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[Cancer Research 60, 7039-7047, December 15, 2000]
© 2000 American Association for Cancer Research

Regular Articles

Identification of a Caspase-2 Isoform that Behaves as an Endogenous Inhibitor of the Caspase Cascade1

Nathalie Droin, Myriam Beauchemin, Eric Solary2 and Richard Bertrand2,3

Research Centre of the University of Montreal Hospital Centre, Notre Dame Hospital, Montreal Cancer Institute, Montreal, Quebec, H2L 4M1, Canada [N. D., M. B., R. B.], and INSERM U517, Faculty of Medicine and Pharmacy, 21033 Dijon, France [N. D., E. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2S-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last {alpha}-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-{alpha}) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The evolutionary conserved process known as apoptosis plays an essential role in normal development, tissue homeostasis, and immune responses, whereas its dysregulation is involved in the pathogenesis of various diseases (1, 2, 3, 4) . Apoptosis is also one of the cell-death mechanisms that can be triggered in cancer cells by cytotoxic drugs (5, 6, 7) . This death process usually requires the activation of a series of cysteine aspartate-specific proteases, referred to as caspases (8) . These enzymes are produced as zymogens, known as procaspases, that require proteolytic cleavage to constitute a catalytically active endopeptidase (9, 10, 11) . Fully mature caspases are heterotetramers that contain two large and two small subunits. In addition to the four subunits that constitute the active enzyme, procaspases contain an amino-terminal prodomain of varying length (12) . The caspase gene family contains 14 mammalian enzymes, of which 11 have been cloned in humans (11) . Some of these enzymes (caspase-1, -4, -5, and -11) are involved mainly in cytokine processing. Other caspases can be subdivided into two groups on the basis of their prodomain length. A simple view of the proteolytic cascade activated during apoptosis involves caspases with a long prodomain (caspase-8, -9, and -10) as initiator enzymes that cleave and activate effector caspases with a short prodomain (caspase-3, -6, and -7). In turn, these latter enzymes cleave a series of key cellular proteins, which results in cell dismantling. Actually, in vitro studies have shown that effector caspases can cleave and activate caspases with a long prodomain, suggesting feedback amplification loops (13) .

Activation of initiator caspases requires adaptor molecules that interact with their prodomains. These prodomains contain either a death effector domain, or DED,4 or a caspase recruitment domain, or CARD. Both the DED and the CARD function as interaction motifs that allow specific protein-protein interaction. Interaction between CARDs in the adaptor molecule APAF-1 and in the prodomain of procaspase-9 mediates the recruitment, oligomerization, and activation of the enzyme that, in turn, activates effector caspases such as caspase-3 and -7 (14, 15, 16, 17) . Similarly, interaction between DEDs in the adaptor FADD/MORT1 and the prodomain of procaspase-8 mediates the recruitment of the enzyme in a death-initiating signaling complex. FADD/MORT1 contains another protein-protein interaction domain known as the DD, also found in the COOH-terminal intracytoplasmic part of plasma membrane death receptors. Interaction between DDs comes after ligand-dependent receptor activation (18, 19, 20, 21, 22, 23) . All these interaction domains have a similar three-dimensional structure consisting of six or seven {alpha}-helices organized in an antiparallel arrangement (24, 25, 26) .

Human procaspase-2L, initially described as Ich-1 L (27) , is a long prodomain-containing enzyme that is activated in many cell types in response to various apoptotic stimuli, including growth factor withdrawal, DNA damaging agents, TNF-{alpha}, and Fas ligation (27, 28, 29, 30, 31) . The role of caspase-2L in the proteolytic cascade that leads to cell death remains poorly understood. The caspase-2L prodomain contains a CARD that interacts with a similar domain of the adaptor molecule RAIDD/CRADD (29 , 32) . This latter molecule also contains a DD that can associate with the DD of the serine/threonine kinase RIP (32) , an enzyme that interacts with TNFR-1 via TRADD (33) . RAIDD/CRADD was proposed to recruit procaspase-2L to TNFR-1 in a manner similar to the recruitment of procaspase-8 to the Fas death receptor via FADD/MORT1 (29 , 32) . However, whereas disruption of the CASP-8 gene in mice completely abolishes TNF-{alpha}-induced cell death, CASP-2 null mice do not exhibit any significant defect in TNF-{alpha} mediated apoptosis, which suggests that caspase-2L does not play a key role as an initiator enzyme in this cell death pathway (34 , 35) . In addition to its recruitment at TNFR-1, procaspase-2L has been shown to be released from mitochondria upon apoptotic stimulation, accumulating in the nucleus in which the protein colocalizes with RAIDD to subnuclear dot-like and filamentous structures, and migrating to the Golgi apparatus where it specifically cleaves golgin-160 (36, 37, 38, 39) . In a cell-free system established to reconstitute the proteolytic cascade activated by cytochrome c released from the mitochondria upon apoptotic stimuli, procaspase-2L was found to be cleaved by effector caspases such as caspase-3, which suggests its possible downstream involvement in the cascade (40) .

Whereas overexpression of procaspase-2L induces apoptosis in various cell types, a truncated isoform has been initially described whose overexpression prevents the death of cells deprived of growth factors (27) . Alternative splicing of CASP gene mRNA has been identified for several other caspases, including caspase-1, -6, -7, -8, -9, and -10 (22 , 23 , 41, 42, 43, 44, 45, 46, 47) . Ectopic transient overexpression of some of these isoforms demonstrated pro-apoptotic activity, because of either intrinsic proteolytic activity or the presence of a DED or a CARD involved in either the recruitment and autoprocessing of procaspases (10) or the formation of insoluble filamentous structures termed death effector filaments (36 , 48) . Conversely, overexpression of alternative spliced isoforms of the CASP-9 gene, which encode procaspase-9b, inhibits the activation of procaspase-9 by competitively interacting with the CARD of APAF-1 (46 , 47) , whereas caspase-8{alpha}3 impedes Fas- and TNFR-1-induced cell death (23) .

