GTMB 7 - Gene Therapy & Molecular Biology

Burek et al: Calcium induced cell deathconsidered to be involved predominantly in apoptoticsignalling (Sadowski-Debbing et al, 2002). In addition tothe role in apoptosis and inflammation, an involvement ofcaspases in other processes, like cell cycle regulation,hematopoesis and signal transduction in the immunesystem have been proposed (Denis et al, 1998; Los et al,2001). All caspases are synthesized as inactive zymogensthat are activated through proteolytic cleavage. Among thecaspase activation pathways, the best described ones arethe death-receptor dependent signalling cascades, withFADD adaptor molecule and caspase-8 as the key players,and the mitochondria/apoptosome dependent pathway thatrelies on Apaf-1 and caspase-9 (Krammer, 2000; Walczakand Krammer, 2000; Zheng and Flavell, 2000; Renz et al,2001). Both pathways are interconnected, thusamplification loops may take place (Sadowski-Debbing etal, 2002). The mitochondrial pathway is largely controlledby Bcl-2 family members. Bcl-2 family proteins exert itspro-and antiapoptotic action partially by influencingcalcium homeostasis of mitochondria and endoplasmicreticulum (ER) (reviewed in Hajnoczky et al, 2003).The family comprises both antiapoptotic andproapoptotic proteins. All antiapoptotic family members(e.g. Bcl-2, Bcl-X L ) share three or four Bcl-2 homology(BH) regions, and they localize to the cytoplasmic side ofintracellular membranes (Bouillet and Strasser, 2002). Theproapoptotic Bcl-2 family members can be further dividedinto two subgroups. Members of the first subgroup, bestrepresented by Bax and Bak (reviewed in Bouillet andStrasser, 2002) have two or three BH regions and appearto be structurally similar to their prosurvival relatives(Suzuki et al, 2000). The second subgroup of proapoptoticBcl-2-related proteins, (e.g. Bad, Bid, Bim) share only theshort BH3 region (reviewed in Bouillet and Strasser,2002). The exact mechanism of apoptosis regulation byBcl-2 family members is not fully understood (Strasser etal, 2000). It is widely believed that Bcl-2 functions topreserve the mitochondrial membrane integrity,mitochondrial and ER calcium homeostasis and preventthe release of cytochrome c and other proapoptoticmolecules from the mitochondria. BH3-only proteinsappear to sense stimuli that cause cellular stress andinitiate the death cascade. Proapoptotic Bax and Bak areessential for cell killing governed by BH3-only proteins,and this form of cell death is antagonized byoverexpresion of Bcl-2 (reviewed in Hajnoczky et al,2003; Marsden and Strasser, 2003).To gain insight into the mechanisms that governcalcium triggered cell death we have used a T-cellleukemiabased model and calcium ionophores asmodulators of intracellular Ca 2+ level. We show here thatthe calcium activated apoptotic pathway rely on yet-to-bedefined,caspase-8-dependent and Bcl-2-inhibitablepathway. Interestingly, the pathway does not rely onFADD-adaptor molecule. Thus, we provide furtherevidences for an intrinsic (death receptor-independent)death pathway that relies on caspase-8.II. Materials and methodsA. Materials and cell cultureAll cell lines were grown in 5% CO 2 at 37°C using aRPMI-1640 medium supplemented with 10% heat-inactivatedfetal calf serum and antibiotics (GIBCO, Eggenstein, Germany).A23187 was purchased from Sigma (Deisenhofen, Germany).The caspase inhibitor zVADfmk (benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone) was purchased from Enzyme SystemsProducts (Dublin, CA), and staurosporine from RocheBiochemicals (Mannheim, Germany). All other chemicals werefrom Merck KG (Darmstadt, Germany) or Roth (Karlsruhe,Germany). Stable transfectants of Jurkat cells overexpressingBcl-2 and Jurkat clone that was deficient in caspase-8 were akind gift of Dr. J. Blenis, (Harvard Medical School, Boston,Massachusetts, USA).B. Cell extracts and immunoblottingThe proteolytic processing of caspase-3 and caspase-8 wasdetected by immunoblotting. Briefly, 5 x 10 5 cells were seeded in6-well plates and treated with the apoptotic stimuli. After theindicated time, cells were washed in cold PBS and lysed in 1%Triton X-100, 50 mM Tris-HCl, pH 7.6 and 150 mM NaClcontaining 3 µg/ml aprotinin, 3 µg/ml leupeptin, 3 µg/mlpepstatin A and 2 mM phenylmethylsulfonyl fluoride (PMSF).Subsequently, the proteins were separated under reducingconditions by 12 % sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and electroblotted to a polyvinylidene difluoridemembrane (Amersham, Braunschweig, Germany). The equalloading of protein was controlled by measuring the proteinconcentration using the Bradford assay (BioRad, Munich,Germany). Membranes were blocked for 1 h with 5% non-fat drymilk powder in TBS and then incubated for 1 h with murinemonoclonal antibodies directed against caspase-3 (TransductionLaboratory, Heidelberg, Germany). Membranes were washedfour times with TBS/0.02% Triton X-100 and incubated with therespective peroxidase-conjugated affinity-purified secondaryantibody for 1 h. Following extensive washing, the reaction wasdeveloped by enhanced chemiluminescent staining using ECLreagents (Amersham).C. Fluorimetric assay of caspase activity -DEVD-ase assayCytosolic cell extracts were prepared by lysing cells in abuffer containing 0.5% NP-40, 20 mM HEPES pH 7.4, 84 mMKCl, 10 mM MgCl 2 , 0.2 mM EDTA, 0.2 mM EGTA, 1 mMDTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatinand 1 mM PMSF. Caspase activity was determined by theincubation of cell lysates with 50 µM of the fluorogenic substrateDEVD-AMC (N-acetyl-Asp-Glu-Val-Aspaminomethylcoumarin,Bachem, Heidelberg, Germany) in 200 µl buffer containing 50mM HEPES pH 7.3, 100 mM NaCl, 10% sucrose, 0.1% CHAPSand 10 mM DTT. The release of aminomethylcoumarin wasmeasured by fluorometry using an excitation wavelength of 360nm and an emission wavelength of 475 nm.D. Measurement of cell death and apoptosisCell death was measured either by the detection ofhypodiploid nuclei (Nicoletti method) (Renz et al, 2001) or bythe uptake of propidium iodide (PI) (Stroh et al, 2002). Briefly,for the measurement of hypodiploid DNA, nuclei were preparedby lysing 10 4 cells in 100 µl of hypotonic lysis buffer (1%sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI). The nuclei174