peptide sorting and MALDI-

% sub-G1 cells 25 MS/MS (matrix assisted laser desorption ionization tandem mass spectrometry) or ESI-MS/MS (electrospray ionization tandem mass spectrometry) for the first 0 and second screens, respectively. control 8h 16h24h 48h 72h Using this differential setup, levels of N termini in Figure 2 Detached cells are dying from apoptosis, whereas control and treated cells were compared and singletons adherent cells are not. Flow cytometry analysis of detached (light or neo-N-termini in the proteome digest of detached bars) and adherent (dark bars) cells after treatment with 200 nM taxol for the indicated periods of time. The fraction of annexin V-, A549 cells specifically pointed to processed proteins PI- and sub-G1-positive cells was measured. Compared to adherent (Van Damme et al., 2005; Vande Walle et al., 2007) cells, for all measurements a larger fraction of the detached cells (Table 1). Two major classes were distinguished: (1) 16 stain positive, except for annexin V at late time points after taxol processed sites C-terminal to aspartic acid (Asp), 8 of treatment (X48 h). Adherent cells show a slow increase in all measured parameters over time but lack the significant fluxes which were previously reported among others in Fas- between 24and 48h observed for detached cells as a result from the treated Jurkat cells and are considered caspase cleavage execution phase of cell death. PI, propidium iodide. sites (Van Damme et al., 2005) and (2) 11 processed sites C-terminal to other residues, 10 of which are hitherto unreported. concentration were measured by flow cytometry as a decrease in red fluorescence of acridine orange (AO) and Caspases process proteins in taxol-treated A549 cells, an increase in green fluorescence of Fluo-4, respectively although their activity cannot completely be inhibited (Figure 3b). To validate our proteomics data, we selected proteins Detached cells treated for 48 h with taxol showed containing hitherto unreported cleavage sites and for activation of procaspase-3 as observed by the presence which antibodies were available (plectin, microtubule- of two processed forms of the large caspase-3 subunit. associated protein 1B (MAP1B) and clathrin light chain Consistent with previous studies, we confirmed that this A (CLCA)) to monitor alterations or destructions of was accompanied by activation of the intrinsic mito- epitopes by proteases using western blots. chondrial apoptotic pathway as observed by the Plectin migrates with an apparent molecular weight cleavage of Bid and release of cytochrome c in the much lower than estimated from its sequence containing cytosol (Huisman et al., 2002; Park et al., 2004) (data 4684 residues (Pytela and Wiche, 1980). For unexplained

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4583 A549 A549 A549 detached cells adherent cells control 200 nM taxol 200 nM taxol

24h 48h 24h 48h

37 kDa →

caspase-3 25 kDa → 20 kDa → 15 kDa →

25 kDa → p30 calpain 20 kDa →

detached cells adherent cells 150 150

120 120

90 90 LMP

counts 60 counts 60

30 30

0 0 100 101 102 103 104 100 101 102 103 104 red fluorescence -AO red fluorescence -AO

60 60

50 50

40 40 2+ 30 30 Ca counts counts 20 20

10 10

0 0 100 101 102 103 104 100 101 102 103 104 green fluorescence − fluo 4 green fluorescence − fluo 4 Figure 3 Protease activity is only present in detached cells. (a) Immunoblots on CHAPS lysates of taxol-treated detached or adherent A549 cells against caspase-3 and calpain small subunit. (b) Flow cytometry measurement of LMP and intracellular Ca2 þ by incubating cells with AO or Fluo-4. Filled histogram: control; gray line: 24 h taxol treatment; dark line: 48 h taxol treatment. After 48 h taxol treatment, activation of caspase-3 and calpains and lysosomal rupture are observed exclusively in detached cells. AO, acridine orange; LMP, lysosomal membrane permeabilization.

reasons, plectin migrated as a doublet. We observed Yet we observed the disappearance of epitopes in both a time-dependent appearance of a plectin fragment forms, which could indicate that processing of the (at ±250 kDa; Figure 4a) which, given the epitope isoform B was such that a neo-N-terminus was created recognized by the monoclonal antibody, may well that was not analysable by our technique. correspond to the expected C-terminal plectin fragment. None of these processing events were detected in Furthermore, this fragment shows a near-perfect over- lysates from adherent cells under taxol treatment, and lap with the predicted C-terminal globular domain of none of them, which, given the nature of the processed plectin for which a more normal electrophoretic sites, most likely resulted from caspase activity, were mobility is expected compared to the intact protein inhibited by zVADfmk (Figure 4a). Even the formation carrying an extensive rod domain. of a C-terminal epitope by cleavage of cytokeratin A similar time-dependent production of fragments or (KRT)18 at Asp-397 (Leers et al., 1999), considered disappearance of precursors was noticed for MAP1B highly diagnostic for caspase activity, was not pre- and CLCA (Figure 4a). For MAP1B, a fragment at vented. Also, cleavage of the classical caspase substrate ±210 kDa was generated in accordance with the size of poly (ADP-ribose) polymerase (PARP) to its p85 the N-terminal fragment formed by a cleavage at fragment was present in this condition (Figure 4a) position 1859 (Table 1). Experiments on the CLCA (Nicholson et al., 1995). These unexpected observations revealed two bands that were probably the intact indicate that zVADfmk did not work or that a isoforms A and B, both recognized by the antibody. signiﬁcant portion of caspase activity was not inhibited The identiﬁed cleavage site is unique for the isoform A. by caspase inhibitors.