Here, we report the identification of two additional isoforms of the CASP-2 gene. These isoforms encode proteins designated caspase-2L-Pro and caspase-2S-Pro because they correspond to caspase-2L and to caspase-2S prodomain, lacking the last {alpha}-helix of their CARD domains. We focused on caspase-2L-Pro, which is shown to function as an endogenous apoptosis inhibitory molecule by interfering with procaspase-2L activation and the proteolytic cascade.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture.
All cell lines were obtained from the American Type Culture Collection (Manassas, VA) and grown in suspension culture in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin in an atmosphere of 95% air and 5% CO2 at 37°C. Cell culture products were purchased from Life Technologies, Inc. (Grand Island, NY). To ensure exponential growth, the cells were resuspended in fresh medium 24 h before each treatment. For DNA labeling, they were grown with [14C]-thymidine (0.03 µCi/ml) for 24 h, then chased overnight in isotope-free medium before to drug treatment.

Drugs and Chemicals.
Radioactive precursors [{alpha}-32P]-dCTP (>3000 Ci/mmol) and [2-14C]-thymidine (59 mCi/mmol) were procured from ICN BioMedicals (Costa Mesa, CA). Etoposide (VP16), CPT, cycloheximide, and recombinant TNF-{alpha} were purchased from the Sigma Chemical Co. (St. Louis, MO), and stock solutions made in Me2SO were stored at -80°C. The agonist anti-human Fas mAb (IgM, clone CH-11) was obtained from Upstate Biotechnologies (Lake Placid, NY). The specific fluorogenic derivative peptides Ac-DEVD-AMC, Ac-LEHD-AFC, Ac-VEID-AFC, z-VDVAD-AFC, and MCA-VDVADGWK(DNP)-NH2 were acquired from Bachem Bioscience Inc. (King of Prussia, PA) and Calbiochem-Novobiochem Corporation (San Diego, CA). All other chemicals were of reagent grade and purchased from Sigma Chemical Co., ICN BioMedicals, Roche Molecular Biochemicals (Montreal, Quebec, Canada), or other local sources.

cDNA Cloning, Site-directed Mutagenesis and Transfection.
Human CASP-2L-pro and CASP-2S-Pro cDNAs were first cloned by RT-PCR from mRNA using CASP-2L- and CASP-2S-specific adaptor primers containing NotI sequences at their respective ATG start and TGA stop codons. All amplified fragments were directly cloned into pTarget vector (Promega, Charbonnières, France) and sequenced according to the manufacturer’s instructions (Pharmacia Biotech, Orsay, France). The open reading frame of CASP-2L-pro cDNA was subcloned into pTarget vector by PCR with CASP-2L-pro-specific primers, PRO sense primer encoding HA epitope tag sequences (5'-ATGTATCCTTATGATGTTCCTGATTATGCTGCCGCTGACAGGGGACGCAGG-3') and PRO antisense primer sequences (5'-TCAGGAGTGCAAGGCTTCACAGAAGGCATCAAAAG CTTGGGG-3'). CASP-2L-pro site-directed mutagenesis was performed on the pTarget-HA-CASP-2L-pro plasmid, using primer PRO sense and primer MUT-2 L antisense (5'-TCAGGAGTGCAAGGCTGCACAGAAGGCAGCAAAAGCTTGGGG-3') containing the mutations. The pTarget-His-CASP-2L vector was generated by PCR using the CASP-2L-specific primers, with the sense primer encoding His repeated sequences. The pCDNA3-FLAG-RAIDD vector was obtained from Dr. V. M. Dixit (University of Michigan Medical School, Ann Arbor, MI).

In transient expression experiments, all transfections were performed by electroporation at 270 V (Gene Pulser; Bio-Rad, Hercules, CA) with 10 µg of each vector, and the cells were incubated for 48 h at 37°C before analysis. To generate stable transfected cell populations, 10 x 106 Namalwa cells were electroporated at 270 V with 10 µg of each plasmid, including empty pTarget vector or pTarget encoding HA-CASP-2L-Pro or HA-CASP-2L-ProMut. The electroporated Namalwa cells were selected during two months in RPMI 1640 media supplemented with 10% FCS and 2 mM L-glutamine in the presence of 1.5 mg/ml G418.

In Vitro Transcription/Translation Experiments and Production of Recombinant Caspase-2L-Pro Protein.
In in vitro transcription/translation studies, procaspase-2L, procaspase-2S and caspase-2L-Pro proteins were generated by TNT®-coupled transcription/translation assay, according to the manufacturer’s instructions (Promega), with purified pTarget-CASP-2L, pTarget-CASP-2S, and pTarget-HA-CASP-2L-pro plasmids. To generate recombinant protein, CASP-2L-pro cDNA was first amplified by PCR with specific adaptor primers containing EcoRI sequences at the ATG start codon and XhoI sequences at the TGA stop codon. The PCR product was inserted in the pCR-TOPO vector (TA cloning system; InVitrogen, San Diego, CA) and then subcloned in the bacterial expression vector pET-30a (+)-His-TAG (Novagen, Madison, WI) at the EcoRI and XhoI sites. Escherichia coli BL21 (DE3) was transformed with purified plasmids and recombinant protein expression induced for up to 15 h by adding 100 µM isopropylthio-ß-galactoside to exponentially growing bacteria at room temperature. The bacteria were collected by centrifugation, resuspended in 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), and samples were sonicated on ice. After centrifugation at 12,000 x g for 20 min, bacterial lysates were applied to a charged and equilibrated Chelating-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) chromatography column. The column was then washed with 10 v of 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), 6 v of 50 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), and the bound protein was eluted with 6 v of 1.0 M imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9). Proteins were concentrated and dialyzed several times in a Centricon Plus-20 centrifugal device (Millipore, Bedford, MA). The purity of recombinant caspase-2L-Pro was measured by SDS-PAGE and Coomassie Blue R-250 staining (not shown).

