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Role of Caspases and CD95/Fas in the Apoptotic Effects of a Nucleotide Analog PMEG in CCRF-CEM Cells

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Role of Caspases and CD95/Fas in the Apoptotic Effects of a Nucleotide Analog PMEG in CCRF-CEM Cells ANTICANCER RESEARCH 30: 2791-2798 (2010) Role of Caspases and CD95/Fas in the Apoptotic Effects of a Nucleotide Analog PMEG in CCRF-CEM Cells HELENA MERTLÍKOVÁ-KAISEROVÁ, IVAN VOTRUBA, MARIKA MATOUŠOVÁ, ANTONÍN HOLÝ and MIROSLAV HÁJEK Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Gilead Sciences and IOCB Research Center, Prague, Czech Republic Abstract. Background/Aim: 9-[2-(phosphonomethoxy)ethyl] (S phase arrest) and induction of apoptosis as observed in guanine (PMEG) is a guanine acyclic nucleotide analog whose several leukemia cell lines (3). Recently, a PMEG prodrug, targeted prodrugs are being investigated for chemotherapy of GS-9219, has been shown to display significant in vivo lymphomas. Its antiproliferative effects have been attributed to antitumor effects in dogs with spontaneous non-Hodgkin's cell cycle arrest and induction of apoptosis, however, the lymphoma (4). The anticancer activity of a PMEG congener, underlying mechanisms remain poorly understood. The 9-[(2-phosphonomethoxy)ethyl]-2,6-diaminopurine objective of this study was to determine the requirements for (PMEDAP), has also been previously described in vivo in caspase and CD95/Fas activation in PMEG-induced apoptosis. spontaneous rat T-cell lymphoma (5, 6). Although apoptosis Additionally, the influence of PMEG on cell cycle regulatory induction appears to play a role in the antitumor activity of proteins was explored. Materials and Methods: CCRF-CEM PMEG, the underlying molecular mechanisms remain largely cells were exposed to PMEG with/without caspase inhibitor or unknown. anti-Fas blocking antibody and assayed for phosphatidyl serine Caspase activation is a frequent hallmark of apoptosis in externalization, mitochondrial depolarization and the cleavage many cell types (7). In most cases, caspase-mediated cell of procaspase 3 and the nuclear protein poly (ADP-ribose) death can be assigned to one of two independent pathways: polymerase (PARP). Results: Despite an observed increase of the intrinsic (mitochondrial) cascade or the extrinsic caspase 3, 8 and 9 proteolytic activity, neither pretreatment of (receptor) pathway. The intrinsic cascade involves mitocho- the cells with cell-permeable caspase inhibitors nor blocking ndrial depolarization, cytochrome c translocation from the death receptor with anti-Fas antibody did prevent apoptosis mito-chondria to cytosol and cleavage of procaspase 9 to induced by PMEG. Conclusion: PMEG-induced apoptosis is its active form, while the extrinsic (receptor) pathway caspase- and CD95/Fas-independent. requires binding of specific ligands to death receptors (CD95/Fas, TNFR) and is accompanied by caspase 8 9-[(2-Phosphonomethoxy)ethyl]guanine (PMEG) is a struc- activation (8). Both pathways merge while activating the tural analog of naturally occurring nucleotide dGMP and a downstream effector caspases (such as 3, 6 or 7) cleaving member of the family of acyclic nucleoside phosphonates various nuclear targets. (ANPs). Its potent antiproliferative properties (1) have been In the present work, the involvement of caspases and the largely attributed to the ability of its active metabolite, role of mitochondrial and CD95/Fas pathways in PMEG- PMEG diphosphate (PMEGpp), to be incorporated into induced apoptosis were studied in a T-cell lymphoblastic cell DNA, inhibit cellular replicative polymerases α, δ and ε, and line (CCRF-CEM). In addition, expression of various act as a DNA chain terminator (2). Perturbations in the DNA cyclins, cyclin-dependent kinases (CDKs) and cyclin-depen- replication process result in an altered cell cycle distribution dent kinase inhibitors (CDKIs) were explored in order to explain the molecular basis of cell cycle effects of PMEG and to aid the understanding of how the cell cycle effects contribute to the overall antitumor activity of PMEG. Correspondence to: Miroslav Hájek, Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nám. 2, 166 10 Materials and Methods Prague 6, Czech Republic. Tel: +420 220183393, Fax: +420 220183560, e-mail: [email protected] Materials. PMEG, was prepared according to Holý (9). The identity and purity of the compound was verified by means of NMR Key Words: Acyclic nucleoside phosphonate, programmed cell spectroscopy. All other chemicals and materials were obtained from death, CCRF-CEM cells, cell cycle. common commercial sources. 