Oncogene Oncogene 4584

Table 1 Protease cleavage sites identiﬁed in taxol-treated detached A549 cells

18 13 SwissProt ac- Identified peptide Start End Cleavage Protein description No. of single O2-labeled No. of single C6-labeled cession site peptides identified by peptides identified by MALDI-MS/MS (highest ESI-MS/MS (highest scorethreshold score) scorethreshold score)

(A) Cleavage sites C-terminal to an Asp residue cells A549 taxol-treated of degradomics Substrate P09496 Ac-GVMNGEYYQESNGPTDSYA 77 102 DAVDk Clathrin light chain A 1 (80–27) 3 (68–30) AISQVDR P63241 Ac-FETGDAGASATFPMQCSALR 7 26 DDLDk Eukaryotic translation initiation factor 5A-1 1 (55–31) P43243 Ac-GQSDENKDDYTIPDEYR 764 780 ENADk Matrin-3 (Van Damme et al., 2005) 1 (57–30) P46821 Ac-SGGKTPGDFSYAYQKPEETTR 1860 1880 LEKDk Microtubule-associated protein 1B 1 (35–30) Q9UNZ2 Ac-LIHDQDEDEEEEEGQR 78 93 SFRDk NSFL1 cofactor p47 1 (47–29) Q9H1E3 Ac-SGPPTKKIR 30 38 YGRDk Nuclear ubiquitous casein and cyclin-depen- 1 (44–29) dent kinases substrate (Van Damme et al., 2005) P55209 Ac-GLVETPTGYIESLPR 58 72 ERLDk Nucleosome assembly protein 1-like 1 1 (59–31) 1 (68–30) (Van Damme et al., 2005) Q15149 Ac-GPAAEAEPEHSFDGLR 2772 2787 DALDk Plectin 1 1 (47–30) 1 (38–31) Impens F P26599 Ac-AGMAMAGQSPVLR 173 185 AAVDk Polypyrimidine tract-binding protein 1 4(53–30) (Van Damme et al., 2005)

P53990 Ac-VGFTDDVKKGGPGR 198 211 DLIDk Protein KIAA0174(Van Damme et al., 2005) 1 (34–30) al et O15355 Ac-NEEAALLHEEATMTIEELLTR 139 159 DDVDk Protein phosphatase 2C isoform gamma 2 (79–31) P35637 Ac-GKEFSGNPIKVSFATR 356 371 DWFDk RNA-binding protein FUS (Van Damme 1 (44–30) et al., 2005) Q13501 Ac-IDVEHGGKR 257 265 IEVDk Sequestosome-1 1 (36–30) O94811 Ac-PSGKGKGKAGR 182 192 ERFDk Tubulin polymerization-promoting protein 1 (33–30) P08670 Ac-FSLADAINTEFKNTR 86 100 DSVDk Vimentin (Van Damme et al., 2005) 1 (40–29) 7 (93–31) Ac-VDVSKPDLTAALR 258 270 VQIDk 1 (34–30)

(B) Sites C-terminal to non-Asp residues P05787 Ac-YTSGPGSR 25 32 SSRSk Keratin, type II cytoskeletal 8 1 (32–31) Ac-LSPLVLEVDPNIQAVR 73 88 NQSLk 1 (59–30) Ac-SPLVLEVDPNIQAVR 7488 QSLL k 1 (52–33) P05783 Ac-SVQAPSYGAR 18 27 RSLGk Keratin, type I cytoskeletal 18 1 (44–33) Ac-VSSAASVYAGAGGSGSR 29 45 GARPk 3 (73–33) Ac-KSQDLAKIMADIR 241 253 VDAPk 1 (82–42) P08727 Ac-KLTMQNLNDR 81 90 AGNEk Keratin, type I cytoskeletal 19 2 (44–33) Q15513 Ac-VSEANSQTELLLR 30 42 LFQKk Protein SPHAR (S-phase response protein) 1 (36–33) Q9NQC3 Ac-AAPVPTAPAAGAPLMDFG 65 91 AGLSk Reticulon-42 (48–33) NDFVPPAPR P26599 Ac-AMAGQSPVLR 176 185 DAGMk Polypyrimidine tract-binding protein 1 1 (48–41) (Van Damme et al., 2005) Q15349 Ac-LEPVLSSNLAQRR 711 723 QAPRk Ribosomal protein S6 kinase alpha-2 1 (34–30)

Abbreviations: Asp, aspartic acid; ESI-MS/MS, electrospray ionization tandem mass spectrometry; MALDI-MS/MS, matrix assisted laser desorption ionization tandem mass spectrometry. SwissProt accession number, sequence of the identiﬁed neo-N-terminal peptide (‘Ac-’ denotes a-amino-acetylation), start and end position of the peptide in the protein sequence, the four amino acids preceding the cleavage site, protein description and the number of spectra by which each peptide was identiﬁed in both analyses (including the highest MASCOT scorethreshold score) are indicated. Sites previously reported in Fas-treated Jurkat cells are indicated. Substrate degradomics of taxol-treated A549 cells F Impens et al 4585 DEVD-ase activity A549 A549 1400 A549 detached cells adherent cells control 200 nM taxol 200 nM taxol 1200 24h 48h 48h 24h 48h 48h 1000 plectin 250 kDa → 800 MAP1B 250 kDa → 600 % of control

125 kDa → CLC 400

50 kDa → M30 200 37 kDa → KRT18

p116 → 0 PARP p85 → control

+ + 50 µM zVADfmk M zVAD M zVAD M zVAD M zVAD M zVAD µ µ µ µ µ 48h taxol 48h taxol + 48h taxol + 25 50 48h taxol + 48h taxol + 48h taxol + 100 200 500