Expression Studies and Southern Blotting.
cDNAs from normal human tissues were obtained from InVitrogen, and genomic DNA from transfected lines was extracted by a salting-out procedure (49) . CASP-2L and CASP-2L-pro cDNAs were amplified with sense primer (5'-ATGGCCGCTGACAGGGGACGC-3') and antisense primer (5'-TCAGGAGTGCAAGGCTTCAC-3'). The presence of pTarget-HA-CASP-2L-pro and pTarget-HA-CASP-2L-ProMut in the transfected lines was monitored by PCR using sense and antisense primers directed to internal vector sequences. PCR was performed in a reaction mixture containing 200 µM 2'-deoxynucleotide 5'-triphosphate mix, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 ng of each primer, and 0.5 units of Taq DNA polymerase (Perkin-Elmer Biosystem, Markham, Ontario, Canada). The reaction mixtures were heated for 5 min at 94°C and amplified for 35 cycles with denaturation at 94°C for 50 s, annealing at 45°C for 50 s, and extension at 72°C for 30 s. The amplified products were electrophoresed on 1.2% agarose gel in Tris-acetate buffer (pH 8.0) and gel-stained with ethidium bromide. For Southern blot analysis, DNA was transferred by capillarity to GeneScreen nylon membranes (Dupont-NEM Research Products, Boston, MA). After DNA denaturation and fixation, blots were hybridized with a [{alpha}-32P]-dCTP labeled EcoRI-EcoRI Casp-2L-Pro DNA restriction fragment. Hybridizations were undertaken overnight at 42°C in a solution containing 2 x SSC, 2 x Denhart, 2% (w/v) SDS, 50% (v/v) formamide, and 100 µg/ml salmon sperm DNA. The blots were washed twice for 20 min at 42°C in 0.2 x SSC and 0.1% (w/v) SDS, then for 45 min at 56°C in 0.02 x SSC and 0.05% (w/v) SDS. DNA was visualized by autoradiography on Kodak X-AR film.

Protein Extraction, Coimmunoprecipitation and Western Blot Analysis.
The antibodies used in this study included anti-human procaspase-2 pAb that recognize the NH-2 terminal domain of procaspase-2 L [Ich-1(Ab-1); Calbiochem-Novabiochem Corporation], anti-HA peptide epitope tag mAb (clone 12CA5; Roche Molecular Biochemicals) and anti-FLAG peptide epitope tag mAb (M2; Upstate Biotechnologies). For protein extraction, the cells were washed twice in PBS, lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl, (pH 8.0), 0.1% Na-SDS, and 0.5% Na-desoxycholate in the presence of protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml pepstatin, 10 µg/ml aprotinin, 2.5 µg/ml trypsin inhibitor, and 5 µg/ml leupeptin) for 30 min, then centrifuged (20 min; 15,000 x g), and supernatants collected. For immunoprecipitations, control and transfected Namalwa cells were collected by centrifugation, washed twice with ice-cold PBS, and then homogenized in lysis buffer [50 mM Tris (pH 7.4); 150 mM NaCl; 1 mM NaVO4; 1% BSA; 1% NP40; 2 mM phenylmethylsulfonyl fluoride; 1 mg/ml aprotinin] at 4°C for 30 min. Extracts were centrifuged and supernatants collected. Specific anti-HA or anti-FLAG monoclonal antibodies were added for 1 h, and immunocomplexes were captured and precipitated with protein A/G-Sepharose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For Western blots, after SDS-PAGE and electrotransfer to nitrocellulose membranes (Amersham Pharmacia Biotech), the membranes were incubated for at least 2 h with 5% nonfat milk in PBST (PBS, 0.1% Tween 20) and for 2–3 h at room temperature with primary antibodies. Horseradish peroxidase-conjugated goat antirabbit or antimouse secondary antibodies were then added for 30–60 min, and proteins revealed by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) and autoradiography.

Caspase Activity Assays.
For control and drug-treated cell extracts, cells were homogenized at 4°C for 30 min in lysis buffer containing 100 mM Hepes (pH 7.5), 5 mM EDTA, 5 mM DTT, 20% glycerol (or 10% sucrose for caspase-6 extracts), and 0.3% NP40, centrifuged (10,000 x g for 15 min at 4°C), and supernatants collected and frozen at -80°C. Typically, 200 µg of extracted proteins per assay were used to monitor caspase activities.

For the cell-free system, cell-free extracts were generated from U937 cells based on methods described previously (40 , 50 , 51) . Briefly, 2–4 x 108 cells were pelleted and washed twice with cold PBS before a single wash with 5 ml of ice-cold cell extract buffer [20 mM Hepes-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 µM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 2 mg/ml aprotinin]. The cells were pelleted, resuspended in 2 v of ice-cold cell extract buffer and transferred to a 2-ml Dounce-type homogenizer. They were allowed to swell under hypotonic conditions for 20 min on ice. They were then disrupted with 20–50 strokes of a B-type pestle. Lysis was confirmed by examination of a small aliquot of the suspension under a light microscope. The lysates were centrifugated at 15,000 x g for 15 min at 4°C, and supernatants were frozen in aliquots at -80°C until required. Caspase activation was initiated by adding 50 µg/ml cytochrome c and 1 mM dATP in 100 µg cell-free extract per assay in the absence or presence of various amounts of purified recombinant caspase-2L-Pro protein.

