Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca2+ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca2+ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca2+ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-010-0533-1) contains supplementary material, which is available to authorized users.

On the cellular level, oxidative stress may cause various responses, including autophagy and cell death. All of these outcomes involve disturbed Ca 2؉ signaling. Here we show that the nuclear enzymes poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 control cytosolic Ca 2؉ shifts from extracellular and intracellular sources associated with autophagy or cell death. The different Ca 2؉ signals arise from the transient receptor potential melastatin 2 (TRPM2) channels located in the cellular and lysosomal membranes. They induce specific stress kinase responses of canonical autophagy and cell death pathways. Autophagy is under the control of PARP1, which operates as an autophagy suppressor after oxidative stress. Cell death is activated downstream of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT, whereas cell survival correlates with the phosphorylation of p38, stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK), and cyclic AMP response element-binding protein (CREB) with its activating transcription factor (ATF-1). Our results highlight an important role for PARP1 and PARP2 in the epigenetic control of cell death and autophagy pathways.

The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca2+ homeostasis using the Fluo-4 dye and compared results with the metabolic cell viability assay Alamar Blue. We investigated a panel of toxins (As2O3, gossypol, H2O2, staurosporine, and titanium(IV)-salane complexes) in four different mammalian cell lines covering three different species (human, mouse, and African green monkey). All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).

DNA damage to cells activates nuclear poly(ADP-ribose)polymerases (PARPs) and the poly(ADP-ribose) (PAR) synthesized is rapidly cleaved into ADP-ribose (ADPR) by PAR glycohydrolase (PARG) action. Naturally appearing tannin-like molecules have been implicated in specific inhibition of the PARG enzyme. This review deals with the in vitro and in vivo effects of tannins on PAR metabolism and their downstream actions in DNA damage signaling.

BackgroundChemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown.MethodsFour different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38.ResultsHere we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton.ConclusionsIn summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2538-0) contains supplementary material, which is available to authorized users.