A549 A549 detached cells control 200 nM taxol A549 A549 Jurkat A549 detached cells adherent cells Jurkat 50 ng/ml 48h 48h 48h 48h 48h control 200 nM taxol 200 nM taxol control α-Fas

plectin 24h 48h 48h 24h 48h 48h 6,5h 24h 250 kDa → 75 kDa → 50 kDa → M30 50 kDa → PTBP1 37 kDa → KRT18 37 kDa → + 25 µM zVADfmk

+ + 25 µM zVDVADfmk β 20 kDa → TIF1- fragment + 50 µM E64d 15 kDa → µ + + 50 µM zVADfmk + 50 M AEBSF + 50 µM pepstatin A

Figure 4 Three unreported Asp-specific processed sites could be confirmed but not inhibited. Immunoblot experiments were performed on CHAPS lysates of taxol-treated detached and adherent A549 cells or Fas-treated Jurkat cells. (a) zVADfmk did not inhibit processing of plectin, MAP1B and CLC and did not prevent the formation of the cytokeratin 18 M30 epitope (M30 KRT18) and cleavage of PARP. (b) Cleavage of two known caspase substrates, PTBP1 and TIF1-b, was prevented by zVADfmk. (c) Measurement of the DEVDase activity in taxol-treated A549 cells: zVADfmk was very efficient in inhibiting the DEVDase activity after taxol treatment, at the concentrations used for the immunoblot experiments further validating its activity. (d) zVADfmk, zVDVADfmk, E-64d, AEBSF and pepstatin A were added to evaluate the effect of inhibition of caspases, caspase-2, cysteine proteases, serine proteases and aspartic proteases, respectively. None of the inhibitors was able to prevent processing of plectin or formation of M30 KRT18. AEBSF, aminoethylbenzenesulfonyl fluoride hydrochloride; CLC, clathrin light chain; KRT, cytokeratin; MAP1B, microtubule-associated protein 1B; zVADfmk, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone; zVDVADfmk, N-benzyloxycarbonyl-Val-Asp(O-Me)-Val-Ala-Asp(O-Me) fluoromethyl ketone.

We ruled out the former, as in taxol-treated detached cells polypyrimidine tract-binding protein 1 (PTBP1) A549 A549 and TIF1-b, two known caspase substrates (Van A549 detached cells adherent cells control 200 nM taxol 200 nM taxol Damme et al., 2005), were cleaved with the generation of correctly sized fragments (also observed in Fas- 24h 48h 24h 48h treated Jurkat cells, see Figure 4b) and these cleavages were inhibited by zVADfmk. Furthermore, in lysates of 50 kDa → taxol-treated cells, zVADfmk concentrations as low as KRT8 25 mM completely abolished general DEVDase activity 37 kDa → (Figure 4c). To exclude the possibility of protein processing being done by caspase-2, a member of the caspase family poorly inhibited by zVADfmk (Garcia- 50 kDa → Calvo et al., 1998), we used its specific inhibitor → KRT18 zVDVADfmk (N-benzyloxycarbonyl-Val-Asp(O-Me)- 37 kDa Val-Ala-Asp(O-Me) fluoromethyl ketone) and showed that plectin processing and formation of the M30 25 kDa → KRT18 epitope were again not inhibited. Additionally, Figure 5 Fragmentation of KRT8 and KRT18 was confirmed by broad-spectrum protease inhibitors such as E64-d immunoblotting proteins from taxol-treated detached A549 cells. (cysteine proteases), AEBSF (serine proteases) and KRT, cytokeratin.

