The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1.

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders.

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling. Double-strand breaks (DSB)5 are potentially the most cytotoxic form of DNA damage in human cells because they lead to genomic rearrangements, cancer predisposition, and perhaps cell death if unrepaired or repaired incorrectly (1). Consequently, the DNA damage response involves parallel modulation of redundant signaling pathways leading to lesion detection, processing, and repair. Ataxia telangiectasia mutated (ATM) is a DNA damage-responding kinase that is rapidly activated after the induction of DSB (2). Within minutes of DNA damage induction, ATM is recruited and activated in the vicinity of DSBs, where it induces the phosphorylation of a number of proteins required for DNA damage response and repair, including proteins of MRN (Mre11/Rad50/NBS1) complex, p53, SMC1 and histone variant H2AX (3). However, the detailed mechanisms of how ATM is activated and regulates its downstream effectors are not fully understood. Although ATM activation is mainly associated with DSB formation as part of the damage detection mechanism following ionizing radiation (IR), several studies indicate that the signaling kinase ATM is also activated in response to the environmental carcinogen N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) (4 -6).Poly(ADP-ribose) polymerases (PARPs) are also constitutive factors of the DNA damage surveillance network, acting through a DNA break sensor function (7). Several observations indicate that poly(ADP-ribosyl)ation also plays an early role in DSB signaling and repair pathways (8 -11). PARP-1 and PARP-2 are highly activated upon binding to DNA strand interruptions and synthesize, within seconds, large amounts of the negatively charged polymer of ADP-ribose (PAR) on several nuclear proteins including themselves, histones, topoisomerase I, and DNA-dependent protein kinase (DNA-PK) (12). The immediate activat...

Poly(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers formed by members of the PARP family of enzymes. Despite its discovery and subsequent partial purification in the 1970s and the cloning of its single gene in the late 1990s, little is known about the role of PARG in cell function. Because of its low abundance within cells and its extreme sensitivity to proteases, PARG has been difficult to study. The existence of several PARG isoforms with different subcellular localizations is still debated today after more than 30 years of intensive research. In this article, we want to summarize and discuss the current knowledge related to PARG, its different forms and subcellular distribution. We also examine the possible biological roles of PARG in modulating chromatin structure, transcription, DNA repair and apoptosis.

Our preliminary results suggest that detection of GCC mRNA in LNs is associated with risk of disease recurrence in patients with untreated stage II colon cancer. A larger validation study is ongoing.

Molecular detection of tumour cells in LNs may have prognostic value in identifying patients diagnosed as having pN0 colon cancer who will relapse following surgery.

Poly(ADP‐ribosyl)ation is a post‐translational modification catalyzed mostly by the 116‐kDa enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1), a nuclear enzyme that transfers an ADP‐ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500‐fold increase in net poly(ADP‐ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP‐ribose) synthesis is PARP‐1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD+ depletion. It has also been proposed that the presence of negatively charged poly(ADP‐ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP‐ribose) in cells using nonisotopic reagents and for identifying the poly(ADP‐ribose) polymerase enzymes present in cells.

Purpose: Recurrence risk assessment to make treatment decisions for early-stage colon cancer patients is a major unmet medical need. The aim of this retrospective multicenter study was to evaluate the clinical utility of guanylyl cyclase C (GCC) mRNA levels in lymph nodes on colon cancer recurrence.Methods: The proportion of lymph nodes with GCC-positive mRNA (LNR) was evaluated in 463 untreated T3N0 patients, blinded to clinical outcomes. One site's (n ¼ 97) tissue grossing method precluded appropriate lymph node assessment resulting in post hoc exclusion. Cox regression models tested the relationship between GCC and the primary endpoint of time to recurrence. Assay methods, primary analyses, and cut points were all prespecified.Results: Final dataset contained 366 patients, 38 (10%) of whom had recurrence. Presence of four or more GCC-positive lymph nodes was significantly associated with risk of recurrence [hazard ratio (HR) ¼ 2.46, 95% confidence interval (CI), 1.07-5.69, P ¼ 0.035], whereas binary GCC LNR risk class (HR ¼ 1.87, 95% CI, 0.99-3.54, P ¼ 0.054) and mismatch repair (MMR) status (HR ¼ 0.77, 95% CI, 0.36-1.62, P ¼ 0.49) were not. In a secondary analysis using a 3-level GCC LNR risk group classification of high (LNR > 0.20), intermediate (0.10 < LNR 0.20), and low (LNR 0.10), high-risk patients had a 2.5 times higher recurrence risk compared with low-risk patients (HR ¼ 2.53, 95% CI, 1.24-5.17, P ¼ 0.011).Conclusions: GCC status is a promising prognostic factor independent of traditional histopathology risk factors in a contemporary population of patients with stage IIa colon cancer not treated with adjuvant therapy, but GCC determination must be performed with methodology adapted to the tissue procurement and fixation technique.