TY - JOUR
AU - Chabouté, Marie-Edith
AB - Abstract In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G2/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G2/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants. ATM/ATR pathways, BY-2 cells, cell cycle, double-strand break response, E2F, paraptotic-like, programmed cell death Introduction Apoptosis is a recent evolutionary mode of programmed cell death (PCD), which evolved to eliminate damaged or unwanted animal cells through activation of the caspase family proteases and phagocytic clearance (Hengartner, 2000). In addition, at least three other non-apoptotic forms of PCD have also been described in metazoans and were designated as autophagy, paraptosis, or mitotic catastrophe, respectively. However, none of them completely fulfils the typical features of apoptotis (Bröker et al., 2005). In plants, an apoptotic strategy does not exist due to the presence of the cell wall that impedes phagocytosis by other cells. Instead, autophagic PCD takes place when the final proteolytic activity resides in the cell’s own autophagosome or lytic vacuole (van Doorn and Woltering, 2005). Plant metacaspases are distant relatives of caspase which have been recently described to play a role during the hypersensitive response (HR; Coll et al., 2010). Strikingly, they do not exhibit the same proteolytic activity as do animal caspases (Vercammen et al., 2007) since caspase-like activities can participate in execution of plant PCD (Hatsugai et al., 2006). Another, very little known type of PCD is paraptosis, which has so far been identified in animals and protists (Sperandio et al., 2000; Jimenez et al., 2009). This process involves cytoplasmic vacuolization and mitochondrial swelling, in the absence of apoptotic markers such as caspase activation, oligonucleosomal DNA cleavage, or nuclear fragmentation (Sperandio et al., 2000, 2004; Jimenez et al., 2009). Despite the elucidation of morphological characteristics, the precise molecular basis of non-apoptotic PCD types remained unknown, especially in plants. In eukaryotes, the phosphatidyl-3 kinase (PI-3K) family members ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related) are the main sensors of DNA damage. While ATM senses DNA double-strand breaks (DSBs), ATR is activated in response to single-stranded DNA (Brown and Baltimore, 2003; Lee and Paull, 2007). In mammals, DNA damage signalling also involves checkpoint kinases such as Chk1 and Chk2, which activate downstream effectors such as the E2F1 or p53 transcriptional factors, ultimately leading to cell cycle checkpoint activation, DNA repair, or apoptosis (Chaturvedi et al., 1999; DeGregori, 2002; Lee and Paull, 2007). Ectopic expression of the transcriptional activator E2F1 induces not only S phase entry in quiescent cells, but also p53-dependent or p53-independent apoptosis, which is enhanced upon DNA damage (Qin et al., 1994; Kowalik et al., 1995; Hsieh et al., 1997). The pro-apoptotic E2F1 function was also described to occur in Drosophila melanogaster and Caenorhabditis elegans, thus confirming that the E2F1 function seems to be conserved in animals (Asano et al., 1996; Schertel and Conradt, 2007). The apoptotic role of E2F1 was affirmed by transcriptional data showing that it controls the expression of numerous core pro-apoptotic genes (Muller et al., 2001). Besides its pro-apoptotic role, E2F1 has been recently shown to contribute directly to DNA repair and genome maintenance (Chen et al., 2011). However, all the molecular mechanisms underlying the cell’s decision to proliferate or to die are not yet fully understood (Buchakjian and Kornbluth, 2010). Plants had to evolve a specific control mechanism for the DNA damage response, among others due to their sessile life. As compared with animals, they developed different strategies to cope with this type of stress. Such an adaptation is the high capacity to regenerate or replace damaged tissues and organs, according to the indeterminate growth of meristems. In comparison with mammals, some players in the DNA damage response are conserved in plants, for example ATM, ATR, and E2Fs (Garcia et al., 2003; Culligan et al., 2004; Roa et al., 2009), but, strikingly, many ATM and ATR targets such as p53, Chk1, or Chk2 are missing (Cools and De Veylder, 2009). In Arabidopsis, the roles of ATM and ATR were demonstrated during DNA repair, cell cycle checkpoint activation (Culligan et al., 2004, 2006; Ricaud et al., 2007), and more recently during DNA damage-induced PCD (Fulcher and Sablowski, 2009; Furukawa et al., 2010). Altogether, in plants the study of cell death induction in response to DNA damage is still emerging and has not yet been investigated during cell cycle progression. In plants, the E2F factors are transcriptional activators or repressors sharing similar complexity with the animal E2Fs (Lincker et al., 2008). E2Fs have been shown to regulate genes implicated in cell cycle control and DNA repair (Chabouté et al., 2000; Lincker et al., 2004; Vandepoele et al., 2005; Roa et al., 2009; Radziejwoski et al., 2011), but their role in the regulation of PCD has never been investigated. In Arabidopsis, the overexpression of the transcriptional activator E2Fa, with its transactivating partner DPa, induces ectopic cell division, endoreduplication, or endomitosis rather than PCD (De Veylder et al., 2002; Henriques et al., 2010). In addition, available microarray data do not suggest any up-regulation of PCD-related genes in Arabidopsis overexpressing E2Fa/DPa, compared with wild-type plants (Vandepoele et al., 2005; Naouar et al., 2009). These results do not exclude a possible role for E2Fa in plant PCD regulation, because a E2Fa/DPa-dependent transcription of PCD-related genes might occur only in response to genotoxic stress, or the PCD response might be mediated by other E2F members. The only tobacco NtE2F factor described is a transcriptional activator (Sekine et al., 1999) similar to AtE2Fa and AtE2Fb (Lincker et al., 2008). NtE2F is up-regulated in response to genotoxins (Lincker et al., 2004), but its role during PCD regulation has not yet been established. To characterize the genotoxic-induced PCD in plants, dividing tobacco BY-2 cells were used as a model for cellular and cell cycle analyses. This could extend previous results obtained in Arabidopsis root meristem, which is confined to a few developmentally specified cells only (Fulcher and Sablowski, 2009; Furukawa et al., 2010). In the present work, a novel type of non-apoptotic PCD induced by the DSB-inducer bleomycin (BLM) was characterized in plants sharing features with paraptotic PCD. Besides its regulation by ATM/ATR, such PCD was mainly induced upon the activation of the G2/M checkpoint, which was modulated by NtE2F overexpression. Materials and methods Cultivation of plant material and BLM treatment Tobacco BY-2 (Nicotiana tabaccum cv. Bright Yellow-2) cells were maintained by weekly subculture in modified Murashige and Skoog medium (Sigma, St Louis, MO, USA) as described (Nagata et al., 1992). Cells were cultivated at 26 °C in darkness on an orbital shaker set at 130 rpm. Before BLM treatment, mid-log phase cells were diluted (1:3). BLM was added to wild-type or NtE2F synchronized cells at specific cell cycle stages after aphidocolin (APC) release (0 h): 1 h for S phase, 5 h for G2, 9 h for M, and 12 h for G1. When NtE2F cells were treated in G2 and M, BLM was added with a 0.5 h and 1 h delay compared with the wild type, respectively. Arabidopsis plants (Arabidopsis thaliana, ecotype Col-0) were grown in vitro under a 14 h photoperiod and at 21 °C/18 °C. Seeds were surface sterilized for 5 min in 70% ethanol and 6 min in 0.3% bleach, and rinsed three times in distilled water. Seeds were sown on Murashige and Skoog medium (Duchefa MO225, Haarlem, The Netherlands) supplemented with 1% sucrose and 1.2% agar (Duchefa 9002-18-0). For BLM treatment, 5-day-old plantlets were transferred onto a medium containing 10−5 M or 10−4 M BLM and cultivated for 24 h for further analysis. Generation of the NtE2F line The open reading frame of NtE2F (BAA86386) was amplified by PCR with specific attb1 and attb2 primers (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGCAGCAGCCACATCATCA-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCAGCTCCCAGTTGTATTTGC-3′) and cloned into the GATEWAY vector pDONR201 (Invitrogen Corporation, Carlsbad, CA, USA). Then, the construct was transferred into the pK7WGF2 vector (Karimi et al., 2002), to express the fusion under the control of the 35S promoter. The green fluorescent proitein (GFP):NtE2F fusion was introduced into the Agrobacterium tumefaciens strain GV3101, which was used for subsequent transformation of BY-2 as described (An et al., 1985). Viability assay, BCECF staining, and ROS detection The FDA (fluorescein diacetate) viability assay was used to detect cell death in BY-2 cells. A 50 μl aliquot of cell suspension was mixed with 50 μl of FDA working solution (1/1000 in distilled water; stock solution 0.2% FDA in acetone, Sigma) and incubated for 2 min before observation by epifluorescence microscopy (Nikon E800, Champigny sur Marne, France or Olympus