PARP inhibition suppresses topoisomerase 1 poisoning induced Caspase-3 dependent cell death in zebrafish embryos
Sophiko Tsikarishvili a, Margarita Karapetian a, Nina Kulikova b, Giorgi Zaalishvili a, *
Abstract
In the present study the role of poly(ADP)ribosylation on rubitecan induced caspase dependent cell death was evaluated. We show that Top1 poisoning by rubitecan induces caspase mediated apoptosis which was reduced by PARP inhibitor olaparib in zebrafish embryo. Collectively our data introduces zebrafish as a valuable model for PARP related biomedical research.
Keywords:
Rubitecan
Olaparib
PARP-1
Caspase-3
Zebrafish embryo
1. Introduction
Poly (ADP ribose) polymerase 1 (PARP-1) is a ~113 kDa DNA damage sensing enzyme ubiquitously expressed in eukaryotic cells. Upon binding to DNA single or double strand breaks PARP-1 catalyzes NADþ dependent covalent attachment of poly(ADP-ribose) (PAR) polymers on itself (automodification) and other target proteins [1]. By doing so PARP-1 helps to recruit and modulate the activity of different proteins engaged in DNA repair pathways [2,3]. While PARP-1 activation is necessary for the elimination of DNA strand breaks, overactivation of PARP-1, observed during irreversible DNA damage conditions, may activate different pathways of regulated cell death (RCD). For instance, it is well established that PARP-1 hyperactivation can trigger parthanatosis e caspaseindependent RCD characterized by bioenergetic catastrophe, coupled to PAR-guided apoptosis-inducing factor (AIF) release from mitochondria and consequent DNA degradation [4]. Also, it has been reported that at the initial steps of apoptosis temporal PARP activation is important for caspase-dependent apoptosis [5]. However, in order to prevent energetical collapse and DNA repair in apoptotic cells, activated caspase-3 and caspase-7 cleave PARP-1 yielding 24 kDa and 89 kDa fragments – the hallmarks of caspasemediated apoptosis [6]. While the role of PARP-1 and PARylation in RCD has been extensively studied in higher vertebrates, the role of PARP-1 in the cell death of lower vertebrates, including zebrafish, has yet to be explored. Zebrafish as a model organism is becoming increasingly important in experimental research. Such characteristics as cost effectiveness, high fecundity, transparency of embryos and similarity of organs and cell types to mammals, make it a valuable model organism for different research fields [7,8]. Humans and zebrafish share 70% of genes out of which 84% are known to be associated with human diseases [9]. Apoptotic process also seems to be highly conserved through mammals and zebrafish, including high homology of the apoptosome and all its main components[10].
Currently, only two reports demonstrated the apoptotic cleavage of PARP-1 in zebrafish using commercially available antibodies against the N-terminal region of mammalian PARP-1. In both cases the obtained Western Blots did not contain full length PARP-1 band [11,12]. Recently, we [13] as well as others [14] have shown that the potent PARP inhibitor olaparib increases mortality and suppresses DNA repair in zebrafish embryos treated by topoisomerase 1 (TOP1) poisons, alkylating agents and X-ray irradiation. Similar results were also obtained for embryos of the ancient fish species (Aciper ruthenus) [15]. The aim of the present study was to evaluate the effects of Top1 poisoning and PARP inhibition on caspase-dependent apoptosis in zebrafish embryos.
2. Materials and Methods
2.1. Antibodies and reagents
Rabbit polyclonal anti zebrafish PARP-1 antibodies (Rb pAbs anti-Zebrafish PARP-1) were generated against zebrafish PARP-1 Nterminal fragment (120-240aa). Service was provided by Proteogenix (https://www.proteogenix.science/). Rb mAb anti-activated Caspasse 3, Alexa Fluor 488-conjugated and HRP- conjugated goat-anti-rabbit Abs were purchased from Abcam. Rubitecan(9-NC) and olaparib were purchased from Santa Cruz Biotechnology (USA). All other reagents used in this study were of molecular biology grade.
2.2. Animals
Danio rerio AB strains were reared in multi-tank aquarium supplied with the central filtration system at standard laboratory conditions at 28.5 C at 14 h of light and 10 h of dark per day. All experiments have been carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the protection of animals for scientific purposes. Danio rerio fry was collected and examined under the stereo-microscope. Fertilized eggs were selected and grown in Danieau’s medium (17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca (NO3)2$4H2O, 1.5 mM HEPES, pH 7.6) at 28.5 C. Live and healthy 24hpf embryos were transferred into 12 well cell culture plates, up to 30 embryos per well in 3 ml of the same medium. Top1 poison 9-NC (final concentration 1 mM and 10 mM) and PARP inhibitor olaparib (final concentration 20 mM) where added directly to the medium from 10 mM stock solutions in dimethyl sulfoxide (DMSO). Final DMSO concentration in all samples was adjusted to 0.3%. Treated embryos were rinsed in fresh Danieau’s medium, dechorionated and immediately proceeded for whole mount immunofluorescence or western blotting.
2.3. Western blotting
For crude extract preparation, 20 embryos were dissociated by pipetting in 200 ml of PBS, 5x Laemlli loading buffer was added and samples were heated at 95 C for 5 min. Samples were subjected to 12% SDS-PAGE (~1.2 embryo per well) and transferred onto a nitrocellulose (NC) membrane. Protein loading and transfer was monitored by Ponceau S staining. NC membranes were incubated with polyclonal rabbit anti-zebrafish PARP-1 Abs (1:2000 dillution) and secondary HRP-conjugated goat-anti rabbit Abs (1:4000 dillution). The bands labelled with the antibodies were visualized using a chemiluminescent luminol reagent (SantaCruz) by exposure onto X-ray films.