Caspase activities were measured by monitoring fluorescence continuously in a dual luminescence fluorometer (LS 50B; Perkin-Elmer) using specific emission and excitation wavelengths for the fluorogenic derivative peptides z-VDVAD-AFC, Ac-DEVD-AMC, Ac-LEHD-AFC, Ac-VEID-AFC, and MCA-VDVADGWK(DNP)-NH2. Reactions were performed in cuvettes and temperature maintained at 37°C in a water-jacketed sample compartment. The assay mixture contained 100 mM Hepes (pH 7.5), 20% (v/v) glycerol [or 10% (v/v) sucrose for caspase-6 substrate], 5 mM DTT, 5 mM EDTA, and 100 µM fluorogenic peptide substrates. Enzyme activities were determined as initial velocities expressed as relative intensity/min/mg.

Quantitation and Analysis of DNA Fragmentation.
The kinetics of DNA fragmentation were measured by a filter DNA elution assay reported previously (52) . DNA was extracted by a salting-out procedure (49) . After electrophoresis in 1.8% agarose gel in Tris-borate-EDTA buffer (pH 8.0), DNA was visualized by ethidium bromide staining.

Yeast Two-Hybrid System.
The MatchMaker LexA 2-hybrid system (Clontech Laboratories, Inc., Palo Alto, CA) was used as described elsewhere (53) . CASP-2L-pro cDNA was subcloned in 2-hybrid plasmid pLexA vector, and Casp-2L, FADD, RAIDD and APAF-1 cDNAs were subcloned in the 2-hybrid plasmid pB42AD. Sequential transformations were performed by the lithium acetate method in yeast strain EGY48, and the cells were plated on histidine-deficient (for pLexA marker), tryptophan-deficient (for pB42AD marker), and histidine/tryptophan/leucine-deficient (for LEU2 reporter gene assay) minimal SD induction/selection media. APAF-1 cDNA was kindly provided by Dr. X. Wang (University of Texas Southwestern Center, Dallas, TX), FADD and RAIDD cDNAs by Dr. V. M. Dixit (University of Michigan Medical School). pLexA-p53, pLexA-Lam, and pB42AD-SV40Large T from Clontech Laboratories served as positive and negative controls.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning of CASP-2L-Pro cDNA.
A set of primers encompassing the open reading frame of Casp-2L consistently generated two amplified fragments of 1308 and 1090 bp by RT-PCR. Both fragments were amplified in several human cell lines (HL60, U937, K562, KCL22, Jurkat, BV173, and HT29) and hybridized with a specific CASP-2L probe (data not shown). Nucleotide sequence analysis revealed that the 1308-bp fragment encoded CASP-2L ORF, whereas the 1090-bp fragment represented a shorter variant form that was designated CASP-2L-pro (Fig. 1A)Citation . CASP-2L-pro contains the first 256 nucleotides of CASP-2L and then differs by an alternative splicing that deletes the next 218 nucleotides. The deletion generates a translational frameshift with a stop codon 18 nucleotides downstream (Fig. 1, A and B)Citation . CASP-2L-pro encodes a protein of 91 amino acids that contains the prodomain of procaspase-2L in which the CARD is deleted of the last {alpha}-helix and lacks the subsequent large and small subunits of procaspase-2L (Fig. 1C)Citation . The calculated molecular weight of caspase-2L-Pro was estimated at Mr 11,000, and the in vitro translated product migrated at Mr 15,000 on SDS gels (Fig. 1D)Citation . A similar variant isoform of CASP-2S was identified by RT-PCR using a set of primers encompassing the open reading frame of CASP-2S and was designated CASP-2S-Pro (data not shown).



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  		Fig. 1. Nucleotide and deduced amino acid sequences of caspase-2L-Pro. A, nucleotide sequence alignment of CASP-2L and CASP-2L-pro. GT- and AG-conserved splice sites are underlined in CASP-2L sequence. B, organization of CASP-2L-pro. GT- and AG-conserved splice sites are surrounded. CASP-2L and CASP-2L-pro stop codons are indicated. C, deduced amino acid sequence of caspase-2L-Pro compared with procaspase-2L. Boxes indicate {alpha}-helices of the caspase recruitment domain. D, Western blots of procaspase-2L, procaspase-2S, and HA-caspase-2L-Pro generated in vitro by TNT®-coupled transcription/translation assay. The translated products were fractionated on 12% SDS-PAGE, detected by anti-caspase-2 or anti-HA antibodies, and revealed by enhanced chemiluminescence.

 
Expression of CASP-2L-Pro.
On the basis of PCR and Southern blot analyses (Fig. 2, A and B)Citation , CASP-2L-pro was found to be constitutively expressed in a series of normal human tissues, including skin, breast, and colon, whereas its expression was virtually undetectable in lung, spleen, brain, testis, and ovary. A protein of Mr 15,000, corresponding to the Mr of caspase-2L-Pro, was also detected in cellular extracts prepared from human B lymphoma Namalwa, monocytic-like leukemic U937 and promyelocytic HL60 cells using polyclonal antibodies raised against the NH-2 terminal domain of procaspase-2L (Fig. 2C)Citation . These antibodies also recognized procaspase-2L as a Mr-48,000 protein (Fig. 2C)Citation . A protein of Mr 15,000 was also detected in HeLa and Jurkat cells, suggesting that caspase-2L-Pro is expressed in many cell types (data not shown).



