The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes

Plant Physiology and Biochemistry 96 (2015) 72e82

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Research article

The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes * _ Aneta Zabka , Konrad Winnicki, Justyna Teresa Polit, Janusz Maszewski dz, Pomorska 141/143, 90-236 Ło dz, Poland Department of Cytophysiology, Faculty of Biology and Environmental Protection, University of Ło

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2015 Received in revised form 17 June 2015 Accepted 17 July 2015 Available online 23 July 2015

DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Allium cepa Apoptosis-like programmed cell death Chromosome aberrations DNA topoisomerase II Etoposide Ellipticine Proteasomes

1. Introduction In Eukaryotes, progression through the cell cycle is accompanied by almost complete dispersion of chromatin during S-phase replication, and by M phase condensation of the nucleoplasm to allow for individualization of chromosomes and segregation of sister chromatids. Many integrated control mechanisms of these alternating processes, based on specific cyclin-dependent kinases (CDKs), cyclins (regulatory subunits of CDK), protein phosphatases and other regulatory factors (De Veylder et al., 2007; Gong and Ferrell, 2010) not only determine the proper sequence of

* Corresponding author. _ E-mail addresses: [email protected] (A. Zabka), [email protected] (K. Winnicki), [email protected] (J.T. Polit), [email protected] (J. Maszewski). http://dx.doi.org/10.1016/j.plaphy.2015.07.016 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.

activities critical for interphase and mitotic events, but also organize an apparatus to protect the next generation of cells against errors in the transmission of genetic information (Dissmeyer et al.,  et al., 2011). 2009; Lipavska Unrepaired DNA single-strand breaks (SSBs) and other lesions in the genetic material generated both by endogenous processes (replication errors, oxidative stress-induced damage to base pairs) and by exposure to exogenous factors (drugs, xenobiotics, ionizing radiation and UV light) may collide with the transcriptional and replication machinery and may be converted into lethal doublestrand breaks (Caldecott, 2007). To preclude such a possibility, any abnormalities or structural damage of the genome activate molecular pathways of the cell cycle checkpoint machinery, which is capable of inhibiting DNA biosynthesis and mitotic chromosome condensation (Jackson and Bartek, 2009; Baranello et al., 2014). Slowing or arrest of the ‘nuclear cycle’ allow the cell time to express

