Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

Published OnlineFirst January 12, 2010; DOI: 10.1158/1541-7786.MCR-09-0373 Molecular DNA Damage and Cellular Stress Responses Cancer Research Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells Alex Lyakhovich1,2 and Jordi Surrallés1,3 Abstract Fanconi anemia (FA) is a rare syndrome characterized by developmental abnormalities, progressive bone marrow failure, and cancer predisposition. Cells from FA patients exhibit hypersensitivity to DNA cross-linking agents and oxidative stress that may trigger apoptosis. Damage-induced activation of caspases and poly ADP ribose polymerase (PARP) enzymes have been described for some of the FA complementation groups. Here, we show the constitutive activation of caspase-3 and PARP cleavage in the FA cells without exposure to exogenous DNA-damaging factors. These effects can be reversed in the presence of reactive oxygen species scavenger N- acetylcystein. We also show the accumulation of oxidized proteins in FA cells, which is accompanied by the tumor necrosis factor (TNF)-α oversecretion and the upregulation of early stress response kinases pERK1/2 and p-P38. Suppression of TNF-α secretion by the extracellular signal-regulated kinase inhibitor PD98059 results in reduction of caspase-3 cleavage, suggesting a possible mechanism of caspases-3 activation in FA cells. Thus, the current study is the first evidence demonstrating the damage-independent activation of caspase-3 and PARP in FA cells, which seems to occur through mitogen-activated protein kinase activation and TNF-α oversecretion. Mol Cancer Res; 8(1); 46–56. ©2010 AACR. Introduction Cells derived from FA patients are hypersensitive to cross- linking agents that are potent inducers of programmed cell Fanconi anemia (FA) is a genetic disorder characterized death (PCD or apoptosis; ref. 6). Studies in cohorts of FA by congenital abnormalities, bone marrow failure, and patients show that PCD is uncommon in the absence of marked cancer susceptibility (1, 2). Currently, 13 FA com- damaging factors and the major cause of mortality is bone plementation groups are known to exist, each of which re- marrow failure (7-9). This can be mediated by several indu- presents a FA subtype abbreviated as A (FANCA), B cers, including dissolved oxygen, superoxide and hydroxyl (FANCB), C (FANCC), D1 (FANCD1/BRCA2), D2 radicals, and other reactive oxygen species (ROS), and re- (FANCD2), E (FANCE), F (FANCF), G (FANCG), I sults in cell growth arrest or cell death (10-12). FA cells (FANCI/KIAA1794), J (FANCJ/BRIP1), L (FANCL), M are sensitive to ROS, thus acquiring abnormalities in several (FANCM), and N (FANCN; refs. 3, 4). With the exception redox status end points (13-15). FANCA and FANCG are of FANCD2 and FANCI, the encoded FA proteins have redox-sensitive proteins that are multimerized to form a nu- no similarities with each other but cooperate in a common clear complex in response to oxidative stress (16). Not sur- FA/BRCA pathway by the formation of subnuclear and prisingly, the survival of human primary FA BM cells can be nuclear complexes, where activation by monoubiquitinyla- ameliorated by reducing oxidative stress (14). tion of FANCD2 and FANCI seems to orchestrate a cas- Hypersensitivity to ROS often acts as a second messenger cade of events in response to DNA damage (5). in cellular signaling and is mediated by specific cysteine pro- teases, known as caspases (17). Sequential activation of caspases, on induction of PCD, has been shown in FA Authors' Affiliations: 1Department of Genetics and Microbiology, hematopoietic progenitor cells in response to mutagenic Universitat Autònoma de Barcelona, Barcelona, Spain; 2Chris Doppler stress (18). Caspases exist within the cell as zymogens (inac- Laboratory on Molecular Cancer Chemoprevention, Medical University tive proforms) that can be cleaved to form active enzymes of Vienna, Vienna, Austria; and 3Centre for Biomedical Research on Rare Diseases, Bellaterra, Spain following the induction of apoptosis (19). One of the first Note: Supplementary data for this article are available at Molecular Can- proteins identified as a substrate for caspases is poly ADP cer Research Online (http://mcr.aacrjournals.org/). ribose (PAR) polymerase (PARP; also known as PARP1 Corresponding Author: Jordi Surrallés, Department of Genetics and Mi- and adenosine diphosphate ribosyltransferase; ref. 20). crobiology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barce- PARP is involved in DNA damage repair and catalyzes the lona), Barcelona, Spain. Phone: 34-93-581-1830; Fax: 34-93-581-2387. synthesis and binding of PAR to DNA strand breaks and E-mail: [email protected] and Alex Lyakhovich, Christian Doppler Laboratory on Molecular Cancer Chemoprevention, 1090 Vienna, Austria. modifying nuclear proteins (21). The ability of PARP to re- Phone: 43-1-40400-6154; Fax: 43-1-40400-4724. E-mail: alex.lyakho- pair damaged DNA is prevented following cleavage of [email protected] PARP by caspase-3 (21). doi: 10.1158/1541-7786.MCR-09-0373 Present research is aimed at studying caspase-3 activity as ©2010 American Association for Cancer Research. well as PARP status in FA cells of several complementation 46 Mol Cancer Res; 8(1) January 2010 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/1541-7786.MCR-09-0373 Caspase-3/PARP Activation in Fanconi groups in the absence of exogenous DNA damage. We have dissolved in DMSO, was used at 50 μmol/L final concentra- shown higher caspase-3 activity and PARP cleavage in FA- tion. ROS inhibitor NAC, from Sigma, was used at 2 mmol/L D2 and other FA complementation groups compared with final concentration. WP9QY (Abgent, Inc.), an inhibitor of their corrected counterparts or in the small interfering TNF-receptors, was used at 1 μmol/L concentration. RNA (siRNA) FANCD2–depleted normal cells versus scRNA-transfected cells. Interestingly, the level of PARP Cells Treatment and Preparation of Cell Lysates cleavage and caspase-3 upregulation could be reduced on For siRNA experiments, we followed previously de- treatment of cells with ROS inhibitor, suggesting that oxi- scribed procedures with the same FANCD2 siRNA se- dative stress is one of the mediators in these events. We also quences (Invitrogen; ref. 22). Whenever necessary, the showed higher level of apoptosis in FA cells with PKC inhibitor bisindolylmaleimide I (GF109203X from corresponding accumulation of oxidized proteins. This Calbiochem) was added directly to the cell medium as was accompanied by tumor necrosis factor (TNF)-α over- DMSO-dissolved stock solution. Cells were washed with secretion and upregulation of early damage-response ki- ice-cold PBS and resuspended in buffer C consisting of nases p-ERK1/2 and p-P38. Moreover, suppression of 20 mmol/L HEPES (pH 7.9), 420 mmol/L KCl, 25% α TNF- secretion by extracellular signal-regulated kinase glycerol, 0.1 mmol/L EDTA, 5 mmol/L MgCl2,0.2% (ERK) inhibitor PD98059 results in the reduction of cas- Nonidet P-40, 1 mmol/L DTT, and a 1:40 volume of pro- pase-3 cleavage, suggesting a possible mechanism of cas- tease inhibitor mixture (Sigma) on ice for 30 min. After pases-3 activation in FA cells. The blocking of TNF centrifugation at 12,000 × g for 10 min at 4°C, the super- receptor decreased caspase-3 activity in D2−/−,butnot natant was collected as the total cell lysate. The pellet was in D2−/−–corrected cells, suggesting that caspase-3 re- resuspended in buffer A [10 mmol/L HEPES (pH 7.9), α sponse is TNF /TNFR1 mediated. The intracellular 10 mmol/L KCl, 1.5 mmol/L MgCl2,20%glycerol, ROS seemed to be TNFα/TNFR1-dependent for D2−/− 1 mmol/L DTT, and a 1:40 volume of protease inhibitor but not for D2−/−–corrected cells, suggesting that overpro- mixture containing 4-(2-aminoethyl)benzenesulfonyl fluo- duction of TNFα, rather than ROS accumulation, causes ride, pepstatin A, E-64, bestatin, leupeptin, aprotinin, and caspase-3/PARP response. Thus, the current study is the sodium EDTA] and homogenized on ice by sonication. first evidence demonstrating the damage-independent acti- After centrifugation at 1,000 × g for 10 min at 4 C, the vation of caspase-3 and PARP in FA cells, and that this ac- supernatant was collected as the cytoplasmic fraction. The tivation probably occurs through mitogen-activated protein resulting nuclear pellet was then washed with buffer A twice kinases (MAPK) activation and TNF-α oversecretion. and resuspended in TNE buffer [10 mmol/L Tris-HCl (pH 7.5), 0.1 mol/L NaCl, 1 mmol/L EDTA]. For de- phosphorylation experiments, 100 units of λ-phosphatase Materials and Methods (Calbiochem) were added to the appropriate cell lysates following incubation for 30 min at 37°C. For the degradation Cell Lines and Culturing assay (Supplementary Fig. S1), siRNA FANCD2–depleted The following human transformed fibroblast cell lines samples were incubated in parallel with the mixture of were used in this study: PD20 (FANCD2−/−), PD20 retro- sodium vanadate and sodium pyrophosphate (both from virally corrected with pMMp-FANCD2 cDNA, FANCA−/ Sigma). To test for IFN response, we analyzed siRNA- − (PD220)–transformed fibroblasts and corrected counter- transfected primary cells or cells stimulated with 2 μg parts (FA consortium),4 FANC-G– and FANC-G– Poly-I-poly-C were followed by Western blot analyses of corrected primary fibroblasts (gift of Dr. D. Schindler, eIF2α/P-eIF2α antibodies or real-time PCR experiments Wurzburg University, Wurzburg, Germany). In addition, (22) to test the expression of the IFN pathway–related we used primary human fibroblasts derived from a healthy gene STAT1. individual and lymphoblastoid cells from a non-FA patient [FANCL (EUFA 868), FANCJ (LFA 177), and FANCA SDS-PAGE and Western Blot Analysis (LFA 82)–transformed lymphoblastoid cells].

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