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4586 pepstatin (aspartate proteases) had no or limited effect for 20 min at 37 1C with 200 nM human m-calpain (Figure 4d). (treated) or 1 mM EDTA (control) (F Impens et al., unpublished data). Taken together, these results therefore indicate that classical calpains play a prominent Several non-Asp sites are found in cytotoxic role in both studied cell death models and that their granule-mediated cell death activity is responsible for the observed cleavage sites in In addition to the Asp-specific sites, 11 non-Asp KRT8 and KRT18. sites were identified in which cleavage occurred C-terminal to various residues, suggesting activities of several broad-spectrum proteases (Table 1). Selected cases were validated by immunoblotting in detached Discussion cells (Figure 5). Lysosomes share a lot of their protein content with Previous reports indicated the activation and involve- more specialized cytotoxic granules, which can be ment of different protease families in the execution of considered as secretory lysosomes in natural killer cells taxol-induced cell death. However, such studies were and CD8 þ cytotoxic T lymphocytes. Next to granule- generally fragmentary and often led to different conclu- specific proteases (for example, granzymes), several sions. To obtain a more general picture, we analysed ubiquitously expressed lysosomal proteases (for example, lysates from A549 cells treated with taxol for 48 h. Our cathepsins) reside in these granules (Smyth et al., 2001). analyses were unique, as both substrates and their Given the occurrence of LMP in our model (Figure 3) cleavage sites were identified, providing two levels of and the overlapping proteolytic content of lysosomes information: substrates link to possibly lost or altered and cytotoxic granules, we searched for common biological functions and cleaved sites may point to the substrates in other cell death models. Therefore, we proteases involved. compared the processed sites found here with those We showed that mitotic arrest, apoptosis and observed during Fas-induced apoptosis in human Jurkat proteolytic activity are exclusively observed in cells that cells (Van Damme et al., 2005), Omi-directed cleavage detached from the culture dish and not in adherent (Vande Walle et al., 2007) and a coculture of purified cells (Figures 1–3). Thus, not discriminating between natural killer cells with Fas-negative chronic myelogen- detached and adherent non-small lung cancer cells upon ous leukemia lymphoblast K562 cells in the presence taxol treatment (Broker et al., 2002, 2004) may lead to of zVADfmk and zDEVDfmk (P Van Damme et al., incomplete results. unpublished data). Almost exclusively in the latter, The protease substrates identified here comprise we found cleavages identical to those in taxol-induced proteins with different functions (Table 1). Cytoskeletal A549 cell death (Table 2). For instance, of the 38 sites proteins form an important though not dominant class. detected in dying K562 cells, 5 were also identified in The intermediate filament components vimentin and the A549 cells: in reticulon-4, PTBP1, KRT8 and KRT18. keratins 8, 18 and 19 are examples and the microtubule- Apart from cleavage of PTBP1 at Met-175 in Fas- interacting proteins MAP1B and TPPP/p25 represent a induced apoptosis in Jurkat cells (Van Damme et al., second cytoskeletal network. Plectin is the cytolinker 2005), these sites were never found in our studied interconnecting the different elements. Our results models. Furthermore, five additional sites were found in suggest disintegration of the cytoskeleton and could KRT8 and KRT18 of K562 cells (Table 2). It is worth explain loss of cellular adhesive capacity. However, mentioning that although we could confirm KRT8 and following 24h of taxol incubation, a stage where cells KRT18 processing by immunoblotting in A549 cells, we have already detached, very little proteolytic fragmenta- failed to do this in cell lysates of apoptotic K562 cells. tion of these cytoskeletal proteins was detected. There- This could be due to the low abundance of these fore, probably not extensive cytoskeletal fragmentation proteins in K562 cells and the overall greater sensitivity but rather the signaling cascades following mitotic arrest of mass spectrometry-based proteomics (Enoksson acting early in the cell death process could be the et al., 2007). primary cause of cell detachment. As suggested above, the striking substrate overlap Several substrates with important nuclear functions between taxol-induced A549 cell death and cytotoxic were identified: in particular, SPHAR (S-phase respon- granule-mediated K562 cell death points to activation of sive protein) and nucleosome assembly protein 1-like 1 a similar protease mechanism, most likely derived from (NAP-1). Loss of SPHAR function reduces the rate of lysosomal or cytotoxic granule origin. Additionally, we DNA synthesis during S phase and causes G2-phase found that calpains cannot be excluded as part of this prolongation (Digweed et al., 1995). NAP-1 is important common activity, as these proteases are activated not for chromatin assembly and cell cycle regulation. This, only in taxol-treated A549 cells (Figure 3) but also in the together with the microtubule-stabilizing activity of coculture model (data not shown). By screening for taxol, could reinforce mitotic arrest of taxol-treated substrates of human m-calpain in A549 cell lysates with cells. Previously, spliceosome components were found to N-terminal COFRADIC, we identified several KRT be the preferred protease targets (Van Damme et al., cleavage sites also reported here (Table 2). For this latter 2005). Here, we only identified RNA binding protein experiment, freeze–thaw lysates of A549 cells with a FUS (FUS) and PTBP1 cleaved at the same site as total protein concentration of 2 mg/ml were incubated found before, strongly suggesting the involvement of

Oncogene Table 2 Identical and additional non-Asp-specific sites identified in NK cell-mediated cell death of K562 cells and an in vitro catalogue of m-calpain substrates SwissProt Identified peptide Start End Cleavage Protein description Identified in Identified in Identified in accession site A549+taxol K562+NK A549 lysate+ m-calpain

P05787 Ac-YTSGPGSR 25 32 SSRSk Keratin, type II cytoskeletal 8 Ac-LSPLVLEVDPNIQAVR 73 88 NQSLk Ac-SPLVLEVDPNIQAVR 7488 QSLL k Ac-LVLEVDPNIQAVR 76 88 LLSPk Ac-VLEVDPNIQAVR 77 88 LSPLk Ac-LEVDPNIQAVR 78 88 SPLVk Ac-VDPNIQAVR 80 88 LVLEk Ac-SQISDTSVVLSMDNSR 237 252 RELQk Ac-SSFGSGAGSSSFSR 441 454 YSLGk Ac-FGSGAGSSSFSR 443 454 LGSSk Ac-SGAGSSSFSR 445 454 SSFGk P05783 Ac-GSVQAPSYGAR 17 27 YRSLk Keratin, type I cytoskeletal 18 Ac-SVQAPSYGAR 18 27 RSLGk Ac-VSSAASVYAGAGGSGSR 29 45 GARPk Ac-SAASVYAGAGGSGSR 31 45 RPVSk Ac-SGGLATGIAGGLAGMG- 60 90 GGMGk GIQNEKETMQSLNDR Ac-TGIAGGLAGMGGIQNE- 65 90 GGLAk KETMQSLNDR Ac-NEKETMQSLNDR 79 90 GGIQk usrt erdmc ftxltetdA4 cells Impens A549 F taxol-treated of degradomics Substrate Ac-KETMQSLNDR 81 90 IQNEk Ac-AQIFANTVDNAR 138 149 EDLRk Ac-KVIDDTNITR 187 196 HGLRk Ac-VDAPKSQDLAKIMADIR 237 253 LTVEk al et Ac-KSQDLAKIMADIR 241 253 VDAPk Ac-AQYDELAR 254261 ADIR k Ac-QSAEVGAAETTLTELR 285 300 VVTTk Ac-SAEVGAAETTLTELR 286 300 VTTQk Ac-AEVGAAETTLTELR 287 300 TTQSk Ac-AAETTLTELR 291 300 AEVGk Ac-LEIDLDSMR 306 314TVQS k P08727 Ac-KLTMQNLNDR 81 90 AGNEk Keratin, type I cytoskeletal 19 Q15513 Ac-VSEANSQTELLLR 30 42 LFQKk Protein SPHAR (S-phase response protein) Q9NQC3 Ac-AAPVPTAPAA- 65 91 AGLSk Reticulon-4 GAPLMDFGNDFVPPAPR P26599 Ac-LAASAAAVDAGMA- 164185 GNLA k Polypyrimidine tract-binding protein 1 MAGQSPVLR Ac-ASAAAVDAGMA- 166 185 LALAk MAGQSPVLR Ac-AMAGQSPVLR 176 185 DAGMk Q15349 Ac-LEPVLSSNLAQRR 711 723 QAPRk Ribosomal protein S6 kinase alpha-2