0250-7005/2010 $2.00+.40 2791 ANTICANCER RESEARCH 30: 2791-2798 (2010) Cell culture and proliferation assay. CCRF-CEM cells (ATCC Cell cycle analysis. Cells (106) were washed twice with PBS and CCL 119) were cultured in RPMI-1640 medium supplemented fixed with 70% ice-cold ethanol for 30 min. Fixed cells were with 10% (v/v) fetal calf serum, 200 μg/ml of streptomycin, 200 washed in PBS, treated with RNase A (500 μg/ml) for 30 min at U/ml of penicillin G and 4 mM glutamine under a humidified 37˚C and incubated with a staining solution containing 0.1% Triton 5 ® atmosphere containing 5% CO2 at 37˚C. A total of 10 cells per X -100 and propidium iodide (100 μg/ml) in PBS for 1 h. Cellular ml were seeded 24 h prior to an experimental treatment. Where a DNA content was determined using a flow cytometer FACSAria 6-day incubation was included, cells were subcultured and given (BD Biosciences, San Jose, CA, USA) and analyzed using ModFit fresh media and fresh compound at day 3. Cell proliferation was LT 3.0 program (Verity Software House, Topsham, ME, USA). assessed using Countess® Automated Cell Counter (Invitrogen Ltd, Paisley, UK). DNA fragmentation assay. A total of 1.5×106 cells were suspended in 500 μl of lysis buffer (100 mM Tris-HCl, pH 8.0, 10 mM EDTA, Quantitative RT-PCR. Total RNA from 106 cells was extracted using 2% SDS) containing 5 μl of proteinase K and incubated for 1 hour RNeasy Mini isolation kit (Qiagen GmbH, Hilden, Germany) at 56˚C. 200 μl of 5 M NaCl and 1/10 volume of 3 M sodium according to the manufacturer's protocol. The purity of RNA samples acetate were added followed by centrifugation at 15,000×g for 5 was characterized by their 260/280 and 260/230 absorption ratios, min. Supernatant was incubated with RNase A for 30 min, RT and which were at least 1.9 and 1.8, respectively. RNA integrity was the DNA was precipitated by adding two volumes of absolute verified using the Lab-on-a-Chip Agilent 2100 Bioanalyzer (Agilent ethanol at –20˚C overnight. The pelleted DNA was washed twice in Technologies, Santa Clara, CA, USA). cDNA was synthesized from 70% ethanol, air-dried and resuspended in 50 μl of TE-buffer (10 0.5 μg RNA using RT2 First Strand kit (SABiosciences, Frederick, mM Tris-HCl, pH 8.0, 1 mM EDTA). 0.6 μg of DNA were MD, USA) according to the manufacturer's protocol. Human Cell subjected to electrophoresis on a 0.7% agarose gel containing Cycle RT2-Profiler PCR array (SABiosciences) was then used for ethidium bromide and visualized under UV light. gene quantification employing DNA Engine Opticon® 2 thermocycler (Biorad, Hercules, CA, USA). Western blot analysis. A total of 107 cells were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Pierce, Determination of caspase activity. A total of 107 cells were lysed in Rockford, IL, USA) and centrifuged at 20,000×g. 30 μg of total 50 mM HEPES buffer pH 7.4, containing 0.3% CHAPS and 5 mM supernatant protein were loaded on a 12% polyacrylamide gel, DTT and incubated for 15 min on ice. The 20,000×g supernatant was electrophoresed and electroblotted onto a PVDF membrane. used for determination of caspase activity using the synthetic Membranes were blocked in 5% non-fat dry milk (Cell Signaling colorimetric substrates: N-Acetyl-Asp-Glu-Val-Asp-p-nitroaniline Technology (CST), Danvers, MA, USA) in TBS containing 0.05% (Ac-DEVD-pNA; Sigma, St. Louis, MO, USA), N-Acetyl-Ile-Glu- of Tween 20 (Pierce) and probed with anti-PARP or anti-cleaved Thr-Acp-p-nitroaniline (Ac-IETD-pNA; Sigma) and Leu-Glu-His- caspase 3 antibodies (CST) and appropriate HRP-conjugated Asp-p-nitroaniline (LEHD-pNA; Merck KGaA, Darmstadt, Germany) secondary antibodies (CST). SuperSignal® West Femto for caspase 3, 8 and 9, respectively. Each microplate well contained Chemiluminescent Substrate (Pierce) was used for signal detection 80 μl of assay buffer (20 mM HEPES pH 7.4, 0.1% CHAPS, 5 mM by CCD camera. DTT, 2 mM EDTA), 10 μl of cell lysate and 10 μl of the respective substrate (final concentration 200 μM). Caspase activity was Statistical evaluation. Unless otherwise indicated, the data are calculated from the slope of recorded absorbance at 405 nm, which presented as the mean±SEM from at least three independent experi- was compared to the standard curve of free pNA (Sigma). ments. Statistical evaluation was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego CA, Flow cytometric determination of mitochondrial membrane potential USA; www.graphpad.com). (ΔΨ). Cells (106) were washed with PBS and resuspended with 1 ml of pre-warmed culture media containing 1 μg/ml of JC-1 dye (5.5’, Results 6.6’ -tetrachloro-1.1’, 3.3’-tetraethylbenzimidazolyl-carbocyanine iodide). The cells were incubated for 15 min at 37˚C after which they were washed twice with PBS and immediately subjected to FACS PMEG induces concentration- and time-dependent apoptosis analysis. The fluorescent cationic dye is accumulated in the in CCRF-CEM lymphoblasts. PMEG-treated cells revealed mitochondria of polarized (‘healthy’) cells emitting red fluorescence an early (24 h) inhibition of cell growth, which became even (590 nm) due to formation of J-aggregates. Depolarized cells are more pronounced at later time points (72 h and 144 h) characterized by the presence of monomeric form of the dye in the (Figure 1).
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