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specific genes and to activate necessary repair factors (Elledge, 1996; Esnault et al., 2010). During transcription and DNA replication some genomic aberrations arise as a consequence of inappropriate activity of enzymes called DNA topoisomerases I and II, which are responsible for solving the problems concerning DNA topology (Jackson and Bartek, 2009). Type II topoisomerase (Topo II) is involved in DNA replication, transcription, chromatin remodeling, condensation and chromosome segregation (Mak et al., 2005; Nitiss, 2009a). In an ATP-dependent reaction, by relaxation of positively and negatively supercoiled DNA, Topo II enables the removal of knots, supercoils and catenates (Champoux, 2001; Vos et al., 2011; Lane et al., 2013). Accordingly, its activity is directed mainly toward decatenation of sister chromatids after the S phase is complete (Charbin et al., 2014). Topo II inhibitors are one of the major groups of cytostatics showing significant anticancer activity. Two categories of chemical agents with different mechanisms of action can block the functions of Topo II: Topo II poisons and catalytic inhibitors (Nitiss, 2009b; Pommier et al., 2010). The first group includes etoposide (VP-16), teniposide (VM-26), and DNA intercalators, such as anthracenedione (mitoxantrone), anthracyclines (doxorubicin and daunorubicin) and aminoacridines (e.g. amsacrine; m-AMSA), which stabilize the cleavable (or ‘cleavage’) complexes and inhibit DNA religation. Another group of Topo II poisons, including ellipticines, azatoxins, quinolones and isoflavones (genistein), exert their inhibitory actions by enhancing the formation of covalent enzyme~ a-Fouce et al., cleaved DNA complexes (Pommier et al., 2010; Balan 2014). Collision of Topo II-DNA cleavable complexes with the advancing replication fork or transcription machinery results in the formation of DNA single- and double-strand breaks (SSBs and DSBs, respectively). Catalytic inhibitors, such as bisdioxopiperazines (ICRF-159, ICRF-193, ICRF-187), fostriecin, aclarubicin, suramin, novobiocin and merbaron (Bassi and Palitti, 2000; Park and Avraham, 2006), block Topo II enzymatic activity without trap~ a-Fouce ping the covalent complexes (Pommier et al., 2010; Balan et al., 2014). Our current study on Allium cepa root meristem cells compares the effects of two DNA Topo II inhibitors e etoposide (ETO; also known as VP-16) and ellipticine (EPC) e on cell cycle progression in primary root meristems of A. cepa. Despite distinct modes of action exerted by these drugs and their evident impact on nuclear integrity, no essential changes between ETO- and EPC-treated seedlings were noticed in the frequency of cells at the G1, S and G2/M phases, nuclear morphology, chromosome structures, tubulin-microtubule systems, distribution of mitosis-specific phosphorylation of histone H3, and the induction of apoptosis-like programmed cell death (ALPCD). However, significant differences between experimental series appeared following incubation of onion roots with ETO and EPC mixed with MG132 (an inhibitor of 26S proteasomes). This result indicates specific dependence of effects formed by Topo II inhibitors on proteasome-mediated degradation of molecular complexes created at the DNA level. 2. Results 2.1. Influence of ETO, EPC, and their mixtures with MG132 on mitotic activity and the incidence of chromosomal aberrations The final concentrations of Topo II inhibitors, 200 mM ETO and 200 mM EPC, were selected in a series of preliminary tests using 1, 10, 20, 50, 100, 200, 300 mM solutions (on the basis of the available literature) and 0.5, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h treatments, the latter period covering the mean cell cycle time in apical root

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meristems of A. cepa (Navarrete et al., 1987). Exploiting the ability to form fluorescent adducts with DNA, covalent intercalation of 200 mM EPC was recorded in the G1, S, and G2 nuclei of A. cepa root meristem cells at all incubation times (0.5, 2, 4, 6, 8, 10, 12, 14, 16, 18 h) by microspectrofluorimetric measurements. As shown in Fig. 1AeK, chromatin fluorescence increased with time, reaching the highest value at the 14 and 16 h treatment time-points, which was followed by a slight decrease of nuclear EPC fluorescence at 18 h of EPC treatment. A considerable number of studies have implicated that DNA topoisomerases undergo proteasome-mediated degradation in cells treated with Topo II poisons [e.g., (Lyu et al., 2007; Azarova et al., 2010)]. In order to address this problem, some experiments were performed using MG132 (carbobenzoxyl-L-leucyl-L-leucyl-Lleucinal), a membrane-permeable hydrophobic peptide aldehyde which is known to strongly inhibit cellular mechanisms of ubiquitin/26S proteasome-dependent protein degradation (Lee and Goldberg, 1996). Accordingly, two samples of onion roots were pretreated for 30 min with MG132 at a concentration of 100 mM, followed by continuous co-treatment with ETO þ MG132 and EPC þ MG132 mixtures. Prior to this study, the effects of MG132 applied solely were evaluated. Despite some increase in mitotic indices (MI, statistically significant with p < 0.05 at time points 2 and 4, when compared to the untreated seedlings; Fig. 2A), no structural or functional abnormalities were observed in Feulgen-stained onion root meristems following successive incubations with only MG132. Although successive ETO treatments also did not change MI values (Fig. 2B), a vast number of chromosomal aberrations appeared, with the earliest cases detected as soon as after 2-h incubation. Later, the percentage of mitotic cells with disturbed chromosome structures increased, reaching the highest value (about 25%) at the 8 h time point. Then, the number of aberrant cells gradually decreased until their almost complete disappearance in 18 h (Fig. 2B). During co-incubation of the MG132-pretreated onion seedlings with the mixture of ETO þ MG132 (0.5-to-18 h treatment periods), mitotic activity remained at a level of 4e10% (Fig. 2C). Although evident symptoms of chromosomal aberrations were also observed as soon as after 2-h incubation (Fig. 2C), the highest number of mitotic abnormalities in ETO þ MG132-treated root tips (ca. 28% of cells) appeared already after 4 h incubation and were still frequent after 6- and 8-h treatments. Starting from the 12 h time point, the number of cells with aberrant chromatin morphology in root meristems exposed to ETO þ MG132 mixture significantly decreased (Fig. 2C). The changes in MI observed after successive incubation periods of A. cepa root meristem cells in 200 mM EPC are shown in Fig. 2D. During the first 8 h of treatment, mitotic activity was similar to the control. At four subsequent time points (10e16 h), MI slightly increased (by about 3e4%). A small number of mitotic cells with chromosomal aberrations were observed in preparations made after a short, 2-h incubation period with EPC (Fig. 2D). With time, similarly as in the case of ETO (Fig. 2B), there was a gradual increase in cells with altered chromatin morphology. The highest amount of the cells showing abnormal mitotic structures (about 23%, as in the case of ETO) was noted after 8-h EPC-treatment. Then, their number gradually declined to about 7% at 18 h of incubation (Fig. 2D). Compared with onion root meristems incubated with only EPC, the combined influence of EPC þ MG132 caused a significant decrease in the number of aberrant M-phase cells (to about 3e5%), with no significant time-related changes in the mitotic activity, except some increase during the earliest incubation periods (Fig. 2E).