Abbreviations: Asp, aspartic acid; NK, natural killer. Oncogene 4587 Substrate degradomics of taxol-treated A549 cells F Impens et al 4588 caspases. Two further interesting substrates are NSFL1/ and, in particular, in models of taxol-induced cell p47 and reticulon-4 (Nogo), both associated with death. membranes or membrane proteins (Kondo et al., 1997; The second group of cleavage sites is characterized by Yan et al., 2006) and whose cleavage could be in line a diverse set of non-Asp residues at the P1 position, with the lysosomal permeability observed after taxol including small hydrophilic, bulky hydrophobic and treatment. Overall, our substrate list may be the source basic residues. Among these, we detected two processing for future experiments in which some of the hypotheses events C-terminal to proline. Several of these sites forwarded here could be verified. overlap with sites that were identified in a model of The 27 identified cleavage sites were divided into two cytotoxic granule-mediated cell death and with groups. The first group contains Asp sites located at an in vitro catalog of m-calpain substrates (Table 2 and caspase consensus motifs. It is important to note here F Impens et al., unpublished data). These findings thus that, as can be assessed by MEROPS (http://merops. suggest the action of a heterogeneous pool of proteases sanger.ac.uk), the ability of human intracellular pro- in which calpains play a prominent role, although teases to cleave after aspartic residues is mainly a feature cathepsins cannot be excluded. Therefore, we believe of caspases and granzyme B. Eight such Asp sites that our finding of an unusual number of common were reported previously (Van Damme et al., 2005), cleavage sites suggests overlapping mechanisms of whereas eight are new. Quite surprisingly, some of the protease release or activation in taxol-induced cell death latter (cleavage of MAP1B (LEKDk), CLC (DAVDk) and immune-based cytotoxicity. and plectin (DALDk)) were not inhibited by the well- known, potent caspase inhibitor zVADfmk (Figure 4a). Interestingly, when A549 lysates were assayed with the Materials and methods M30 KRT18-specific antibody in the presence of this inhibitor, the specific KRT18 fragment appeared, Materials indicating that cleavage at a site highly diagnostic for Taxol (from Taxus yunnanensis; Sigma-Aldrich, St Louis, MO, caspase activity was not blocked (Figure 4a). Likewise, USA), zVADfmk (Sigma-Aldrich), zVDVADfmk (R&D the remaining PARP cleavage was observed under these Systems, Minneapolis, MN, USA) and E64-d (loxistatin; conditions. When the same lysates were assayed with Biotrend Chemikalien, Cologne, Germany) were dissolved in anti-PTBP1 and anti-TIF1-b antibodies, inhibition of dimethyl sulfoxide and stored at 20 1C. A fresh solution in processing occurred (Figure 4b), indicating that our water was made before use for AEBSF (aminoethylbenzene- previous results were not due to inadequately working sulfonyl fluoride hydrochloride; Sigma-Aldrich). Pepstatin A (Serva, Heidelberg, Germany) was dissolved in 10% acetic acid pancaspase inhibitors but rather to kinetic effects in dimethyl sulfoxide. Protease inhibitor cocktail tablets were leading to failure of inhibition of certain members of from Roche Diagnostics (Mannheim, Germany). Protein the caspase family. The additional lower band observed concentrations were measured by the Bio-Rad DC Protein with the M30 KRT18 antibody in conditions with Assay Kit from Bio-Rad (Mu¨ nchen, Germany). Antibodies zVADfmk (Figure 4d) also points to such effects, as against the following proteins were used: caspase-3 (rabbit it suggests efficient inhibition of caspase cleavage at polyclonal, 1/4000; Stressgen, Ann Arbor, MI, USA), calpain Asp-238 but not at Asp-397 detected by the antibody small subunit (mouse monoclonal, 1/1000; Calbiochem, (assuming that cleavage at one of the non-Asp sites Nottingham, UK), PARP (mouse monoclonal, 1/3000; near the N terminus of the protein (Table 2) is Biomol, Plymouth, PA, USA), plectin (mouse monoclonal, responsible for the lower molecular weight band). Such 1/300; BD Biosciences, San Jose, CA, USA), MAP1B (mouse monoclonal, 1/750; Abcam plc, Cambridge, UK), CLC (mouse remaining caspase activity might explain the often monoclonal, 1/1000; Abcam), M30 KRT18 CytoDEATH contradictory results concerning caspase dependency in (mouse monoclonal, 1/1000; Alexis, Lausen, Switzerland), taxol-induced cell death (Broker et al., 2002, 2004; TIF1-b (KAP1, rabbit polyclonal, 1/2000; Abcam), PTBP1 Huisman et al., 2002; Ofir et al., 2002; Park et al., 2004; (mouse monoclonal, 1/500; Abcam), KRT8 (mouse mono- Ehrlichova et al., 2005; Lu et al., 2005; Day et al., clonal, 1/1500; Abcam), KRT18 (mouse monoclonal, 1/1500; 2006; Pineiro et al., 2007): in the presence of zVADfmk, Abcam). Secondary antibodies (1/2000 dilution) against mouse together with non-Asp-specific proteases, this activity and rabbit antibodies were from Abcam and from DAKOpatts will still contribute to cell death in which some (Copenhagen, Denmark) against goat antibodies. caspase cleavage events are prevented (for example, PTBP1 and TIF1-b), whereas others are still observed Cell culture, SILAC labeling and taxol treatment of A549 cells (for example, PARP and M30 KRT18; Figure 4). The A549 cells (ATCC, Manassas, VA, USA) were grown in F12- reason that caspase inhibitors are not eliminating all K medium (Invitrogen, Carlsbad, CA, USA) supplemented of their targeted protease activity can be due to the with 10% fetal calf serum (Lonza, Verviers, Belgium), 50 IU/ fact that in our model other classes of massively ml penicillin and 50 mg/ml streptomycin (Invitrogen). Trypsin- activated proteases, such as calpains and cathepsins, Versene (EDTA) (Lonza) was used to detach cells, except for might compete for their binding (inhibition of the proteome analyses where Versene (EDTA) solution (Lonza) was used to detach control cells. SILAC labeling cathepsins by zVADfmk has been described; Schotte was performed by growing cells for at least six population et al., 1999). Therefore, these results again illustrate doublings in arginine-free F12-K medium (Invitrogen), sup- that conclusions based on (unspecific) protease inhibi- plemented with 10% dialysed fetal calf serum (Invitrogen), tors should be judged with care in models where 50 IU/ml penicillin, 50 mg/ml streptomycin and 0.3 mM 12 13 many different proteases are activated simultaneously light ( C6, from normal F12-K medium) or heavy ( C6)