2.4. Whole mount immunofluorescence analysis and quantitation
Whole mount Immunofluorescence was performed as described in Ref. [16]. Briefly, embryos were fixed in 4% PFA/1xPBS, incubated with primary rabbit anti-activated caspase-3 Abs (1:500 dillution) for 2 h and then overnight with Alexa Flour 488 conjugated goat anti rabbit Abs (1:1000 dillution). Embryos with low autofluorescence background were selected, mounted on a drop of 4% methylcellulose in depression slide and analyzed under BX 41 Olimpus fluorescence microscope. Images were taken with CCD camera at the same magnification, exposure and gain. Caspase-3 positive signals in zebrafish eye were quantitated using ImageJ software. Briefly, embryo’s eye area was selected, the background was subtracted, threshold was set manually and the signals were counted by Analyze Particle tool. At the same time zebrafish embryo’s eye area was also measured for developmental delay statistics. In total three independent experiments were performed and averages per experiment, per group containing at least 10 embryos were calculated. Statistical significance between groups was determined by two-tailed, unpaired Student’s T-test.
3. Results and discussion
Since commercially available antibodies failed to detect full length PARP-1 in zebrafish, we generated polyclonal antibody specific to N-terminal domain of zebrafish PARP-1 (120-240aa) which was able to detect full length PARP-1 in zebrafish embryos as well as in mammalian cells crude extracts Fig. 1a. Previously we have reported [13] that zebrafish embryos’ treatment with micromolar concentrations of 9-NC induces DNA damage in a dosedependent manner, therefore the embryos treated with 1 mM (induces moderate DNA damage) and 10 mM 9-NC (induce extensive damage) were tested for the apoptotic cleavage of PARP-1 by Western blotting analysis. As shown in Fig. 1b 24 kDa PARP-1 fragment was detected in embryos treated for 5 and 7 h but not in those treated for 3 h, or in untreated ones. As far as we know, this is the first time, when the full length PARP-1 and its apoptotic fragment have been detected by Western blotting in zebrafish embryos. Thus PARP-1 could be proposed as a specific marker for caspase-dependent apoptosis in zebrafish embryos, as it is established for mammals. These results also suggest that 70 kDa and 25 kDa bands observed in previous studies [11,12] were nonspecific or, the antibodies used could detect the fragments but not the full length PARP-1.
For in situ detection of apoptotic cells in 9-NC treated zebrafish embryos, whole mount immunofluorescence using anti-activated caspase-3 Abs has been performed. Caspase 3 positive cells were observed through the body of 9-NC treated embryos, showing predominant localization in the brain, eyes and notochord area Fig. 2a. A similar distribution of TUNEL positive cells after treatment with campthotecin was observed previously [17].
At the next stage of our research, to evaluate the effect of PARylation on 9-NC induced caspase-dependent apoptosis, embryos were treated with 1 mM and 10 mM 9-NC in combination with PARP1 inhibitor olaparib. Due to the presence of high density caspase positive cells in the head area of 9-NC treated embryos, we focused on the quantification of individual signals in the embryo’s eye. As shown on Fig. 2b in untreated embryos as well as in embryos treated with olaparib alone only a few caspase-3 positive signals were detected, while the amount of caspase positive cells were dramatically increased in the embryos treated with both 1 mM and 10 mM 9-NC. Interestingly, olaparib significantly reduced the presence of caspase positive signals in 10 mM 9-NC treated embryos but not in 1 mM 9-NC treated embryos. However, olaparib failed to reduce the eye’s developmental delay in 10 mM 9-NC treated embryos Fig. 2c.
Our finding that PARP-1 inhibition in 9-NC treated embryos causes a decrease in caspase-3 positive cells without affecting developmental delay is in agreement with previously obtained data from different model systems [18,19] and clearly indicates that PARP-1 and PARylation regulates the RCD pathways in zebrafish embryo. According to our results, two main scenarios of olaparib effect on the execution of apoptotic program can be proposed in zebrafish: first scenario relies on the fact, that PARP-1 and PARylation are essential for the repair of Top1 cleavage complexes. Namely, PARP-1 is required for tyrosyl-DNA phosphodiesterase (TDP1) activation which is responsible for removal of the residual peptide remaining after Top1 proteosomal degradation, critical step in repair of cleavage complexes induced by Top1 poisons [20]. Therefore, inhibition of PARylation could leave a significant portion of the Top1 cleavage complexes unrepaired, inducing a transcription machinery collapse and thus preventing induction of the transcription dependent apoptosis. This scenario is supported by our data showing on the one hand, the same amount of caspase-3 positive signals in 1 mM and 10 mM 9-NC treated embryos and, on the other hand, more significant effect of PARP inhibitor in 10 mM 9NC treated embryos compared to 1 mM 9-NC treated embryos. Second scenario, which does not contradict the first one, proposes that PAR regulates activity of p53 which itself is a key player in DNA damage induced apoptosis in mammals as well as in zebrafish [16,17,21e23]. Collectively, our data introduces zebrafish as a valuable in vivo model to study the role of PARP-1 and PAR in RCD pathways and to investigate in situ cellular response to the combinatory effect of PARP and Top1 inhibitors (a promising strategy for cancer treatment) [24].
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