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Fig. 1. Fluorescence of EPC-DNA complexes in cell nuclei after incubation of A. cepa seedlings with 200 mM EPC: 30 min (A), 2 h (B), 4 h (C), 6 h (D), 8 h (E), 10 h (F), 12 h (G), 14 h (H), 16 h (I), 18 h (J); bar ¼ 50 mm. (K) Mean intensity of fluorescence of EPC-DNA complexes in cell nuclei from the drug-treated root meristems (expressed as mean pixel value [pv] spanning the range from 0 to 255).

2.2. ETO, EPC, and their mixtures with MG132 induce abnormal mitotic phenotypes Incubations of A. cepa seedlings with either ETO, EPC or with their mixtures containing MG132 gave rise to the appearance of Mphase cells with entangled and sticky chromosomes during proand metaphase (Fig. 3). As a consequence, all experimental treatments resulted in chromosomal constrictions, breaks, and chromosome bridges during ana- and telophase, often leading to asymmetric segregation of chromosomes (Fig. 3; anaphase, telophase A and B). This, in turn, brought about an uneven distribution of genetic material into two post-telophase nuclei (with various DNA contents) and different levels of chromatin condensation (Fig. 3; post-telophase). Despite all similarities, our results also show some specific effects of ETO and EPC e the first drug producing more fragmented (acentric) or pulverized chromosomes (Fig. 3; ETO, ETO þ MG132; see anaphase and telophase A) while the other rendering them much more condensed (Fig. 3; EPC; see prophase and EPC þ MG132; see metaphase).

2.4. ETO and EPC contribute to the defects in the mitotic spindle Normal organization of the microtubular system in the control cells and its changes induced by ETO and EPC treatments were demonstrated using immunofluorescence of b-tubulin (Fig. 5). Exposure of onion root meristems to each of these drugs resulted in marked alterations in microtubule arrays and, consequently, in an uneven separation of sister chromatids into two daughter cells. During prometaphase, almost 4% and 3% of ETO- and EPC-treated cells, respectively, showed abnormalities in the organization of the mitotic spindle, as evidenced by the accumulation of large amounts of microtubular structures on one of the cell poles. Specifically after treatment with EPC, about 75% of anaphase cells revealed deformations of the mitotic spindle. By the end of the M phase, both Topo II inhibitors (already after 8 h incubation) brought about a significant shift in the position of the phragmoplast, as a result of asymmetric segregation of chromosomes (Fig. 5). 2.5. ETO and EPC have an effect on immunolocalization of phosphorylated histone H3 (Ser10)