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4589 arginine.HCL (Cambridge Isotope Laboratories, Andover, Immunoblotting MA, USA). Harvested cells were lysed on ice for 15 min in 0.8% CHAPS, After treatment of subconfluent A549 cells with 200 nM 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic taxol, detached cells were harvested by spinning down cell acid; pH 7.4), 100 mM NaCl, 0.5 mM EDTA supplemented culture medium at 1500 g for 5 min at 4 1C. The collected cells with a complete protease inhibitor cocktail tablet (1/100 ml). were washed once with ice-cold phosphate-buffered saline Lysates were centrifuged for 15 min at 16 000 g at 4 1C and (4 1C) to remove media components and recentrifuged under pellets were discarded. Equal amounts of total protein were identical conditions. Adherent cells were washed once on plate separated on 4–12% Criterion XT gels in MOPS buffer (Bio- with ice-cold phosphate-buffered saline (4 1C) to remove any Rad) at 150 V and transferred overnight in 50 mM Tris 50 mM remaining detached cells and then harvested by trypsinization boric acid at 10 V onto a polyvinylidene difluoride membrane. and washed as described for detached cells. Wash steps were Protein detection was performed using standard protocols repeated twice for the proteome experiments. for membrane blocking, antibody incubation and film When taxol induction was started, control cells were development. typically passaged frac14; and cultured for another 48 h. As the dimethyl sulfoxide concentration in cell culture due to Protein extraction and COFRADIC isolation of N-terminal induction with 200 nM taxol was below 0.0005% and had no peptides cytotoxic effect or influence on cell growth, the vehicle was For both proteomic screens, A549 cells were treated for 48 h added only to control cells in the highest concentration when with 200 nM taxol. Detached cells as well as untreated control experiments with protease inhibitors were performed. cells were harvested as described above. Cells were lysed on ice for 15 min in the CHAPS lysis buffer as described above. After 1 Cell culture and Fas treatment of Jurkat cells centrifugation at 16 000 g for 15 min at 4 C, the total protein Experiments were performed as described before (Van Damme concentration in the supernatant was measured and the et al., 2005). protocol was continued with equal amounts of total protein material of control and treated sample (2 mg for both screens). For the first analysis, the unlabeled control and treated sample Cell cycle analysis and measurement of hypoploid DNA content were prepared separately until oxygen-18 labeling (Staes et al., For cell cycle analysis, the protocol of Krishan (1990) was 2004), whereas for the second analysis, SILAC-labeled followed. Isolated nuclei stained with PI were measured by proteome samples were mixed directly after lysis. Isolation flow cytometry upon excitation at 488 nm (FACScalibur of N-terminal peptides by COFRADIC was performed as using CELLQuest software from BD Biosciences). Measure- described previously (Gevaert et al., 2003; Van Damme et al., ment of the hypoploid DNA content was performed the 2005). same way, except that whole cells were analysed (cells were frozen at 80 1C in the presence of 20 mg/ml PI in phosphate- MS/MS analysis buffered saline before FACS measurement). The result of MALDI-MS/MS was performed on oxygen-16/18-labeled one experiment is shown as the average value of at least three samples as described before, except for some minor changes biological replicates. Trends were confirmed in an independent in the automated MS/MS measurements using the Bruker analysis. Ultraflex II TOF/TOF MALDI mass spectrometer under control of Bruker’s WarpLC software (Gevaert et al., 2006): after MS measurement, 3937 MS/MS spectra were recorded Annexin V and PI measurement from peptides present as singletons or as significantly regulated The annexin V FITC Apoptosis Detection kit from Calbio- couples (as assessed by the WARP-LC 1.0 software). LC-ESI- chem was used. A549 cells were treated with taxol as described MS/MS (liquid chromatography ESI-MS/MS) analysis was above. Samples were prepared and measured by flow performed on SILAC-labeled samples as described before cytometry according to the manufacturer’s instructions. The (Ghesquiere et al., 2006). Here, 24022 MS/MS spectra were result of one experiment is shown as the average value of at recorded. least two biological replicates. MASCOT peptide identification Measurement of LMP and intracellular calcium Fragmentation spectra were searched using a local version of A549 cells were treated with taxol as described above. Cells MASCOT (http://www.matrixscience.com). Spectra were were loaded by the addition of AO and Fluo-4(Invitrogen) searched in the human SwissProt database (version 49.0 directly to cell culture media (5 mg/ml AO, 12.5 mM Fluo-4) (MALDI-MS/MS) or version 50.4(ESI-MS/MS), downloaded 30 min before measurement by flow cytometry upon excitation from ftp://ftp.ebi.ac.uk/pub/databases/uniprot/knowledgebase/ at 488 nm (FACScalibur using CELLQuest software). LMP uniprot_sprot.dat.gz) and the human International Protein was measured as a decrease in red AO fluorescence (Zhao Index (IPI) database (version 3.14(MALDI-MS/MS) or et al., 2000), whereas an increase in intracellular Ca2 þ was version 3.19 (ESI-MS/MS), downloaded from ftp://ftp. measured as an increase in green Fluo-4fluorescence (Pineiro ebi.ac.uk/pub/databases/IPI/current/ipi.HUMAN.dat.gz) as et al., 2007). well as in their N-terminally truncated peptide databases made by DBtoolkit (Martens et al., 2005). Peptide identifications were performed as described before (Van Damme et al., 2005; Measurement of DEVDase activity Vande Walle et al., 2007). For the MALDI-MS analysis, we The caspase-glo 3/7 assay from Promega was used according identified 270 spectra belonging to 224unique peptides in 193 to the manufacturer’s instructions. A549 cells were treated unique proteins. For the ESI-MS/MS analysis, we identified with taxol in 96-well plates, and therefore no distinction was 2227 spectra belonging to 1219 unique peptides in 897 unique made between detached and adherent cells. The result of one proteins. All peptide identifications are made publicly available experiment is shown as the average value of four biological via the PRIDE database (http://www.ebi.ac.uk/pride/) (experi- replicates and was confirmed in an independent analysis. ment numbers 2408 and 2409).