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  		Fig. 2. Expression of caspase-2L-Pro in human tissues and cancer cell lines. A, PCR analysis of CASP-2L and CASP-2L-pro expression in normal human tissues. The PCR products were analyzed on agarose gel stained by ethidium bromide after electrophoresis. CASP-2L and CASP-2L-pro fragments are indicated by arrows. The 205-bp fragment observed in the ovary is a nonspecific PCR product. B, Southern blot analysis of PCR products using a [{alpha}-32P]-dCTP EcoRI-EcoRI Casp-2L-Pro DNA restriction fragment as probe. C, Western blot analysis of procaspase-2 L and caspase-2L-Pro in Namalwa, U937, and HL60 cell lines. Two hundred-µg proteins were loaded per lane. After electrophoresis and transfer, procaspase-2L and caspase-2L-Pro were detected by caspase-2 polyclonal antibodies raised against the prodomain of caspase-2L and revealed by enhanced chemiluminescence.

 
Caspase-2L-Pro Specifically Interacted with Procaspase-2L and RAIDD.
Previous studies revealed that procaspase-2 L interacted with the adaptor protein RAIDD/CRADD, an interaction that was suppressed by deletion of the prodomain of procaspase-2L (29 , 32) . In a yeast 2-hybrid assay, caspase-2L-Pro interacted with both procaspase-2L and RAIDD/CRADD, whereas it did not interact with FADD/MORT1 or APAF-1 (Fig. 3A)Citation . These interactions were also observed in cotransfected Namalwa cells where pTarget-HA-CASP-2L-pro was transiently transfected with either pCDNA3-FLAG-RAIDD or pTarget-His-CASP-2L. Immunoprecipitations with anti-FLAG or anti-HA monoclonal antibodies were followed by immunoblottings with anti-FLAG (Fig. 3B)Citation and anti-procaspase-2 antibodies (Fig. 3C)Citation . The caspase-2L-Pro/procaspase-2L interaction appeared weaker than the caspase-2L-Pro/RAIDD interaction, both in the yeast 2-hybrid assay and in cotransfected cells.



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  		Fig. 3. Caspase-2L-Pro interacts with procaspase-2L and RAIDD. A, in a yeast 2-hybrid assay, EGY 48 yeast cells containing caspase-2L-Pro fused to the LexA DNA-binding domain were grown in selective medium in the presence of various proteins fused to the B42 DNA activation domain, as indicated. The positive control in the two-hydrid assay represents pLexA p53 and pB42 SV40 T, and the negative control represents pLexA lamin C and pB42 SV40 T. B, Namalwa cells were transiently transfected by electroporation with empty pTarget, pCDNA3-FLAG-RAIDD, or cotransfected with pCDNA3-FLAG-RAIDD and pTarget-HA-CASP-2L-pro, as indicated. Coimmunoprecipitation experiments (IP) on transfected Namalwa cells were performed with anti-Flag or anti-HA antibodies, as indicated, and blotting was revealed by anti-Flag antibodies before enhanced chemiluminescence. In the control (C), coimmunoprecipitation experiments were undertaken by anti-HA antibodies and A/G-Sepharose beads. C, Namalwa cells were transiently transfected by electroporation with empty pTarget or cotransfected with pTarget-HIS-CASP-2 L and pTarget-HA-CASP-2L-pro. Immunoprecipitation experiments (IP) were performed by anti-HA antibodies (HA), and blotting was revealed by anti-caspase-2 antibodies before enhanced chemiluminescence. In the control (C), coimmunoprecipitation experiments were conducted with anti-HA antibodies (diluted) and A/G-Sepharose beads.

 
Caspase-2L-Pro Interfered with Caspase Activities in Vitro.
To determine whether caspase-2L-Pro could modulate the activation of other caspases, we used a cell-free system, described previously, in which the caspase cascade is activated by the addition of cytochrome c and dATP (40 , 50 , 51) . Cell-free extracts were prepared from untreated U937 cells, and caspase activities were monitored with a series of fluorogenic peptide substrates, suggesting activation of these caspases, in the absence or presence of recombinant caspase-2L-Pro protein. The addition of recombinant caspase-2L-Pro induced dose-dependent inhibition of z-VDVAD-AFC (caspase-2 L substrate), Ac-DEVD-AMC (caspase-3, -7, and -2L substrate), Ac-VEID-AFC (caspase-6 substrate) and Ac-LEHD-AFC (caspase-9 substrate) hydrolysis (Fig. 4)Citation . Recombinant Bcl-xL protein deleted of its COOH-terminal anchor transmembrane domain was used as a negative control in cellular extracts obtained from apoptotic U937 cells. The addition of recombinant Bcl-xL protein (up to 20 µg) had no inhibitory effect on caspase activities (data not shown; Ref. 54 ).



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  		Fig. 4. Caspase-2L-Pro interferes with caspase activities in a cell-free system assay. U937 cell-free extracts were incubated with cytochrome c/dATP in the absence or presence of different amounts of purified recombinant caspase-2L-Pro protein. Cleavage of Ac-LEHD-AFC (caspase-9 substrate), Ac-DEVD-AMC (caspase-3, -7, and -2L substrate), z-VDVAD-AFC (caspase-2L substrate) and Ac-VEID-AFC (caspase-6 substrate) was monitored. Enzyme activities were determined as initial velocities expressed as relative intensity/min/mg and the results expressed as the percentage of relative enzyme activity according to the formula [(Vi-Vo/Va-Vo) x 100], where Vi is the initial velocity measured in activated extract in the presence of various fixed amounts of inhibitors, Vo is the basal initial velocity measured in unactivated control extract, and Va is the initial velocity measured in activated extracts in the absence of inhibitors. Data points are representative of two independent experiments.