Provis AX 70, Hamburg, Germany) using a GFP filter (excitation, 460–500 nm; emission 510–560 nm). Viable cells cleave FDA into a fluorogenic product detected as a green signal, whereas dead cells do not give a signal. Arabidopsis plantlets were incubated in 1000-fold diluted 10 mg ml−1 water–propidium iodide (Sigma) solution for 3 min, rinsed in water, and immediately observed using a red fluorescent protein (RFP) filter (Zeiss Axiovert 100M with Zeiss LSM 510 Software, Le Pecq, France). The integrity of the vacuoles was determined after 5 min of double staining with 6 mM BCECF [(2′,7′-bis-2-carboxyethyl)-5-(and-6) carboxyfluorescein; Sigma] and 0.05% Evans blue as described (Matsuoka et al., 1997). For reactive oxygen species (ROS) detection, cells were incubated with DCFH-DA (2′,7′-dichlorfluorescein-diacetate; Sigma) at a final concentration of 2 μM. The images were captured after 120 s at a single 600 ms exposure (Basler A101c Camera, Buckinghamshire, UK). The relative ROS accumulation is presented as mean pixel intensity per cell measured with ImageJ software. Five hundred cells were counted in at least three independent experiments. TUNEL assay and DNA laddering TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay was performed according to the manufacturer’s protocol (TMR red in situ cell death detection kit, Roche Diagnostics GmbH, Heidelberg, Germany) with slight modifications. Just before the TUNEL reaction, two steps were added. First, the samples of positive controls were washed in DNase buffer (40 mM TRIS-HCl pH 7.9; 10 mM NaCl; 6 mM MgCl2; 10 mM CaCl2) and subsequently treated with 40 μg ml−1 DNase I (Invitrogen) for 20 min at 37 °C. Secondly, all the samples were then washed for 5 min in 25 mM TRIS pH 6.6. For DNA laddering, genomic DNA was isolated from 200 mg (fresh weight) of BY-2 cells using the Apoptotic DNA-ladder kit as a standard (Roche Diagnostics GmbH, Mannheim, Germany) and run on agarose gels according to the manufacturer’s instructions. Mitotic index (MI) determination, nuclear morphology, synchronization, and flow cytometry Cells were permeabilized with 0.1% Triton and the nuclei were stained with 20 μg ml−1 4',6-diamidino-2-phenylindole (DAPI; Sigma). The MI was determined as the percentage of mitotic cells among 500 cells analysed by fluorescence microscopy (Nikon E800 or Olympus Provis AX 70). In parallel, the nuclear morphology of at least 300 nuclei was analysed in at least three independent experiments. BY-2 cells were synchronized as described, with slight modification (Kumagai-Sano et al., 2006). After 24 h culture of stationary cells in MS medium containing 15 μM APC (Sigma), the drug was released from the cells by subsequent washing with 2 litres of 3% sucrose. Finally, the cells were resuspended in fresh MS medium. For flow cytometric analysis, 100 μl of CysStain UV ploidy (Partec, Münster, Germany) was added to 100 mg fresh weight of BY-2 cells, which were immediately chopped with a sharp razor blade. The resulting suspension of the nuclei was filtered through a 30 μm CellTrics filter (Partec, Görlitz, Germany) and analysed with the aid of a Cyflow-R ploidy analyser (Partec, Münster, Germany) or BD LSR II Flow Cytometer (BD Biosciences, Belgium). In each experiment, at least 10 000 nuclei were counted. The data from three independent experiments were processed with FlowJo 7.2.5 software (Ashland, OR, USA). RNA extraction, cDNA synthesis, and real-time quantitative PCR Total RNA was isolated from 50 mg fresh weight of BY-2 culture using TRIzol (Invitrogen) according to the manufacturer’s protocol, and then the RNA samples were treated with DNase I (Fermentas International Inc., Glen Burnie, MD, USA). A 1 μg aliquot of RNA was reverse transcribed to cDNA with 0.5 μg of random hexamers p(dN)6 (Roche Diagnostics GmbH, Mannheim, Germany) and RevertAid™ M-MuLV Reverse Transcriptase (Fermentas International Inc.). Real-tme qPCR was performed with Light Cycler 480 SYBR Green I Master (Roche), 1 μM specific primers (RNR1a, 5′-TTTCTTCTCTGCCTGCTGG-3′ and 5′-TCATTGCTGATACACCATTTAC-3′; RNR1b, 5′-TTAGTGCTCTATGCAGAAGGA-3′ and 5′-CAATAGAGCAATACGAGCTAC-3′; 18S, 5′-GGTCTTCAACGAGGAATTCC-3′ and 5′-CGACTTCTCCTTCCTCTCAA-3′) and 1 μl of cDNA in a total volume of 10 μl per reaction. Triplicates of each sample were run and analysed with the Light Cycler 480 System (Roche Diagnostics) according to the manufacturer’s instructions. The stability of 18S gene expression used for the normalization of all samples was verified with the 2−ΔCt method. Quantification of relative expression for each gene was calculated using 2−ΔΔCt as described previously (Livak and Schmittgen, 2001). The data represent the mean values of three independent experiments. Fluorescence in situ hybridization (FISH) BY-2 cells were fixed in ethanol:acetic acid (3:1) for 10 min and washed in 1× phosphate-buffered saline (PBS; 2× 10 min). The cells were transferred into a drop of 60% acetic acid loaded on a slide coated with poly-L-lysine (Sigma) and were squashed under the cover slip. After removal of the cover slip, slides were digested with RNase A (100 μg ml−1 in 2× SSC, 1 h at 37 °C; Invitrogen) and pepsin (50 μg ml−1 in 0.01 M HCl, 10 min, room temperature; Sigma). The hybridization mix (30 μl per slide) contained 15 ng of custom-synthesized peptide nucleic acid (PNA) probe (C3TA3)2 labelled with Cy3 (Applied Biosystems, Carlsbad, CA, USA), 5% dextran sulphate, and 60% formamide in 2× SSC. The heat-denatured hybridization mixture was applied on slides, covered with plastic cover slips, and the slides were subjected to heat denaturation and gradual lowering of the temperature to 37 °C. After hybridization at 37 °C for 18 h, slides were washed with 2× SSC at 42 °C and 0.1× SSC at 42 °C, 5 min each, and mounted in Vectashield (Vector Laboratories, Peterborough, UK) with 1 μg ml−1 DAPI. Images of BY-2 cell nuclei were registered using an Olympus AX 70 fluorescent microscope equipped with filter sets for Cy3 and DAPI and an AxioCam MRm camera (Carl Zeiss). Results Induction of paraptotic-like PCD in tobacco BY-2 cells after DNA damage To ascertain whether and how BLM can induce PCD in cultured BY-2 cells, mid-log phase cells were submitted to different doses of BLM, and cell viability was analysed in the following 72 h using the FDA test. Upon BLM treatment, the cell mortality progressively increased in a dose- and time-dependent manner, while the mortality of control cells was essentially negligible (Fig. 1). To distinguish whether cell death occurred by PCD or in a non-programmed manner, the presence of several PCD hallmarks was investigated: cytoplasm shrinkage, chromatin condensation, DNA laddering, and ROS accumulation. Finally, vacuolization and vacuolar disintegration were analysed, leading to leakage of hydrolytic enzymes into the cytoplasm during autophagic PCD execution in plants (van Doorn and Woltering, 2005). Fig. 1. View largeDownload slide Time- and concentration-dependent effect of BLM on cell mortality. Mid-log BY-2 cells were transferred to MS medium supplemented with 10−5 M or 10−4 M BLM and cultivated for 72 h. At the time indicated, the mortality of both control and BLM-treated cells was evaluated by using FDA staining. The percentage of dead cells (±SD) represents the mean values of five independent experiments. Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Fig. 1. View largeDownload slide Time- and concentration-dependent effect of BLM on cell mortality. Mid-log BY-2 cells were transferred to MS medium supplemented with 10−5 M or 10−4 M BLM and cultivated for 72 h. At the time indicated, the mortality of both control and BLM-treated cells was evaluated by using FDA staining. The percentage of dead cells (±SD) represents the mean values of five independent experiments. Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). First, BLM-treated cells were stained with Evans blue to analyse cytoplasm shrinkage in dead cells. This cytoplasm shrinkage was found in 87±4% and 92±3% of dead cells treated for 72 h with 10−5 M and 10−4 M BLM (P < 0.01), respectively (Fig. 2A), whereas control cells exhibited normal morphology. Formation of apoptotic bodies containing the fragments of cytoplasm and organelles could not be observed. Fig. 2. View largeDownload slide Cytoplasm shrinkage, vacuolization, and vacuolar collapse in BLM-treated BY-2 cells. Mid-log cells were inoculated in MS medium with or without 10−5 M or 10−4 M BLM. After 72 h, the cytoplasm shrinkage was analysed in Evans blue-stained cells by light microscopy (A). Vacuolization was observed with a light microscope equipped with DIC (B). Vacuolar collapse was evaluated in the cell suspension, after double staining with Evans blue and BCECF-AM according to the staining pattern (see text) (A and C). Type I, a living cell with an intact vacuole (*); type II, a living cell with collapsed vacuole (¤); and type III, a dead cell (¥). The scale bars indicate 1 μm. Changes in the populations of BLM-treated cells were evaluated at the times indicated, and the results represent the mean (±SD) from three independent experiments (D). Pictures represent typical examples. Bar=50 μm. Fig. 2. View