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4590 Abbreviations Acknowledgements

AO, acridine orange; CLC, clathrin light chain; COFRADIC, FI is a Research Assistant of the Fund for Scientific COmbined FRactional DIagonal Chromatography; KRT, Research—Flanders (Belgium). This work was supported by cytokeratin; LMP, lysosomal membrane permeabilization; grants to JV and KG from the Inter University Attraction MAP1B, microtubule-associated protein 1B; PCD, pro- Poles (IAP-Phase VI), the Concerted Research Actions of the grammed cell death; PTBP, polypyrimidine tract-binding Ghent University and the Fund for Scientific Research— protein; SILAC, stable isotope labeling by amino acids in cell Flanders (Belgium). We thank Professor J Tavernier for culture. providing the flow cytometry facilities.

References

Bidere N, Lorenzo HK, Carmona S, Laforge M, Harper F, Dumont C caspase-independent routes in the non-small cell lung cancer cell line et al. (2003). Cathepsin D triggers Bax activation, resulting in NCI-H460. Clin Cancer Res 8: 596–606. selective apoptosis-inducing factor (AIF) relocation in T lympho- Janssen K, Pohlmann S, Janicke RU, Schulze-Osthoff K, Fischer U. cytes entering the early commitment phase to apoptosis. J Biol Chem (2007). Apaf-1 and caspase-9 deficiency prevents apoptosis in a Bax- 278: 31401–31411. controlled pathway and promotes clonogenic survival during taxol Blagosklonny MV, Fojo T. (1999). Molecular effects of paclitaxel: treatment. Blood 110: 3662–3672. myths and reality (a critical review). Int J Cancer 83: 151–156. Karmakar S, Banik NL, Patel SJ, Ray SK. (2007). Garlic compounds Broker LE, Huisman C, Ferreira CG, Rodriguez JA, Kruyt FA, induced calpain and intrinsic caspase cascade for apoptosis Giaccone G. (2002). Late activation of apoptotic pathways plays a in human malignant neuroblastoma SH-SY5Y cells. Apoptosis 12: negligible role in mediating the cytotoxic effects of discodermolide 671–684. and epothilone B in non-small cell lung cancer cells. Cancer Res 62: Kondo H, Rabouille C, Newman R, Levine TP, Pappin D, Freemont P 4081–4088. et al. (1997). p47 is a cofactor for p97-mediated membrane fusion. Broker LE, Huisman C, Span SW, Rodriguez JA, Kruyt FAE, Nature 388: 75–78. Giaccone G. (2004). Cathepsin B mediates caspase-independent cell Krishan A. (1990). Rapid DNA content analysis by the propidium death induced by microtubule stabilizing agents in non-small cell iodide-hypotonic citrate method. Methods Cell Biol 33: 121–125. lung cancer cells. Cancer Res 64: 27–30. Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B Day TW, Najafi F, Wu CH, Safa AR. (2006). Cellular FLICE-like et al. (1999). Immunocytochemical detection and mapping of a inhibitory protein (c-FLIP): a novel target for Taxol-induced cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol apoptosis. Biochem Pharmacol 71: 1551–1561. 187: 567–572. de Bruin EC, Meersma D, de Wilde J, den Otter I, Schipper EM, Leist M, Jaattela M. (2001). Four deaths and a funeral: from caspases Medema JP et al. (2003). A serine protease is involved in the to alternative mechanisms. Nat Rev Mol Cell Biol 2: 589–598. initiation of DNA damage-induced apoptosis. Cell Death Differ 10: Li R, Moudgil T, Ross HJ, Hu HM. (2005). Apoptosis of non-small- 1204–1212. cell lung cancer cell lines after paclitaxel treatment involves the Digweed M, Gunthert U, Schneider R, Seyschab H, Friedl R, Sperling BH3-only proapoptotic protein Bim. Cell Death Differ 12: 292–303. K. (1995). Irreversible repression of DNA synthesis in Fanconi Lopez-Otin C, Overall CM. (2002). Protease degradomics: a new anemia cells is alleviated by the product of a novel cyclin-related challenge for proteomics. Nat Rev Mol Cell Biol 3: 509–519. gene. Mol Cell Biol 15: 305–314. Lu KH, Lue KH, Liao HH, Lin KL, Chung JG. (2005). Induction of Ehrlichova M, Koc M, Truksa J, Naldova Z, Vaclavikova R, Kovarr caspase-3-dependent apoptosis in human leukemia HL-60 cells by J. (2005). Cell death induced by taxanes in breast cancer cells: paclitaxel. Clin Chim Acta 357: 65–73. cytochrome c is released in resistant but not in sensitive cells. Martens L, Vandekerckhove J, Gevaert K. (2005). DBToolkit: Anticancer Res 25: 4215–4224. processing protein databases for peptide-centric proteomics. Bioin- Enoksson M, Li J, Ivancic MM, Timmer JC, Wildfang E, Eroshkin A formatics 