 
Transient Transfection of CASP-2L-Pro Induced Low Level of Apoptosis in Namalwa Cells.
Several point mutations in the prodomain sequence of procaspase-2L, including L41F, G78R, L89A, F85A, F82A, or D83A/E87A, were found to prevent procaspase-2L interaction with RAIDD, whereas D83A or E87A substitutions had no effect on this interaction (29) . By directed mutagenesis, we generated the CASP-2L-pro double mutant carrying D83A/E87A substitutions (HA-CASP-2L-ProMut, Fig. 5ACitation ). In transient cell death assays, transfection of wild-type CASP-2L-pro increased the level of DNA fragmentation and apoptosis in Namalwa cells, compared with cells transfected with the empty vector (Fig. 5B)Citation . In contrast, CASP-2L-ProMut transfection did not increase the level of DNA fragmentation and apoptosis in these cells (Fig. 5B)Citation , revealing the importance of a functional CARD domain for this effect.



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  		Fig. 5. Transient expression of caspase-2L-Pro induces apoptosis. A, schematic representation of HA-caspase-2L-Pro and HA-caspase-2L-ProMut carrying D83A and E87A. B, Namalwa cells were transfected with pTarget, pTarget-HA-CASP-2L-pro or pTarget-HA-CASP-2L-ProMut, and DNA fragmentation was quantitated by filter DNA elution assays 48 h after transfection. The results are expressed as the percentage of DNA fragmentation. Data points are the means of three independent experiments (n = 9); bars, SD.

 
Stable Expression of Caspase-2L-Pro Inhibited Apoptosis Induced by Various Stimuli.
Mixed populations of Namalwa cells transfected with empty pTarget, pTarget-HA-CASP-2L-pro and pTarget-HA-CASP-2L-ProMut vectors, were selected under 1.5 mg/ml Geniticin. PCR analysis of genomic DNA, using specific primers directed to vector sequences, demonstrated the presence of the insert in the selected cell population (Fig. 6A)Citation . Western blot analysis revealed the presence of endogenous procaspase-2L and exogenous HA-caspase-2L-Pro wild-type and double mutant in the transfected lines (Fig. 6B)Citation . To evaluate the effect of their expression, cells were exposed to various apoptotic stimuli, including cell death receptor agonists (anti-Fas mAb, TNF-{alpha}) and DNA topoisomerase I- (camptothecin, CPT) and II- (etoposide, VP16) inhibitors. The kinetics of DNA fragmentation were monitored by filter DNA elution assay and visualized by agarose gel electrophoresis. Overexpression of wild-type caspase-2L-Pro delayed the occurrence of DNA fragmentation induced by agonist Fas mAb, VP16, TNF-{alpha}, and CPT compared with empty vector-transfected cells, an effect that was partially or totally prevented by the presence of caspase-2L-ProMut (Fig. 6, C and D)Citation .



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  		Fig. 6. Caspase-2L-Pro overexpression inhibits apoptosis induced by different stimuli. Stable Namalwa variant cells transfected with empty, HA-CASP-2L-pro, and HA-CASP-2L-ProMut vectors were selected as bulk culture under 1500 µg/ml Geneticin, as indicated. A, PCR analysis of genomic DNA showing HA-CASP-2L-pro, and HA-CASP-2L-ProMut insertion in these mixed cell populations. B, Western blot analysis of procaspase-2L, HA-caspase-2L-Pro, and HA-caspase-2L-ProMut expression in these mixed cell populations. Proteins were detected by caspase-2 polyclonal antibodies and revealed by enhanced chemiluminescence. C, kinetics of DNA fragmentation were monitored by filter DNA elution assay in cells treated continuously with 50 ng/ml Fas mAb and 0.8 µg cycloheximide (upper left panel) or 50 ng/ml TNF-{alpha} and 0.8 µg cycloheximide (lower left panel), or after 30-min treatment with 20 µM VP16 (upper right panel) or 1.0 µM CPT (lower right panel). The results are expressed as the percentage of DNA fragmentation. Data points are the means of two independent experiments (n = 6); bars, SD. Symbols: pTarget ({circ}), pTarget-HA-CASP-2L-pro ({blacksquare}), and pTarget-HA-CASP-2L-ProMut ({square}) Namalwa variant cells selected under 1.5 mg/ml Geneticin. D, agarose gel electrophoresis of DNA extracted at the indicated times after VP16 treatment (20 µM; 30 min), from pTarget, pTarget-HA-CASP-2L-pro, and pTarget-HA-CASP-2L-ProMut Namalwa variant cells selected under 1.5 mg/ml Geneticin.

 
Caspase-2L-Pro Interfered with Caspase Activation in Etoposide-treated Cells.
The kinetics of caspase activities were monitored in transfected cells after VP16 treatment. Overexpression of caspase-2L-Pro interfered with activation of various caspases involved in the hydrolysis of Ac-LEHD-AFC (caspase-9 substrate), Ac-DEVD-AMC (caspase-3, -7 and -2L substrate), MCA-VDVADGWK(DNP)-NH2 (caspase-2L substrate) and Ac-VEID-AFC (caspase-6 substrate). In accordance with the kinetics of DNA fragmentation, inhibition of caspase activities observed in Namalwa cells overexpressing wild-type caspase-2L-Pro was either less significant [MCA-VDVADGWK(DNP)-NH2 hydrolysis] or absent (Ac-DEVD-AMC, Ac-VEID-AFC and Ac-LEHD-AFC hydrolysis) in cells overexpressing the D83A/E87A mutant (Fig. 7)Citation .



