Inhibition of Caspase-3-mediated Poly(ADP-ribose) Polymerase (PARP) Apoptotic Cleavage by Human PARP Autoantibodies and Effect on Cells Undergoing Apoptosis

Abstract

Autoantibodies directed to nuclear antigens are serological hallmarks of autoimmune rheumatic diseases such as systemic lupus erythematosus. Although much more is known about the molecular identity and functions of targeted self-antigens, with few exceptions, evidence that autoantibodies to these targets have a particular function and contribute directly to the pathological process is lacking. Here we show that human autoantibodies reacting with the zinc fingers of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA totally prevent the cleavage of poly(ADP-ribose) polymerase by caspase-3, a process that normally occurs during early apoptosis. Furthermore, these antibodies, which are frequent in certain autoimmune rheumatic and bowel diseases, affect the characteristic features of apoptosis and increase cell survival ex vivo. This new observation is important, because failure to remove autoimmune or abnormal cells can give rise to prolonged autoimmune stimulation and tumor formation.Poly(ADP-ribose) polymerase (PARP 1 ; EC 2.4.2.30) is a 116-kDa nuclear enzyme that detects and binds DNA strand breaks produced by various genotoxic agents. DNA breaks activate PARP, which in turn catalyzes the synthesis of poly(ADPribose) from its substrate, the respiratory coenzyme NAD ϩ , at the site of breakage (1, 2). The ADP-ribose polymer is primarily attached to PARP itself (automodification) and to a few other acceptor proteins (heteromodification) involved in chromatin architecture and DNA metabolism, thus affecting their activity. The function of PARP is thought to be related to a number of nuclear processes that involve nicking and resealing of DNA strands, including transcription and DNA repair. PARP is probably associated with a multifunctional complex comprising factor x-ray cross-complementing-1, DNA polymerase ␤, and DNA ligase III, which are involved in the base excision repair pathway. PARP knock-out mice display a marked genomic instability and an extreme sensitivity to genotoxic agents (3, 4), and it has been shown that PARP-deficient cell lines performed very limited DNA repair during the first hours after DNA damage by alkylating agents (5).PARP has a modular organization (1) comprising an N-terminal DNA binding domain, which acts as a molecular nick sensor and contains two zinc finger motifs called F1 and F2, which are involved in the recognition of DNA breaks during DNA repair (6) and a bipartite nuclear localization signal, a central regulating segment, which contains the automodification site, and the C-terminal catalytic domain, which binds NAD ϩ (Fig. 1). The presence of IgG antibodies reacting with the N-terminal F1 and F2 zinc fingers (7, 8) and, in a very few cases, with the PARP catalytic domain (9) has been described in the serum of patients with various systemic autoimmune diseases. In particular, a high prevalence of antibodies reacting with synthetic peptides corresponding to zinc fingers F1 and F2 (residues 18 -59 and 122-165, respectively) was observed in patients with certain...

Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant

“…Active caspase-8 also causes the release of mitochondrial cytochrome c (Li et al, 1998;Luo et al, 1998) thereby linking the two pathways. After activation, both caspase-8 and caspase-9 activate caspase-3, which in turn cleaves and activates other caspases and many cellular proteins including fodrin, protein kinase C d (PKC d), poly (ADP-ribose) polymerase (PARP), gelsolin, DNA fragmentation factor-45 (DFF45) (Decker et al, 2000;Kunstle et al, 1997;Nicholson and Thornberry, 1997;Wolf and Green, 1999), and Sp1 (Piedrafita and Pfahl, 1997). While proteolysis of these and other caspase substrates induces the hallmark of apoptotic cell death, recent reports have also provided evidence for caspaseindependent apoptosis (Borner and Monney, 1999;Johnson, 2000).…”

Resveratrol‐induced apoptosis in MCF‐7 human breast cancer cells involves a caspase‐independent mechanism with downregulation of Bcl‐2 and NF‐κB

“…32,33 Among the proteins cleaved by caspase-3, PARP is considered relevant for its role in DNA repair and as a marker for caspase activity. 34,35 In previous studies, we suggested that the ERa-dependent PI3K pathway could mediate apoptosis induction by RES in MCF-7 cells. In the present work, we analyzed how RES interferes with downstream signaling in the PI3K pathway.…”

“…32,33 Among the proteins cleaved by caspase-3, PARP is considered relevant for its role in DNA repair and as a marker for caspase activity. 34,35 Grant sponsor: Fondo Europeo de Desarrollo Regional-Comision Interministerial de Ciencia y Técnologia (FEDER-CICYT); Grant number: 1FD97-0934; Grant sponsor: Junta de Extremadura; Grant number: 2PR01A092.* Abbreviations: Cyt c, cytochrome c; DHDCFDA, 5(and 6)-carboxy-2 0 ,7 0 -dihydrodichlorofluorescein diacetate; DC(m), mitochondrial membrane potential; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ERa, estrogen receptor-a; GSK-3, glycogen synthase kinase-3; I-kB, NF-kB inhibitor; IKK, I-kB-dependent kinase; MMP, matrix metalloproteinase; NF-kB, nuclear factor-kB; NO, nitric oxide; PARP, poly (ADP-ribose) polymerase; PI3K, phosphoinositide 3-kinase; PKB/AKT, protein kinase B/AKT; RES, resveratrol; ROS, reactive oxygen species; TRAIL, tumor necrosis factor-a-related apoptosis-inducing ligand. …”

“…32,33 Among the proteins cleaved by caspase-3, PARP is considered relevant for its role in DNA repair and as a marker for caspase activity. 34,35 Grant sponsor: Fondo Europeo de Desarrollo Regional-Comision Interministerial de Ciencia y Técnologia (FEDER-CICYT); Grant number: 1FD97-0934; Grant sponsor: Junta de Extremadura; Grant number: 2PR01A092.…”

β2-microglobulin induces apoptosis in HL-60 human leukemia cell line and its multidrug resistant variants overexpressing MRP1 but lacking Bax or overexpressing P-glycoprotein

“…However, treatment with 20 mg/ml b 2 m for 48 h did not induce cleavage of this enzyme to its active 35 and 37 kDa forms in HL-60 cells and its drug resistant variants (Figure 10b). Poly (ADP-ribose) polymerase (PARP) is a 116 kDa nuclear enzyme that binds DNA strand breaks produced by various genotoxic agents, and it is implicated in conserving the integrity of the genome (Oliver et al, 1998;Decker et al, 2000). This enzyme has therefore been shown as a caspase substrate, and cleavage of PARP from its 116 kDa to an 85 kDa form can be detected in caspase-dependent apoptosis induced by various stimuli (Nicholson and Thornberry, 1997;Decker et al, 2000).…”