21: 3584–3585. et al. (2007). Identification of proteolytic cleavage sites by Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, quantitative proteomics. J Proteome Res 6: 2850–2858. Gallant M et al. (1995). Identification and inhibition of the ICE/ Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes CED-3 protease necessary for mammalian apoptosis. Nature 376: M et al. (2001). Cathepsin B acts as a dominant execution protease 37–43. in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol Ofir R, Seidman R, Rabinski T, Krup M, Yavelsky V, Weinstein Y 153: 999–1010. et al. (2002). Taxol-induced apoptosis in human SKOV3 ovarian Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, and MCF7 breast carcinoma cells is caspase-3 and caspase-9 Thornberry NA. (1998). Inhibition of human caspases by independent. Cell Death Differ 9: 636–642. peptide-based and macromolecular inhibitors. J Biol Chem 273: Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey 32608–32613. A et al. (2002). Stable isotope labeling by amino acids in cell culture, Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas SILAC, as a simple and accurate approach to expression proteo- GR et al. (2003). Exploring proteomes and analyzing protein mics. Mol Cell Proteomics 1: 376–386. processing by mass spectrometric identification of sorted N-terminal Park SJ, Wu CH, Gordon JD, Zhong XL, Emami A, Safa AR. (2004). peptides. Nat Biotechnol 21: 566–569. Taxol induces caspase-10-dependent apoptosis. J Biol Chem 279: Gevaert K, Pinxteren J, Demol H, Hugelier K, Staes A, Van Damme J 51057–51067. et al. (2006). Four stage liquid chromatographic selection of Pineiro D, Martin ME, Guerra N, Salinas M, Gonzalez VM. (2007). methionyl peptides for peptide-centric proteome analysis: the Calpain inhibition stimulates caspase-dependent apoptosis induced proteome of human multipotent adult progenitor cells. J Proteome by taxol in NIH3T3 cells. Exp Cell Res 313: 369–379. Res 5: 1415–1428. Pytela R, Wiche G. (1980). High molecular weight polypeptides Ghesquiere B, Van Damme J, Martens L, Vandekerckhove J, Gevaert (270 000–340 000) from cultured cells are related to hog brain K. (2006). Proteome-wide characterization of N-glycosylation events microtubule-associated proteins but copurify with intermediate by diagonal chromatography. J Proteome Res 5: 2438–2447. filaments. Proc Natl Acad Sci USA 77: 4808–4812. Huisman C, Ferreira CG, Broker LE, Rodriguez JA, Smit EF, Schiff PB, Horwitz SB. (1980). Taxol stabilizes microtubules in mouse Postmus PE et al. (2002). Paclitaxel triggers cell death primarily via fibroblast cells. Proc Natl Acad Sci USA 77: 1561–1565.

Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4591 Schotte P, Declercq W, Van Huffel S, Vandenabeele P, Beyaert R. in vivo protein processing during Fas-induced apoptosis. Nat (1999). Non-specific effects of methyl ketone peptide inhibitors of Methods 2: 771–777. caspases. FEBS Lett 442: 117–121. Vande Walle L, Van Damme P, Lamkanfi M, Saelens X, Vandekerc- Smyth MJ, Kelly JM, Sutton VR, Davis JE, Browne KA, Sayers TJ khove J, Gevaert K et al. (2007). Proteome-wide identification of et al. (2001). Unlocking the secrets of cytotoxic granule proteins. HtrA2/Omi substrates. J Proteome Res 6: 1006–1015. J Leukoc Biol 70: 18–29. Wood DE, Newcomb EW. (1999). Caspase-dependent activation of Staes A, Demol H, Van Damme J, Martens L, Vandekerckhove J, calpain during drug-induced apoptosis. J Biol Chem 274: 8309–8315. Gevaert K. (2004). Global differential non-gel proteomics by Yan R, Shi Q, Hu X, Zhou X. (2006). Reticulon proteins: emerging quantitative and stable labeling of tryptic peptides with oxygen-18. players in neurodegenerative diseases. Cell Mol Life Sci 63: 877–889. J Proteome Res 3: 786–791. Zhao J, Kim JE, Reed E, Li QQ. (2005). Molecular mechanism of Torres K, Horwitz SB. (1998). Mechanisms of Taxol-induced antitumor activity of taxanes in lung cancer (Review). Int J Oncol cell death are concentration dependent. Cancer Res 58: 27: 247–256. 3620–3626. Zhao M, Eaton JW, Brunk UT. (2000). Protection against oxidant- Van Damme P, Martens L, Van Damme J, Hugelier K, Staes A, mediated lysosomal rupture: a new anti-apoptotic activity of Bcl-2? Vandekerckhove J et al. (2005). Caspase-specific and nonspecific FEBS Lett 485: 104–108.

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