largeDownload slide Cytoplasm shrinkage, vacuolization, and vacuolar collapse in BLM-treated BY-2 cells. Mid-log cells were inoculated in MS medium with or without 10−5 M or 10−4 M BLM. After 72 h, the cytoplasm shrinkage was analysed in Evans blue-stained cells by light microscopy (A). Vacuolization was observed with a light microscope equipped with DIC (B). Vacuolar collapse was evaluated in the cell suspension, after double staining with Evans blue and BCECF-AM according to the staining pattern (see text) (A and C). Type I, a living cell with an intact vacuole (*); type II, a living cell with collapsed vacuole (¤); and type III, a dead cell (¥). The scale bars indicate 1 μm. Changes in the populations of BLM-treated cells were evaluated at the times indicated, and the results represent the mean (±SD) from three independent experiments (D). Pictures represent typical examples. Bar=50 μm. However, a high proportion of cells exhibited an extensive vacuolization, mainly after 10−4 M BLM treatment. BLM also reduced the number of cytoplasmic strands due to apparent vacuolar fusion to form one central vacuole occupying the majority of the cellular inner space (Fig. 2B). To monitor the integrity of the tonoplast and its relationship to cell viability, BCECF-AM and Evans blue double staining was used. BCECF-AM is cleaved in the fluorogenic product BCECF which was localized to the vacuolar lumen and excluded from the cytoplasm and nuclei of viable cells. Conversely, Evans blue was accumulated in dead cells, whereas it was actively exported out of viable cells. Thus, three different cell categories can be discerned according to their staining patterns by both dyes (Higaki et al., 2007): type I, intact viable cells with intact plasma membrane and tonoplast had Evans blue-negative and BCECF-positive signals in the vacuole; type II, viable cells with intact plasma membrane and collapsed vacuole exhibited Evans blue-negative and BCECF-positive signals in the cytosol and the nucleus; and type III, dead cells with disintegrated vacuolar and plasma membranes showed Evans blue-positive and BCECF-negative signals (Fig. 2A, C). During 72 h of BLM treatment, the number of type I cells progressively decreased while the number of type III cells increased. The number of type II cells that were alive but contained a collapsed vacuole was relatively high after BLM treatment (Fig. 2D). In the non-treated control, 99% of cells belonged to type I (data not shown). Importantly, the relatively high number of type II cells means that cells can survive for a certain period even with a disintegrated vacuole. Therefore, it was suggested that the vacuolar collapse is not a crucial step during execution of BLM-induced PCD as was, for example, reported to happen during elicitor-induced or developmental PCD (Obara et al., 2001; Higaki et al., 2007). Vacuolar collapse may be linked to internucleosomal DNA fragmentation in BY-2 cells (Hatsugai et al., 2004; Kuthanova et al., 2008b). However, when BLM was supplied during 3 d or 7 d to BY-2 cells, no apoptotic DNA fragmentation was observed using TUNEL or DNA laddering assays, respectively (Supplementary Fig. S1 available at JXB online), even though most of the cells were dead (data not shown). Both vacuole rupture-mediated and apoptotic-like PCD could depend on caspase-like activities. However, PCD induction by BLM was insensitive to the most commonly used caspase inhibitors, z-YVAD-FMK and z-DEVD-FMK, when compared with the positive control treated with 50 μM 6-benzylaminopurine (6-BAP; Supplementary Fig. S2), which is a potent inducer of apoptotic-like PCD in BY-2 cells (Mlejnek and Prochazka, 2002; Danon et al., 2004; Hatsugai et al., 2004). In parallel, by DAPI staining, chromatin morphology was analysed after 24 h of BLM treatment in the early phases of cell death induction (Fig. 3A). At 10−4 M BLM, the chromatin was highly condensed into one or several bodies in 81±1% of the cells. In contrast, the control exhibited normal nuclear morphology, with large nucleoli being surrounded by uniformly stained chromatin. After 10−5 M BLM treatment, no significant difference in nuclear morphology was observed, albeit that a fraction of cells (5±5%) exhibited more granular chromatin (data not shown). Fig. 3. View largeDownload slide BLM-induced chromatin condensation and ROS accumulation in BY-2 cells. Cells were transferred into medium supplemented with 10−5 M or 10−4 M BLM. After 24 h cultivation, cells were stained with DAPI (A) or H2FDA (B) and analysed by fluorescence microscopy as described in the Materials and methods. Fluorescence of cleaved H2FDA was quantified with ImageJ Software as mean pixel intensity per cell (±SD) (C). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Pictures represent typical examples. Bar=50 μm. Fig. 3. View largeDownload slide BLM-induced chromatin condensation and ROS accumulation in BY-2 cells. Cells were transferred into medium supplemented with 10−5 M or 10−4 M BLM. After 24 h cultivation, cells were stained with DAPI (A) or H2FDA (B) and analysed by fluorescence microscopy as described in the Materials and methods. Fluorescence of cleaved H2FDA was quantified with ImageJ Software as mean pixel intensity per cell (±SD) (C). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Pictures represent typical examples. Bar=50 μm. Finally, in light of the crucial role played by ROS in PCD and DNA damage (Gadjev et al., 2008; Roldan-Arjona and Ariza, 2009), ROS production was monitored using DCFH-DA, which is a fluorescent detector of a broad range of oxidizing reactions that may be increased during intracellular oxidative stress (Hempel et al., 1999). After 24 h of BLM treatment, DCFH fluorescence apparently increased as compared with control cells, which exhibited only a very weak signal (Fig. 3B). DCFH fluorescence was more pronounced at 10−5 M than at 10−4 M, most probably due to different kinetics of ROS accumulation after both BLM treatments (Fig. 3C). Taken together, these results demonstrate that BLM-induced PCD is a unique type of PCD, which does not fit to the criteria for apoptotic-like PCD or for autophagic PCD, but rather shares characteristics with paraptotic-like PCD. Caffeine inhibits BLM-induced PCD Previous work has reported that DSB-induced PCD is ATR and/or ATM dependent in Arabidopsis (Fulcher and Sablowski, 2009; Furukawa et al., 2010). To define the role of DNA damage signalling during the BLM-induced PCD in tobacco cells, the ATM/ATR inhibitor caffeine was used. Although caffeine affects other cellular processes such as cytokinesis (Yasuhara, 2005), it is also successfully used for ATM/ATR inhibition in mammals (Sarkaria et al., 1999). In plants, caffeine is also able to override the DNA damage G2/M checkpoint arrest (Hartley-Asp et al., 1980; Gonzalez-Fernandez et al., 1985), but seems not to affect the replication checkpoint control in S phase (Amino and Nagata, 1996; Pelayo et al., 2001; Weingartner et al., 2003). The possibility that caffeine is active also towards ATR in tobacco BY-2 cells cannot be ruled out, and thus caffeine may inhibit both the ATM and ATR pathways. Mid-log phase BY-2 cells were inoculated into MS medium supplemented with 5 mM caffeine 1 h prior BLM addition. As a positive downstream target of ATM/ATR signalling, the well-characterized transcriptional induction of the RNR1 gene (Roa et al., 2009) was investigated. In tobacco, the two NtRNR1a and b genes were shown to be induced by DSBs (Lincker et al., 2004, 2008) and, as expected, the BLM-induced RNR1a expression was significantly reduced by caffeine (P < 0.01) (Supplementary Fig. S3 at JXB online). However, this RNR1a down-regulation was only partial, suggesting that under the experimental conditions used either caffeine did not completely inhibit ATM/ATR activities, or there might exist another ATM/ATR-independent regulation of NtRNR1a. To analyse the effect of ATM/ATR inhibition on paraptotic-like PCD, the MI (the percentage of cells in mitosis), chromatin condensation, vacuolar dynamics, and, finally, induction of cell death were evaluated. As expected, BLM caused a rapid arrest of cell division already after 24 h of treatment (Supplementary Fig. S4 at JXB online). Fluorescence-activated cell sorting (FACS) analysis performed on nuclei from BLM-arrested cells showed an increase of 4C nuclei versus control nuclei, and a concomitant reduction of 2C nuclei, which suggested an activation of the G2/M checkpoint but also of the G1/S checkpoint (Fig. 4A). Fig. 4. View largeDownload slide DNA content measurements and effects of the ATM inhibitor caffeine on PCD induction and nuclear morphology in BLM-treated BY-2 cells. Cells were inoculated into MS medium supplemented with BLM (10−5 M or 10−4 M) in the presence or absence of 5 mM caffeine and cultivated for 72 h. DNA content was measured using flow cytometry after 24 h of BLM treatment (A). Quantification of the mitotic index (B), chromatin condensation (D), and vacuolar disintegration (E, F) represents the mean values of three independent experiments (±SD). Nuclear morphology and mitotic catastrophe (MC) were analysed by fluorescent microcopy 24 h after DAPI staining (C). Pictures represent typical examples. Quantification of chromatin condensation was performed after 72 h of BLM treatment (D). The percentage of type II cells relative to the total number of living cells was evaluated during 72 h of BLM treatment. The number of control cells with a disintegrated vacuole was not affected by caffeine treatment (data not shown) (E and F). The effect of caffeine on cell death induction was quantified after 72 h of BLM treatment by using the FDA test (G). Data represent the mean values (±SD). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Bar=5 μm. Fig. 4. View largeDownload slide DNA content measurements and effects of the ATM inhibitor caffeine on PCD induction and nuclear morphology in BLM-treated BY-2 cells. Cells were inoculated into MS medium supplemented with BLM (10−5 M or 10−4 M) in the presence or absence of 5 mM caffeine and cultivated for 72 h. DNA content was measured using flow cytometry after 24 h of BLM treatment (A). Quantification of the mitotic index (B), chromatin condensation (D), and vacuolar disintegration (E, F) represents the mean values of three independent experiments (±SD). Nuclear morphology and mitotic catastrophe (MC) were analysed by fluorescent microcopy 24 h after DAPI staining (C). Pictures represent typical examples. Quantification of chromatin condensation was performed after 72 h of BLM treatment (D). The percentage of type II cells relative to the total number of living cells was evaluated during 72 h of BLM treatment. The number of control cells with a disintegrated vacuole was not affected by caffeine treatment (data not shown) (E and F). The effect of caffeine on cell death induction was quantified after 72 h of BLM treatment by using the FDA test (G). Data represent the mean values (±SD). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Bar=5 μm. Compared with control cells treated only with BLM (10−5 M and 10−4 M), the addition of 5 mM caffeine induced an increase of the MI (11±4% and 13.5±4%, P < 0.05, respectively), and the appearance of cells exhibiting mitotic-like catastrophes [33% (n = 86) and 92% (n = 102) of the mitotic cells, respectively] (Fig. 4C). These mitotic catastrophes were observed neither in untreated cells nor in cells treated with BLM or caffeine alone. The induction of PCD, together with chromatin condensation (Fig. 4D) and vacuolar disintegration (Fig. 4E, F), was significantly reduced, but only in response to 10−4 M BLM (P < 0.01) (Fig. 4G). Thus, the inhibition of the ATM/ATR pathways may suppress G2/M checkpoint activation, leading to entry into mitosis even with damaged DNA. The detection of numerous mitotic catastrophes linked to DNA damage may be a result of the caffeine-mediated inhibition of cytokinesis (Yasuhara, 2005). These results strongly suggest that paraptotic-like PCD induced by high BLM doses is at least partly ATM/ATR dependent. PCD and genome integrity in BY-2 cells is altered by pRB/E2F pathway deregulation A BY-2 cell line overexpressing the GFP:NtE2F fusion under the control of the constitutive 35S promoter was generated in order to study the role of the tobacco NtE2F activator factor on BLM-induced PCD (called NtE2F cells or the NtE2F line). This fusion protein was localized in the nucleoplasm (Fig. 5A), and it was proved that GFP does not alter proper NtE2F transcriptional function using the NtRNR1b promoter as a target (J. Lang et al., unpublished results). In addition, this was confirmed by increased expression of the E2F target gene NtRNR1b in non-treated and BLM-treated NtE2F cells compared with the wild type (Fig. 5B). GFP:NtE2F overexpression did not modify the mortality of untreated and 10−5 M-treated cells, but significantly decreased PCD in 10−4 M-treated cells (P > 0.05) (Fig. 5C). At high BLM concentration, GFP:NtE2F not only reduced PCD, but almost totally inhibited chromatin condensation (Fig. 5D). Similarly, vacuolar collapse (type II cells) was not affected by 10−5 M BLM, but it was significantly decreased to ∼50% at 10−4 M BLM (P < 0.05) (Fig. 5E, F). These data show that vacuolar collapse and PCD induction are partially suppressed by GFP:NtE2F overexpression only in response to high doses of BLM. Fig. 5. View largeDownload slide NtE2F overexpression affects BLM-induced cell death and vacuolar collapse. BY-2 cells were stably transformed with the 35S::GFP:NtE2F construct, and GFP-positive clones, named NtE2F, were selected (A). Mid-log wild-type and NtE2F cells were transferred into MS medium with 10−5 M and 10−4 M BLM. After 24 h, the expression of NtRNR1b was analysed using real-time qPCR (B). After 72 h, the number of dead cells was quantified using the FDA viability test (C); chromatin condensation was analysed and quantified after nuclear DAPI staining (D). The number of vacuole-collapsed cells (type II, see text) in the culture was observed after BCECF-AM and Evans blue double staining during 72 h (E and F). Data represent the mean values (±SD). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Bar=18 μm. (This figure is available in colour at JXB online.) Fig. 5. View largeDownload slide NtE2F overexpression affects BLM-induced cell death and vacuolar collapse. BY-2 cells were stably transformed with the 35S::GFP:NtE2F construct, and GFP-positive clones, named NtE2F, were selected (A). Mid-log wild-type and NtE2F cells were transferred into MS medium with 10−5 M and 10−4 M BLM. After 24 h, the expression of NtRNR1b was analysed using real-time qPCR (B). After 72 h, the number of dead cells was quantified using the FDA viability test (C); chromatin condensation was analysed and quantified after nuclear DAPI staining (D). The number of vacuole-collapsed cells (type II, see text) in the culture was observed after BCECF-AM and Evans blue double staining during 72 h (E and F). Data represent the mean values (±SD). Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Bar=18 μm. (This figure is available in colour at JXB online.) Because deregulation of the pRB/E2F pathway in animal cells leads to increased genome instability (Pickering and Kowalik, 2006; Srinivasan et al., 2007), the effect of NtE2F overexpression on chromatin integrity and mitosis progression was examined. A total of 27±3% (P < 0.01) of 10−5 M BLM-treated NtE2F cells displayed DAPI-stained micronuclei, which were never observed to such an extent in 10−4 M BLM-treated cells or control cells, where their number did not exceed 2±0.5%. This suggested that micronuclei appeared as a result of abnormal mitotic division with lagging chromosomal fragments during anaphase and telophase. This was confirmed by FISH (Fig. 6), using a probe specific to telomeric sequences. Another hallmark of compromised genome integrity in NtE2F cells, even without genotoxins, was the formation of anaphase bridges found in 37±2% (P < 0.01) of telophase NtE2F cells, compared with control cells (1% of anaphase bridges). Fig. 6. View largeDownload slide Formation of micronuclei in NtE2F cells after BLM treatment. Wild-type and NtE2F cells were transferred to MS medium with BLM. The FISH experiment was performed on interphasic nuclei (left) and during telophase (right) 72 h after 10−5 M BLM treatment. Aliquots of BY-2 cells were hybridized with a telomere-specific probe labelled with Cy3 (red) and stained with DAPI (blue). Arrows indicate micronuclei; arrowheads indicate chromosomal bridges. Representative pictures of each sample are shown. Bar=10 μm. Fig. 6. View largeDownload slide Formation of micronuclei in NtE2F cells after BLM treatment. Wild-type and NtE2F cells were transferred to MS medium with BLM. The FISH experiment was performed on interphasic nuclei (left) and during telophase (right) 72 h after 10−5 M BLM treatment. Aliquots of BY-2 cells were hybridized with a telomere-specific probe labelled with Cy3 (red) and stained with DAPI (blue). Arrows indicate micronuclei; arrowheads indicate chromosomal bridges. Representative pictures of each sample are shown. Bar=10 μm. GFP:NtE2F overexpression modulates the G2/M DNA damage checkpoint for PCD induction To investigate further how GFP:NtE2F overexpression affects the DNA damage checkpoint, mitotic entry and FACS analysis were investigated in synchronized cells where BLM (10−5M) was applied at different cell cycle stages (Fig. 7A). APC, a specific inhibitor of DNA polymerase α and δ, reversibly blocks the cells in early S phase (Litvak and Castroviejo, 1985). Wild-type cells reached the maximum of mitosis at 9 h after APC release (0 h) and then progressed into G1, whereas NtE2F cells exhibited the peak of mitosis at 10 h, indicating a slightly slower cell cycle progression. Therefore, BLM treatments of synchronized NtE2F cells at G2 and M phases were modified accordingly (see the Materials and methods). Fig. 7. View largeDownload slide Mitotic index progression, DNA content, and cell death induction in wild-type (WT) and NtE2F cells after 10−5 M BLM treatment. WT and NtE2F cells were synchronized with APC blocking the cells at the entry of S phase. After the release of APC (0 h), BLM was added at different time points (S, G2, M, and G1; arrowheads) within the cell cycle (A). Mitotic index progression was analysed during 12 h after APC release (B). Flow cytometry analysis revealed the DNA content of the cells in the culture 24 h after APC release (C). At 72 h after the APC washing, the number of dead cells (±SD) was analysed with the aid of the FDA viability test (D). Representative values of three independent experiments are presented, due to some variable synchronization rates between each experiment. Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). Fig. 7. View largeDownload slide Mitotic index progression, DNA content, and cell death induction in wild-type (WT) and NtE2F cells after 10−5 M BLM treatment. WT and NtE2F cells were synchronized with APC blocking the cells at the entry of S phase. After the release of APC (0 h), BLM was added at different time points (S, G2, M, and G1; arrowheads) within the cell cycle (A). Mitotic index progression was analysed during 12 h after APC release (B). Flow cytometry analysis revealed the DNA content of the cells in the culture 24 h after APC release (C). At 72 h after the APC washing, the number of dead cells (±SD) was analysed with the aid of the FDA viability test (D). Representative values of three independent experiments are presented, due to some variable synchronization rates between each experiment. Asterisks indicate the values that are significantly different from the control (*P < 0.05; **P < 0.01). As expected, wild-type cells treated with 10−5 M BLM in S or G2 phases did not enter mitosis (Fig. 7B). In contrast, NtE2F cells treated under the same conditions partially entered mitosis, with a more pronounced effect when BLM was added in G2. In addition, as compared with the wild type, the MI dropped more slowly in NtE2F cells when BLM was added in M phase. In order to correlate the lack of entry into mitosis with G2/M checkpoint activation, FACS analyses were performed 24 h after APC release. Wild-type cells treated with BLM in S or G2 mainly exhibited a 4C DNA content, whereas cells treated in the M or G1 phase essentially presented a 2C DNA content as for control cells (Fig. 7C). This confirmed that BLM applied in S or G2 wild-type cells led to G2/M checkpoint activation. In contrast, treatment of NtE2F cells in any cell cycle phase led to a major accumulation of 2C DNA-containing cells. These data indicate that ectopic GFP:NtE2F expression impairs G2/M checkpoint activation in response to 10−5 M BLM, thereby leading to cell accumulation in G1 (Fig. 7C). Previous studies have demonstrated that PCD induction in BY-2 can be regulated throughout the cell cycle (Herbert et al., 2001; Kuthanova et al., 2008a). Therefore, dead cells were counted in synchronized culture after 3 d of 10−5 M BLM treatment applied during cell cycle progression. Wild-type cells exhibited the highest mortality when BLM was applied in G2 (Fig. 7D), whereas the mortality of NtE2F cells was significantly reduced (P < 0.01). Partial inhibition of cell death in NtE2F cells was also found during S or M phase treatments (P < 0.05), but to a lesser extent (Fig. 7D). These data indicate that when applied in G2, 10−5 M BLM induces maximal cell death which is significantly impeded by GFP:NtE2F overexpression. After a 10−4 M BLM treatment applied in G2, cell death induction was down-regulated by NtE2F overexpression (Fig. 8C), but entry into mitosis was not modified (Fig. 8A). Similarly, FACS analysis showed that most of the cells had 4C DNA content in both the NtE2F and wild-type lines (Fig. 8B), suggesting that G2/M checkpoint activation was not altered by NtE2F overexpression in response to 10−4 M BLM. Thus, at high BLM concentration, NtE2F overexpression does not modify G2/M checkpoint activation but only modulates the subsequent PCD induction. Fig. 8. View largeDownload slide Mitotic index, DNA content, and cell death induction in synchronized wild-type (WT) and E2F cells treated with 10−4 M BLM in G2 phase. The mitotic index in synchronized WT and NtE2F cells after BLM treatment during G2 phase (A). (B) Flow cytometry analysis of WT and NtE2F cells. The DNA content was measured 24 h after APC release. (C) Cell death induction in WT and NtE2F cells after BLM treatment during G2 phase. Quantification of cell death was performed 72 h after APC release. Asterisks indicate the values that are significantly different from the control (**P < 0.01). Fig. 8. View largeDownload slide Mitotic index, DNA content, and cell death induction in synchronized wild-type (WT) and E2F cells treated with 10−4 M BLM in G2 phase. The mitotic index in synchronized WT and NtE2F cells after BLM treatment during G2 phase (A). (B) Flow cytometry analysis of WT and NtE2F cells. The DNA content was measured 24 h after APC release. (C) Cell death induction in WT and NtE2F cells after BLM treatment during G2 phase. Quantification of cell death was performed 72 h after APC release. Asterisks indicate the values that are significantly different from the control (**P < 0.01). Discussion This study provides the first evidence of a non-apoptotic PCD in higher plant cells that most closely matches to paraptotic-like PCD. The implication of the NtE2F activator factor during this process was determined using a novel model system based on synchronizable tobacco BY-2 cells treated with BLM. In contrast to mammals, Drosophila, or Caenorhabditis, overexpression of NtE2F fused to GFP prevents BLM-induced PCD, which is at least partly dependent on ATM/ATR pathways. It should be underlined that BLM-triggered PCD appeared to be strongly dependent on G2/M checkpoint activation, which could be modulated by GFP:NtE2F overexpression. BLM induces non-apoptotic PCD The current studies have shown that genotoxic compounds can induce PCD in various plant model systems. Apoptotic-like PCD after UV-C or camptothecin treatments was characterized by typical apoptotic hallmarks such as oligonucleosomal DNA fragmentation, involvement of caspase-like activities, fragmentation of the nucleus, or oxidative burst (De Jong et al., 2000; Danon et al., 2004; Gao et al., 2008). Other authors, using zeocin or γ-irradiation as PCD inducers in Arabidopsis root tips, were not so focused on PCD morphology, thus its classification is more than difficult (Fulcher and Sablowski, 2009; Furukawa et al., 2010). In tobacco BY-2 cells, BLM-induced PCD was characterized by some typical apoptotic features such as cell shrinkage, chromatin condensation, and ROS accumulation; however, apoptotic oligonucleosomal DNA fragmentation was not detected either by TUNEL assay or by DNA electrophoresis. Moreover, specific caspase inhibitors did fail to block BLM-induced cell death as described, for example, for UV-C- or 6-BAP-induced PCD in Arabidopsis mesophyll or BY-2 cells, respectively (Mlejnek and Prochazka, 2002; Danon et al., 2004). These data suggest that caspase activities are not implicated in the BLM-induced PCD. Therefore, this type of PCD does not present a classical apoptotic character, in spite of the apoptotic-like features mentioned above. Vacuolar collapse was described as a key step of autophagic PCD, resulting in a leakage of vacuolar hydrolases such as vacuolar processing enzymes (VPEs) into the cytoplasm. This mechanism ensures a rapid cell death to prevent the pathogen spreading, as was shown for vacuole-mediated PCD during the HR induced by plant elicitors such as cryptogein or by virus infection (Hatsugai et al., 2004; Kuroyanagi et al., 2005; van Doorn and Woltering, 2005). Similarly, vacuole-mediated cell death during xylem development in Zinnia also occurs within a few minutes after vacuolar disintegration (Obara et al., 2001). Thus, if vacuolar collapse mediates PCD, the number of cells with disintegrated vacuoles but still alive (type II) should be very limited. Taking into account that PCD processing was relatively slow (days compared with minutes or hours during HR or developmental PCD) and that PCD did not appear immediately after vacuolar rupture, it is very likely that BLM-induced cell death is distinct from the previously reported vacuole-mediated PCD (Obara et al., 2001; Hatsugai et al., 2004). Moreover, as the VPE-specific inhibitor z-YVAD-FMK did not modify the amount of dead cells, this group of vacuolar hydrolases, being crucial for vacuole-mediated PCD, is most probably not involved in the BLM-induced PCD. Until now, paraptotic PCD was considered as an alternative non-apoptotic form of PCD in animals, for example in some tumours or during neurodegenerative diseases (Bröker et al., 2005; Fombonne et al., 2006; Zhang et al., 2011). This type of cell death is accompanied by vacuolization, phosphatidylserine exposure to the extracellular side of the plasma membrane, absence of oligonucleosomal DNA cleavage, pyknosis, and insensitivity to caspase inhibitors or a requirement for transcription (Sperandio et al., 2000, 2004; Wang et al., 2004). However, not all these hallmarks are always present during paraptotic PCD, showing that a wider range of paraptotic PCD mechanisms may exist (Sun et al., 2010). Taking into account all morphological and biochemical analyses, BLM-induced cell death most resembled paraptosis, mainly due to the apparent inefficiency of caspase