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  		Fig. 7. Kinetics of caspase activities after VP16 treatment. The kinetics of Ac-LEHD-AFC (caspase-9 substrate), Ac-DEVD-AMC (caspase-3,-7 and-2L substrate), MCA-VDVADGWK(DNP)-NH2 (caspase-2L substrate), and Ac-VEID-AFC (caspase-6 substrate) hydrolysis were monitored at the indicated times after VP16 treatment (20 µM; 30 min). Enzyme activities were determined as initial velocities expressed as relative intensity/min/mg, and the results are expressed as percent relative enzyme activity according to the formula [ (Vt/Vo) x 100] where Vt is the initial velocity measured in cell extracts prepared at specified times after drug treatment, and Vo is the initial velocity measured in cell extracts prepared from untreated control cells. Data points are representative of two independent experiments. Symbols are for pTarget ({circ}), pTarget-HA-CASP-2L-pro ({blacksquare}), and pTarget-HA-CASP-2L-ProMut ({square}) Namalwa variant cells selected under 1.5 mg/ml Geneticin.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several endogenous proteins that either directly or indirectly interfere with caspase activation have been identified in human cells. These proteins include six IAPs, known as NIAP, cIAP-1/HIAP-2/hMIHB, cIAP-2/HIAP-1/hMIHC, X-IAP, Survivin, and Apollon, that are structurally characterized by at least one BIR domain (55) . The majority of IAPs inhibit some members of the caspase family, either directly or indirectly, although they also demonstrated caspase-independent inhibitory mechanisms such as those mediated by activation of the transcriptional factor NF-{kappa}B (56, 57, 58) and those that involved a mitotic spindle checkpoint (59) . Another protein that interferes negatively with caspase activation is cellular FLICE-inhibitory protein (c-FLIP, also called Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT/Usurpin), an inactive homologue of caspase-8 and caspase-10 that may act as a competitive inhibitor by impeding the binding of these caspases to the cytosolic domain of death receptors (60) . In addition, the alternative spliced isoform of the CASP-9 gene, which encodes a short variant of procaspase-9, interferes negatively with procaspase-9 activation, whereas one isoform of CASP-8 (caspase-8{alpha}3) impedes Fas- and TNFR-1-mediated cell death (23 , 46 , 47) . In the present study, we noted that an alternative spliced isoform of the CASP-2 gene, which encodes a prodomain-only form of the caspase in which the CARD is deleted of the last {alpha}-helix, also behaves as an endogenous inhibitor of caspases.

Other CASP genes that have been shown to encode several isoforms include CASP-1, -6, -7, -8, -9, and -10 (22 , 23 , 41, 42, 43, 44, 45, 46, 47) . These isoforms are most likely splice variants (61) . Their expression depends on the tissue or the cell line examined. For example, caspase-1{epsilon} isoform is highly expressed in peripheral blood neutrophils and placenta (41) and the short variant isoform of the CASP-9 gene is detected mainly in skeletal muscle (46 , 47) ; whereas the various isoforms of the CASP-10 gene are expressed in several fetal tissues, mainly skeletal muscle, lung, and kidney, but are virtually undetectable in adult tissues (45) . We have described previously the heterogeneous expression of CASP-2L and CASP-2S isoforms in various acute myelogenous leukemia samples (62) . Here, we show that endogenous expression of CASP-2L-pro varies widely in normal human adult tissues, from very low levels in the lung, spleen, brain, testis, and ovary to higher expression levels in the skin and breast. The expression of caspase-2L-Pro also varies among diverse human leukemic cell lines. These results suggest that the physiological role of the various caspase isoforms in modulating apoptotic pathways might be highly tissue-specific.

Among the various caspase variants, caspase-2S was demonstrated to prevent apoptosis of Rat-1 cells when deprived of growth factors (27) , caspase-8{alpha}3 impeded Fas- and TNFR-1-mediated cell death (23) , and the recently described short variant of caspase-9, which lacks the catalytic site, was reported to negatively interfere with various apoptotic stimuli by competitively interacting with the CARD of APAF-1, thus preventing procaspase-9/APAF-1 interaction (46 , 47) . Conversely, the short isoform of caspase-10 designated as caspase-10/c, which is essentially a prodomain-only form of the caspase and also lacks proteolytic activity, can amplify the TNF-{alpha}-mediated death signal by inducing the formation of perinuclear filamentous structures (45) . The CASP-1 gene was reported to encode five isoforms, including three whose overexpression induces apoptosis, whereas the two others increase the survival of Baculovirus-infected cells (41) . In addition, stable overexpression of the Mr-11,500 prodomain of caspase-1 in HeLa cells was recently found to enhance Fas-mediated apoptosis upstream of caspase-8 activation (63) . However, the biological significance of caspase variants remains mostly unclear.

Like several caspase isoforms, the caspase-2L-Pro variant of caspase-2 lacks any catalytic site. With a yeast 2-hybrid system, we demonstrated that caspase-2L-Pro can interact with the adaptor protein RAIDD and, to a lesser extent, with procaspase-2L. The prodomain of procaspase-2 contains a single copy of CARD that was shown to interact with the CARD present in RAIDD, probably through electrostatic bond between the two domains (26) . RAIDD also contains a death domain in its COOH-terminal region that interacts with a death domain present in RIP, a serine/threonine kinase that is recruited to TNFR-1 and required for TNF-mediated activation of NF-{kappa}B (64) . It remains unclear whether interaction between RAIDD and procaspase-2 L is necessary for activation of caspase-2L and the downstream caspase cascade in response to TNF or other cytokines (12) . If this is the case, the interaction of caspase-2L-Pro with RAIDD could prevent interaction of procaspase-2L with the adaptor molecule, which could account for the decreased sensitivity of cells overexpressing caspase-2L-Pro to TNF-mediated cell death. Caspase-2L-Pro could also interfere with the formation of RAIDD-procaspase-2 L complexes that have been observed in the nucleus after treatment with various apoptosis inducers (38) . Regardless of the mechanism, mutations of caspase-2L-Pro that prevent RAIDD/procaspase-2L interaction suppress the protective effect of caspase-2L-Pro toward apoptosis induced by TNF-{alpha} and other death stimuli.

Although weaker than its interaction with RAIDD, interaction of caspase-2L-Pro with procaspase-2L could interfere with the procaspase-2L oligomerization that mediates its activation. Several recent studies have established that procaspase oligomerization can induce caspase activation (10 , 65 , 66) . Homodimerization of procaspase-1 or procaspase-2, which occurs before caspase processing, has been shown to require their respective caspase prodomain (67, 68, 69) . However, the prodomain-prodomain interaction alone is not sufficient to stabilize the dimers of Nedd2 precursors, the mouse homologue of human procaspase-2L. In addition to the prodomain, dimerization of Nedd2 precursor requires the COOH-terminus of the protein (69) . Thus, interaction of caspase-2L-Pro with the prodomain of procaspase-2L may not be sufficient to induce autoprocessing of the proenzyme but could prevent its homodimerization. This homodimerization is required for processing of the precursor caspase and activation, because prodomain-less caspase-2 does not undergo significant processing into active subunits (69) .

In transient cell killing assays, ectopic expression of caspase-2L-Pro induced very low levels of cell death by apoptosis. Transient expression of the prodomain of procaspase-8 has also been shown to induce low levels of apoptosis in both 293T and MCF-7 cell lines (23) . A similar observation was made by transiently expressing CARD- (29) and DED- (70 , 71) containing adaptor proteins in various cell lines. Overexpression of caspase-10/c (45) as well as that of the prodomain of caspase-1 (72) induced high levels of apoptosis in MCF-7 and 293T cell lines, respectively. Expression of caspase-10/c above an undefined concentration threshold results in the formation of insoluble filamentous cytosolic or subnuclear structures (45) . These structures apparently act as a scaffold to recruit and facilitate the autoactivation of cytoplasmic or nuclear procaspases (38 , 48) . A truncated RAIDD molecule consisting of the residues 1–80, which lacked helix 6 of the CARD, was unable to form filaments in transfected cells, suggesting that an intact CARD structure was required for filament formation (38) . Because caspase-2L-Pro does not contain the last {alpha}-helix, the formation of filamentous structures might not account for the limited toxicity of caspase-2L-Pro observed in transient transfection experiments.

Selected cells that stably overexpress caspase-2L-Pro showed significant inhibition of agonist Fas Ab-, TNF{alpha}-, etoposide- and camptothecin-induced apoptosis, indicating that caspase-2L-Pro acted as an inhibitory protein. Our observation that mutation of amino acids involved in caspase-2L-Pro interaction with RAIDD suppressed its anti-apoptotic activity is not sufficient to formally implicate RAIDD-procaspase-2L interactions in cell death pathways induced by these stimuli. Wild-type caspase-2L-Pro interfered with various caspase activities in both etoposide-treated cells and a cell-free system. Because no interaction was detected between caspase-2L-Pro and APAF-1 in the yeast 2-hybrid assay, caspase-2L-Pro may not impede the formation of APAF-1/procaspase-9 complex. We cannot rule out the possibility that caspase-2L-Pro interacts with other proteins in these pathways, e.g., caspase-3 or the recently described APAF-1 homologue designated CARD-4/NOD-1, a protein that interacts with and activates procaspase-9 (73 , 74) .

In conclusion, caspase-2L-Pro is a caspase variant that is expressed in several normal human tissues and cancer cell lines and negatively interferes with death receptor- and drug-mediated apoptosis by preventing caspase activation. In some cell types, caspase-2L-Pro could contribute to the prevention of procaspase-2L autoactivation by interfering either with its prodomain, thereby preventing dimerization of the proenzyme, or with an adaptor molecule such as RAIDD to prevent its recruitment. This caspase isoform could also prevent premature caspase activation by participating in the complex process that generates active subunits. Regardless of the mechanism of action of caspase-2L-Pro, these results indicate that regulation of CASP gene transcription and alternative splicing could play an important role in the modulation of apoptotic pathways, including those triggered by anticancer drugs and cytokines.


   ACKNOWLEDGMENTS
 
We acknowledge Ovid DaSilva (Research Center of the University of Montreal Hospital Center) for the editorial work on this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Medical Research Council of Canada (MT-15019) to R. B. The E. S. group is a labelized group from the Ligue Nationale Contre le Cancer. INSERM U517 receives the support of the Conseil Régional de Bourgogne and ARERS. R. B. is a chercheur-boursier du Fond de la recherche en santé du Québec. N. D. received support from the Ministère de l’Education Nationale, de l’Enseignement et de la Recherche, the Société Française d’Hématologie, and the French Ministère des Affaires Etrangères. Back

2 The two senior authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Research Centre of the University of Montreal Hospital Centre, Notre Dame Hospital, Montreal Cancer Institute, 1560 Sherbrooke Street East, Room Y-5634, Montreal, Quebec, H2L 4M1, Canada. Phone: (514) 281-6000, extension 6615; Fax: (514) 896-4689; E-mail: richard.bertrand{at}umontreal.ca Back

4 The abbreviations used are: DED, death effector domain; CARD, caspase recruitment domain; CPT, 20S-camptothecin lactone; DD, death domain; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin; Ac-LEHD-AFC, acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin; Ac-VEID-AFC, acetyl-Val-Glu-Ile-Asp-7-amino-4-trifluoromethylcoumarin; MCA-VDVADGWK(DNP)-NH2, 7-methoxycoumarin-4yl-acetyl-Val-Asp-Val-Ala-Asp-Gly-Trp-Lys-dinitrophenol-NH2; RT-PCR, reverse transcription-polymerase chain reaction; TNF-{alpha}, tumor necrosis factor-{alpha}; TNFR-1, tumor necrosis factor receptor-1; VP16, etoposide; z-VDVAD-AFC, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin; mAb, monoclonal antibody, IAP, inhibitor of apoptosis; pAb, polyclonal antibody; HA, hemagglutinin. Back

Received 6/ 5/00. Accepted 10/25/00.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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