inhibitors, lack of DNA fragmentation, and extensive vacuolization. However, the signalling involved in this alternative PCD remains to be elucidated in plants in order to confirm whether BLM-induced PCD is strictly homologous to animal paraptosis. Paraptotic-like PCD is inhibited by caffeine and GFP:NtE2F overexpression When caffeine, an inhibitor of the PI-3Ks ATM and ATR, was applied, a significant decrease of PCD, chromatin condensation, and vacuolar disintegration was observed, but only at a high BLM concentration. These data suggest that BLM-induced paraptotic-like PCD is at least partially dependent on ATM/ATR kinases. This phenomenon has already been reported for Arabidopsis meristematic cells that were subjected to DSB inducers (zeocin and γ-irradiation) (Fulcher and Sablowski, 2009; Furukawa et al., 2010), but the novelty of these results is that ATM/ATR could regulate a paraptotic-like PCD in plant cells. This hypothesis finds further support in results obtained with Arabidopsis, where caffeine inhibits the ATM/ATR-dependent PCD in root meristematic cells as observed in atm (Supplementary Fig. S5 at JXB online). These observations may indicate some conservation of the ATM/ATR control of DSB-induced PCD in higher plants. A low concentration of BLM induced only a minor decrease in cell viability, which was not affected by caffeine. There are two possible explanations: this PCD is mediated either by residual ATM/ATR activity in the case of incomplete caffeine inhibition, or by some other pathways, independent of ATM and ATR. Indeed, the existence of such a pathway may be supported by Furukawa’s findings that DNA damage-induced PCD is not totally inhibited in the atm atr double mutant (Furukawa et al., 2010). In contrast to animals, where E2F1 is a positive regulator of apoptosis through transcriptional regulation of pro-apoptotic genes (p53, Apaf1, and caspases) (Muller et al., 2001), NtE2F may have no pro-cell death function in plants since its overexpression prevents cell death induction. However, it cannot be ruled out that some other uncharacterized tobacco E2F factors may have a pro-cell death role. Such a discrepancy in E2F-mediated PCD between plants and animals may be due mainly to evolutionary constraints. Indeed, sedentary plants, in view of their inherent developmental plasticity, can generally tolerate much higher doses of DNA damage and therefore they may not need such strict regulation of PCD as for animal apoptosis (Yokota et al., 2005). This could be illustrated by decreased genomic integrity induced by NtE2F overexpression at low BLM concentrations, but without a subsequent increase of PCD. One has to keep in mind that these experiments based on GFP:NtE2F overexpression could have a limited biological relevance. Nevertheless, this approach could be considered as a tool to study DNA damage checkpoint regulation in relation to PCD. BLM-induced PCD requires G2/M checkpoint activation Even though cell cycle-dependent PCD has already been studied using various models and conditions of PCD induction (Herbert et al., 2001; Kadota et al., 2004), the results are hardly comparable. Cadmium was shown to promote cell death when applied in all cell cycle phases of synchronized BY-2 cells, but to induce DNA fragmentation and apoptotic-like PCD only when applied prior to mitotic entry (Kuthanova et al., 2008b). In contrast, BLM-induced DNA fragmentation was never observed, even when BLM was applied during G2 phase (data not shown). Complementary experiments using a broad range of genotoxic compounds to induce PCD will now be required to define the character and the underlying mechanism of G2/M-related PCD in plant cells. BLM treatment of synchronized cells activates both G1/S and G2/M checkpoints, suggesting the induction of DNA repair pathways; however, a certain cell fraction undergoes PCD. This cell death induction mainly resulted from the BLM concentration-dependent activation of the G2/M checkpoint in order to remove heavily DNA-damaged cells. Simultaneously, BLM treatment triggers an increased expression of NtRNR1b, which is in agreement with previous data showing the key role of RNR during DNA repair (Wang and Liu, 2006; Roa et al., 2009) (Fig. 9A, B). The requirement for G2/M checkpoint activation for PCD induction upon high BLM doses is further supported by the fact that stationary cells that are mainly in G1 phase exhibit low PCD induction under the same conditions of genotoxic treatment (Fig. 5C; Supplementary Fig. S6 at JXB online). Fig. 9. View largeDownload slide Model of PCD regulation in wild-type (A, B) and NtE2F (C, D) cells upon 10−5 M (A, C) and 10−4 M (B, D) BLM treatment. Thick arrows indicate preferential pathways under the given conditions. Fig. 9. View largeDownload slide Model of PCD regulation in wild-type (A, B) and NtE2F (C, D) cells upon 10−5 M (A, C) and 10−4 M (B, D) BLM treatment. Thick arrows indicate preferential pathways under the given conditions. When NtE2F is overexpressed, besides its capacity to induce DNA repair due to NtRNR1b up-regulation as reported previously (Lincker et al., 2008), it also modulates DNA damage G2/M checkpoint activation in a BLM dose-dependent manner. With a low BLM concentration, G2/M checkpoint activation is partially overridden, thus leading to mitotic entry with unrepaired DNA detected as micronuclei and decreased PCD as well (Fig. 9C). These data do not preclude that DNA repair might exist in G1/S. Using a high BLM concentration (10−4 M), NtE2F overexpression does not impair G2/M checkpoint activation, as clearly documented by the accumulation of 4C nuclei and the absence of formation of micronuclei. However, even at this high BLM concentration, NtE2F overexpression significantly reduced the mortality, most probably by increased repair efficiency due to E2F-mediated up-regulation of DNA repair genes, as shown for NtRNR1b (Fig. 9D). In tobacco BY-2 cells, G2/M transition is linked to increased CDKB1 activity and protein levels, suggesting a role for CDKB1 in G2/M checkpoint regulation (Porceddu et al., 2001; Sorrell et al., 2001). The expression and activity of the mitotic CDKB1 have been shown to be induced by both AtE2Fa and AtE2Fb (Boudolf et al., 2004; Magyar et al., 2005; Sozzani et al., 2006). It is therefore tempting to suggest that bypass of the BLM-induced G2/M checkpoint induced by NtE2F overexpression might be related to increased CDKB1 activity controlled by NtE2F, which is a close homologue of AtE2Fa and b (Lincker et al., 2008). Is paraptotic-like PCD an evolutionarily conserved ancestral mode of cell death in eukaryotes? In conclusion, based on morphological criteria, BY-2 cells have the capacity to undergo non-apoptotic cell death with paraptotic-like features, which is impaired by E2F overexpression and ATM/ATR inhibition. It seems that paraptotic-like PCD could be conserved among animals (Sperandio et al., 2000), protists (Jimenez et al., 2009), and plants (this study), and therefore might represent an ancestral form of PCD, notably as compared with the evolutionarily more recent apoptosis. In contrast to apoptotic PCD, the molecular basis of paraptotic cell death is very limited. For example, mitogen-activated protein kinases (MAPKs) regulate paraptosis in animals and algae (Sperandio et al., 2004; Jimenez et al., 2009) and, interestingly, their plant homologues participate in a general stress-activated pathway including DNA damage response (Bulavin et al., 2001; Ulm et al., 2001; Reinhardt et al., 2007). These data raise an interesting question, namely whether the MAPK pathway might be involved in the BLM-induced PCD. An identification of such general paraptotic regulators is needed to demonstrate the conservation of paraptosis among eukaryotes. We thank Dr A. Tissier for providing the atm mutant, and F. Cvrčková and T.J. Bach for critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Youth and Sport of the Czech Republic (MSM 0021620858), Charles University (GAUK 43-259157), and by an EDS bridge scholarship from Collège Doctoral Européen, Strasbourg. References Amino SI,  Nagata T.  Caffeine-induced uncoupling of mitosis from DNA replication in tobacco BY-2 cells,  Journal of Plant Research ,  1996, vol.  109 (pg.  219- 222) Google Scholar CrossRef Search ADS   An G,  Watson BD,  Stachel S,  Gordon MP,  Nester EW.  New cloning vehicles for transformation of higher plants,  The EMBO Journal ,  1985, vol.  4 (pg.  277- 284) Google Scholar PubMed  Asano M,  Nevins JR,  Wharton RP.  Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs,  Genes and Development ,  1996, vol.  10 (pg.  1422- 1432) Google Scholar CrossRef Search ADS PubMed  Boudolf V,  Vlieghe K,  Beemster GT,  Magyar Z,  Torres Acosta JA,  Maes S,  Van Der Schueren E,  Inze D,  De Veylder L.  The plant-specific cyclin-dependent kinase CDKB1;1 and transcription factor E2Fa-DPa control the balance of mitotically dividing and endoreduplicating cells in Arabidopsis,  The Plant Cell ,  2004, vol.  16 (pg.  2683- 2692) Google Scholar CrossRef Search ADS PubMed  Bröker LE,  Kruyt FA,  Giaccone G.  Cell death independent of caspases: a review,  Clinical Cancer Research ,  2005, vol.  11 (pg.  3155- 3162) Google Scholar CrossRef Search ADS PubMed  Brown EJ,  Baltimore D.  Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance,  Genes and Development ,  2003, vol.  17 (pg.  615- 628) Google Scholar CrossRef Search ADS PubMed  Buchakjian MR,  Kornbluth S.  The engine driving the ship: metabolic steering of cell proliferation and death,  Nature Reviews Molecular Cell Biology ,  2010, vol.  11 (pg.  715- 727) Google Scholar CrossRef Search ADS PubMed  Bulavin DV,  Higashimoto Y,  Popoff IJ,  Gaarde WA,  Basrur V,  Potapova O,  Appella E,  Fornace AJJ.  Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase,  Nature ,  2001, vol.  411 (pg.  102- 107) Google Scholar CrossRef Search ADS PubMed  Chaboute ME,  Clement B,  Sekine M,  Philipps G,  Chaubet-Gigot N.  Cell cycle regulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements,  The Plant Cell ,  2000, vol.  12 (pg.  1987- 2000) Google Scholar CrossRef Search ADS PubMed  Chaturvedi P,  Eng WK,  Zhu Y, et al.  Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway,  Oncogene ,  1999, vol.  18 (pg.  4047- 4054) Google Scholar CrossRef Search ADS PubMed  Chen J,  Zhu F,  Weaks RL,  Biswas AK,  Guo R,  Li Y,  Johnson DG.  E2F1 promotes the recruitment of DNA repair factors to sites of DNA double-strand breaks,  Cell Cycle ,  2011, vol.  10 (pg.  1- 8) Google Scholar CrossRef Search ADS PubMed  Coll NS,  Vercammen D,  Smidler A,  Clover C,  Van Breusegem F,  Dangl JL,  Epple P.  Arabidopsis type I metacaspases control cell death,  Science ,  2010, vol.  330 (pg.  1393- 1397) Google Scholar CrossRef Search ADS PubMed  Cools T,  De Veylder L.  DNA stress checkpoint control and plant development,  Current Opinion in Plant Biology ,  2009, vol.  12 (pg.  23- 28) Google Scholar CrossRef Search ADS PubMed  Culligan K,  Tissier A,  Britt A.  ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana,  The Plant Cell ,  2004, vol.  16 (pg.  1091- 1104) Google Scholar CrossRef Search ADS PubMed  Culligan KM,  Robertson CE,  Foreman J,  Doerner P,  Britt AB.  ATR and ATM play both distinct and additive roles in response to ionizing radiation,  The Plant Journal ,  2006, vol.  48 (pg.  947- 961) Google Scholar CrossRef Search ADS PubMed  Danon A,  Rotari VI,  Gordon A,  Mailhac N,  Gallois P.  Ultraviolet-C overexposure induces programmed cell death in Arabidopsis, which is mediated by caspase-like activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death,  Journal of Biological Chemistry ,  2004, vol.  279 (pg.  779- 787) Google Scholar CrossRef Search ADS PubMed  DeGregori J.  The genetics of the E2F family of transcription factors: shared functions and unique roles,  Biochimica et Biophysica Acta ,  2002, vol.  1602 (pg.  131- 150) Google Scholar PubMed  De Jong AJ,  Hoeberichts FA,  Yakimova ET,  Maximova E,  Woltering EJ.  Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases,  Planta ,  2000, vol.  211 (pg.  656- 662) Google Scholar CrossRef Search ADS PubMed  De Veylder L,  Beeckman T,  Beemster GT, et al.  Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor,  EMBO Journal ,  2002, vol.  21 (pg.  1360- 1368) Google Scholar CrossRef Search ADS PubMed  Fombonne J,  Padron L,  Enjalbert A,  Krantic S,  Torriglia A.  A novel paraptosis pathway involving LEI/L-DNase II for EGF-induced cell death in somato-lactotrope pituitary cells,  Apoptosis ,  2006, vol.  11 (pg.  367- 375) Google Scholar CrossRef Search ADS PubMed  Fulcher N,  Sablowski R.  Hypersensitivity to DNA damage in plant stem cell niches,  Proceedings of the National Academy of Sciences, USA ,  2009, vol.  106 (pg.  20984- 20988) Google Scholar CrossRef Search ADS   Furukawa T,  Curtis MJ,  Tominey CM,  Duong YH,  Wilcox BW,  Aggoune D,  Hays JB,  Britt AB.  A shared DNA-damage-response pathway for induction of stem-cell death by UVB and by gamma irradiation,  DNA Repair ,  2010, vol.  9 (pg.  940- 948) Google Scholar CrossRef Search ADS PubMed  Gadjev I,  Stone JM,  Gechev TS.  Programmed cell death in plants: new insights into redox regulation and the role of hydrogen peroxide,  International Review of Cell and Molecular Biology ,  2008, vol.  270 (pg.  87- 144) Google Scholar PubMed  Gao C,  Xing D,  Li L,  Zhang L.  Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure,  Planta ,  2008, vol.  227 (pg.  755- 767) Google Scholar CrossRef Search ADS PubMed  Garcia V,  Bruchet H,  Camescasse D,  Granier F,  Bouchez D,  Tissier A.  AtATM is essential for meiosis and the somatic response to DNA damage in plants,  The Plant Cell ,  2003, vol.  15 (pg.  119- 132) Google Scholar CrossRef Search ADS PubMed  Gonzalez-Fernandez A,  Hernandez P,  Lopez-Saez JF.  Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage,  Mutation Research ,  1985, vol.  149 (pg.  275- 281) Google Scholar CrossRef Search ADS PubMed  Hartley-Asp B,  Andersson HC,  Sturelid S,  Kihlman BA.  G2 repair and the formation of chromosomal aberrations—I. The effect of hydroxyurea and caffeine on maleic hydrazide-induced chromosome damage in Vicia faba,  Environmental and Experimental Botany ,  1980, vol.  20 (pg.  119- 129) Google Scholar CrossRef Search ADS   Hatsugai N,  Kuroyanagi M,  Nishimura M,  Hara-Nishimura I.  A cellular suicide strategy of plants: vacuole-mediated cell death,  Apoptosis ,  2006, vol.  11 (pg.  905- 911) Google Scholar CrossRef Search ADS PubMed  Hatsugai N,  Kuroyanagi M,  Yamada K,  Meshi T,  Tsuda S,  Kondo M,  Nishimura M,  Hara-Nishimura I.  A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death,  Science ,  2004, vol.  305 (pg.  855- 858) Google Scholar CrossRef Search ADS PubMed  Hempel SL,  Buettner GR,  O’Malley YQ,  Wessels DA,  Flaherty DM.  Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123,  Free Radical Biology and Medicine ,  1999, vol.  27 (pg.  146- 159) Google Scholar CrossRef Search ADS PubMed  Hengartner MO.  The biochemistry of apoptosis,  Nature ,  2000, vol.  407 (pg.  770- 776) Google Scholar CrossRef Search ADS PubMed  Henriques R,  Magyar Z,  Monardes A,  Khan S,  Zalejski C,  Orellana J,  Szabados L,  de la Torre C,  Koncz C,  Bogre L.  Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1–E2F pathway activity,  EMBO Journal ,  2010, vol.  29 (pg.  2979- 2993) Google Scholar CrossRef Search ADS PubMed  Herbert RJ,  Vilhar B,  Evett C,  Orchard CB,  Rogers HJ,  Davies MS,  Francis D.  Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line,  Journal of Experimental Botany ,  2001, vol.  52 (pg.  1615- 1623) Google Scholar PubMed  Higaki T,  Goh T,  Hayashi T,  Kutsuna N,  Kadota Y,  Hasezawa S,  Sano T,  Kuchitsu K.  Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells,  Plant and Cell Physiology ,  2007, vol.  48 (pg.  1414- 1425) Google Scholar CrossRef Search ADS PubMed  Hsieh JK,  Fredersdorf S,  Kouzarides T,  Martin K,  Lu X.  E2F1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction,  Genes and Development ,  1997, vol.  11 (pg.  1840- 1852) Google Scholar CrossRef Search ADS PubMed  Jimenez C,  Capasso JM,  Edelstein CL,  Rivard CJ,  Lucia S,  Breusegem S,  Berl T,  Segovia M.  Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase,  Journal of Experimental Botany ,  2009, vol.  60 (pg.  815- 828) Google Scholar CrossRef Search ADS PubMed  Kadota Y,  Watanabe T,  Fujii S,  Higashi K,  Sano T,  Nagata T,  Hasezawa S,  Kuchitsu K.  Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells,  The Plant Journal ,  2004, vol.  40 (pg.  131- 142) Google Scholar CrossRef Search ADS PubMed  Karimi M,  Inze D,  Depicker A.  GATEWAY vectors for Agrobacterium-mediated plant transformation,  Trends in Plant Science ,  2002, vol.  7 (pg.  193- 195) Google Scholar CrossRef Search ADS PubMed  Kowalik TF,  DeGregori J,  Schwarz JK,  Nevins JR.  E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis,  Journal of Virology ,  1995, vol.  69 (pg.  2491- 2500) Google Scholar PubMed  Kumagai-Sano F,  Hayashi T,  Sano T,  Hasezawa S.  Cell cycle synchronization of tobacco BY-2 cells,  Nature Protocols ,  2006, vol.  1 (pg.  2621- 2627) Google Scholar CrossRef Search ADS PubMed  Kuroyanagi M,  Yamada K,  Hatsugai N,  Kondo M,  Nishimura M,  Hara-Nishimura I.  Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana,  Journal of Biological Chemistry ,  2005, vol.  280 (pg.  32914- 32920) Google Scholar CrossRef Search ADS PubMed  Kuthanova A,  Fischer L,  Nick P,  Opatrny Z.  Cell cycle phase-specific death response of tobacco BY-2 cell line to cadmium treatment,  Plant, Cell and Environment ,  2008, vol.  31 (pg.  1634- 1643) Google Scholar CrossRef Search ADS   Kuthanova A,  Opatrny Z,  Fischer L.  Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells?,  Journal of Experimental Botany ,  2008, vol.  59 (pg.  2233- 2240) Google Scholar CrossRef Search ADS PubMed  Lee JH,  Paull TT.  Activation and regulation of ATM kinase activity in response to DNA double-strand breaks,  Oncogene ,  2007, vol.  26 (pg.  7741- 7748) Google Scholar CrossRef Search ADS PubMed  Lincker F,  Philipps G,  Chaboute ME.  UV-C response of the ribonucleotide reductase large subunit involves both E2F-mediated gene transcriptional regulation and protein subcellular relocalization in tobacco cells,  Nucleic Acids Research ,  2004, vol.  32 (pg.  1430- 1438) Google Scholar CrossRef Search ADS PubMed  Lincker F,  Roa H,  Lang J,  Sanchez-Calderon L,  Smetana O,  Cognat V,  Keller M,  Mediouni C,  Houlné G,  Chaboute ME.  Plant E2F factors in cell cycle development and DNA damage response. In: Yoshida K, ed,  Control of cellular physiology by transcription factors E2F. Trivandrum, India: Research Signpost ,  2008(pg.  17- 31) Litvak S,  Castroviejo M.  Plant DNA polymerases,  Plant Molecular Biology ,  1985, vol.  4 (pg.  311- 314) Google Scholar CrossRef Search ADS PubMed  Livak KJ,  Schmittgen TD.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method,  Methods ,  2001, vol.  25 (pg.  402- 408) Google Scholar CrossRef Search ADS PubMed  Magyar Z,  De Veylder L,  Atanassova A,  Bako L,  Inze D,  Bogre L.  The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division,  The Plant Cell ,  2005, vol.  17 (pg.  2527- 2541) Google Scholar CrossRef Search ADS PubMed  Matsuoka K,  Higuchi T,  Maeshima M,  Nakamura K.  A vacuolar-type H+-ATPase in a nonvacuolar organelle is required for the sorting of soluble vacuolar protein precursors in tobacco cells,  The Plant Cell ,  1997, vol.  9 (pg.  533- 546) Google Scholar PubMed  Mlejnek P,  Prochazka S.  Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells,  Planta ,  2002, vol.  215 (pg.  158- 166) Google Scholar CrossRef Search ADS PubMed  Muller H,  Bracken AP,  Vernell R,  Moroni MC,  Christians F,  Grassilli E,  Prosperini E,  Vigo E,  Oliner JD,  Helin K.  E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis,  Genes and Development ,  2001, vol.  15 (pg.  267- 285) Google Scholar CrossRef Search ADS PubMed  Nagata T,  Nemoto Y,  Hasezawa S.  Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants,  International Review of Cytology ,  1992, vol.  132 (pg.  1- 30) Naouar N,  Vandepoele K,  Lammens T, et al.  Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes,  The Plant Journal ,  2009, vol.  57 (pg.  184- 194) Google Scholar CrossRef Search ADS PubMed  Obara K,  Kuriyama H,  Fukuda H.  Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia,  Plant Physiology ,  2001, vol.  125 (pg.  615- 626) Google Scholar CrossRef Search ADS PubMed  Pelayo HR,  Lastres P,  De la Torre C.  Replication and G2 checkpoints: their response to caffeine,  Planta ,  2001, vol.  212 (pg.  444- 453) Google Scholar CrossRef Search ADS PubMed  Pickering MT,  Kowalik TF.  Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation,  Oncogene ,  2006, vol.  25 (pg.  746- 755) Google Scholar CrossRef Search ADS PubMed  Porceddu A,  Stals H,  Reichheld JP,  Segers G,  De Veylder L,  Barroco RP,  Casteels P,  Van Montagu M,  Inze D,  Mironov V.  A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants,  Journal of Biological Chemistry ,  2001, vol.  276 (pg.  36354- 36360) Google Scholar CrossRef Search ADS PubMed  Qin XQ,  Livingston DM,  Kaelin WGJ,  Adams PD.  Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis,  Proceedings of the National Academy of Sciences, USA ,  1994, vol.  91 (pg.  10918- 10922) Google Scholar CrossRef Search ADS   Radziejwoski A,  Vlieghe K,  Lammens T, et al.  Atypical E2F activity coordinates PHR1 photolyase gene transcription with endoreduplication onset,  EMBO Journal ,  2011, vol.  30 (pg.  355- 363) Google Scholar CrossRef Search ADS PubMed  Reinhardt HC,  Aslanian AS,  Lees JA,  Yaffe MB.  p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage,  Cancer Cell ,  2007, vol.  11 (pg.  175- 189) Google Scholar CrossRef Search ADS PubMed  Ricaud L,  Proux C,  Renou JP,  Pichon O,  Fochesato S,  Ortet P,  Montane MH.  ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis,  PLoS One ,  2007  2, e430 Roa H,  Lang J,  Culligan KM,  Keller M,  Holec S,  Cognat V,  Montane MH,  Houlne G,  Chaboute ME.  Ribonucleotide reductase regulation in response to genotoxic stress in Arabidopsis,  Plant Physiology ,  2009, vol.  151 (pg.  461- 471) Google Scholar CrossRef Search ADS PubMed  Roldan-Arjona T,  Ariza RR.  Repair and tolerance of oxidative DNA damage in plants,  Mutation Research ,  2009, vol.  681 (pg.  169- 179) Google Scholar CrossRef Search ADS PubMed  Sarkaria JN,  Busby EC,  Tibbetts RS,  Roos P,  Taya Y,  Karnitz LM,  Abraham RT.  Inhibition of ATM and ATR kinase activities by radiosensitizing agent, caffeine,  Cancer Research ,  1999, vol.  59 (pg.  4375- 4382) Google Scholar PubMed  Schertel C,  Conradt B.  C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions,  Development ,  2007, vol.  134 (pg.  3691- 3701) Google Scholar CrossRef Search ADS PubMed  Sekine M,  Ito M,  Uemukai K,  Maeda Y,  Nakagami H,  Shinmyo A.  Isolation and characterization of the E2F-like gene in plants,  FEBS Letters ,  1999, vol.  460 (pg.  117- 122) Google Scholar CrossRef Search ADS PubMed  Sorrell DA,  Menges M,  Healy JM, et al.  Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar Bright Yellow-2 cells,  Plant Physiology ,  2001, vol.  126 (pg.  1214- 1223) Google Scholar CrossRef Search ADS PubMed  Sozzani R,  Maggio C,  Varotto S,  Canova S,  Bergounioux C,  Albani D,  Cella R.  Interplay between Arabidopsis activating factors E2Fb and E2Fa in cell cycle progression and development,  Plant Physiology ,  2006, vol.  140 (pg.  1355- 1366) Google Scholar CrossRef Search ADS PubMed  Sperandio S,  de Belle I,  Bredesen DE.  An alternative, nonapoptotic form of programmed cell death,  Proceedings of the National Academy of Sciences, USA ,  2000, vol.  97 (pg.  14376- 14381) Google Scholar CrossRef Search ADS   Sperandio S,  Poksay K,  de Belle I,  Lafuente MJ,  Liu B,  Nasir J,  Bredesen DE.  Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix,  Cell Death and Differentiation ,  2004, vol.  11 (pg.  1066- 1075) Google Scholar CrossRef Search ADS PubMed  Srinivasan SV,  Mayhew CN,  Schwemberger S,  Zagorski W,  Knudsen ES.  RB loss promotes aberrant ploidy by deregulating levels and activity of DNA replication factors,  Journal of Biological Chemistry ,  2007, vol.  282 (pg.  23867- 23877) Google Scholar CrossRef Search ADS PubMed  Sun Q,  Chen T,  Wang X,  Wei X.  Taxol induces paraptosis independent of both protein synthesis and MAPK pathway,  Journal of Cellular Physiology ,  2010, vol.  222 (pg.  421- 432) Google Scholar CrossRef Search ADS PubMed  Ulm R,  Revenkova E,  di Sansebastiano GP,  Bechtold N,  Paszkowski J.  Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis,  Genes and Development ,  2001, vol.  15 (pg.  699- 709) Google Scholar CrossRef Search ADS PubMed  van Doorn WG,  Woltering EJ.  Many ways to exit? Cell death categories in plants,  Trends in Plant Science ,  2005, vol.  10 (pg.  117- 122) Google Scholar CrossRef Search ADS PubMed  Vandepoele K,  Vlieghe K,  Florquin K,  Hennig L,  Beemster GT,  Gruissem W,  Van de Peer Y,  Inze D,  De Veylder L.  Genome-wide identification of potential plant E2F target genes,  Plant Physiology ,  2005, vol.  139 (pg.  316- 328) Google Scholar CrossRef Search ADS PubMed  Vercammen D,  Declerq W,  Vandenabeele P,  Van Breusegem F.  Are metacaspases caspases?,  Journal of Cell Biology ,  2007, vol.  179 (pg.  375- 380) Google Scholar CrossRef Search ADS PubMed  Wang Y,  Li X,  Wang L,  Ding P,  Zhang Y,  Han W,  Ma D.  An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression,  Journal of Cell Science ,  2004, vol.  117 (pg.  1525- 1532) Google Scholar CrossRef Search ADS PubMed  Wang C,  Liu Z.  Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development,  The Plant Cell ,  2006, vol.  18 (pg.  350- 365) Google Scholar CrossRef Search ADS PubMed  Weingartner M,  Pelayo HR,  Binarova P,  Zwerger K,  Melikant B,  de la Torre C,  Heberle-Bors E,  Bogre L.  A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint,  Journal of Cell Science ,  2003, vol.  116 (pg.  487- 498) Google Scholar CrossRef Search ADS PubMed  Yasuhara H.  Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast,  Plant and Cell Physiology ,  2005, vol.  46 (pg.  1083- 1092) Google Scholar CrossRef Search ADS PubMed  Yokota Y,  Shikazono N,  Tanaka A,  Hase Y,  Funayama T,  Wada S,  Inoue M.  Comparative radiation tolerance based on the induction of DNA double-strand breaks in tobacco BY-2 cells and CHO-K1 cells irradiated with gamma rays,  Radiation Reseach ,  2005, vol.  163 (pg.  520- 525) Google Scholar CrossRef Search ADS   Zhang JS,  Li DM,  He N,  Liu YH,  Wang CH,  Jiang SQ,  Chen BQ,  Liu JR.  A paraptosis-like cell death induced by delta-tocotrienol in human colon carcinoma SW620 cells is associated with the suppression of the Wnt signaling pathway,  Toxicology ,  2011, vol.  285 (pg.  8- 17) Google Scholar CrossRef Search ADS PubMed  © The Author [2012]. 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TI - Non-apoptotic programmed cell death with paraptotic-like features in bleomycin-treated plant cells is suppressed by inhibition of ATM/ATR pathways or NtE2F overexpression
JF - Journal of Experimental Botany
DO - 10.1093/jxb/err439
DA - 2012-01-20
UR - https://www.deepdyve.com/lp/oxford-university-press/non-apoptotic-programmed-cell-death-with-paraptotic-like-features-in-lxPtEaCeoL
SP - 2631
EP - 2644
VL - 63
IS - 7
DP - DeepDyve
ER -