2.3. Influence of ETO and EPC on nuclear DNA contents On the basis of cytophotometric measurements of Feulgen DNAstained nuclei, the percentages of cells with 2C, 2-4C and 4C levels were estimated in the control meristems (Fig. 4A, B; black histogram profiles) and in the root tips of seedlings incubated for 18 h with ETO and EPC (Fig. 4A, B; dark gray histograms). As compared to the control, slightly increased numbers of cells in G1and S-phase and, consequently, decreased numbers of G2-phase cells were observed in plants treated either with ETO or EPC (Fig. 4A, B). Quite similar results were obtained in the experiments on onion root meristem cells treated using ETO þ MG132 and EPC þ MG132 mixtures (data not shown).

Considering our earlier data indicating considerable changes in histone H3 phosphorylation induced under DNA stress conditions _ (Zabka et al., 2012), ETO- and EPC-treated root meristem cells of A. cepa were immunolabelled using an anti-H3S10Ph antibody (Fig. 6). As compared with the control cells (two upper rows in Fig. 6), a vast number of ETO- and EPC-treated cells show marked changes in the H3S10Ph distribution patterns. In early prophase cells, both after treatment with ETO (Fig. 6; middle rows) and EPC (Fig. 6, two bottom rows), H3S10Ph signals were localized not only in the pericentric regions of condensing chromosomes, but also in their more distant parts (underneath nuclear envelope). At later stages of M-phase (in meta- and anaphase), H3S10Ph

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2.6. Apoptotic-like programmed cell death after treatments with ETO, EPC, ETO þ MG132, and EPC þ MG132 mixtures When compared with the control (Fig. 7A), onion root meristems incubated with ETO or ETO þ MG132 mixture (Fig. 7B, C, and F) and with either EPC or EPC þ MG132 mixture (Fig. 7D, E, and G) revealed cell nuclei having altered chromatin structures and marked symptoms of degeneration, characteristic of AL-PCD. During the first 14 h of treatment with ETO, the number of interphase cells showing AL-PCD was relatively low (less than 1%; Fig. 7F). Only after the 14 h incubation with ETO, there was a slight increase in nuclei with chromatin destruction (up to about 1.5%). In contrast to the effects observed in ETO-treated onion root cells, incubation with ETO þ MG132 resulted first in a sharp increase in the number of cells with AL-PCD at 4 h time-point, which was followed by a gradual decrease in the frequency of cells with developed symptoms of degeneration (Fig. 7F). In onion seedlings exposed to EPC, the frequency of interphase cells with AL-PCD has been steadily raising over the 4e18 h timespan, reaching about 4% in the last experimental period (18 h; Fig. 7G). Up to the 10 h period of treatment with EPC þ MG132 mixture, the changing incidence of cells showing AL-PCD revealed close similarity to the effects of EPC. Since then their percentage considerably decreased (12e16 h) and raised again following 18 h incubation (Fig. 7G). 3. Discussion

Fig. 2. The effect of incubations with MG132 (A), ETO (B), ETO þ MG132 mixture (C), EPC (D), and EPC þ MG132 mixture (E) on mitotic index (% ± S.D.; gray diagrams) and percentage of aberrant M-phase cells (% ± S.D.; black diagrams). Compared to the control (C), statistically significant change in MI values (p < 0.05) is marked by an asterisk.

immunofluorescence was dispersed along the chromosomes, while in telophase, phosphorylation of H3 histones disappeared almost completely (Fig. 6; left column). No specific changes in the distribution of histone H3 phosphorylation sites were noticed in onion root meristem cells treated using ETO þ MG132 and EPC þ MG132 mixtures.

ETO, a semisynthetic derivative of podophyllotoxin is one of the most widely used anti-cancer drugs (Montecucco and Biamonti, 2007). Inhibition of Topo II with ETO leads to the formation of DNA-Topo II-ETO complexes, formation of SSBs, DSBs, and ultimately to cell death (Nakada et al., 2006; Smart et al., 2008; Quennet et al., 2011). At low concentrations, ETO produces mainly Topo II-linked SSBs, while at higher concentrations, the number of DSBs was found to increases dramatically (Bromberg et al., 2003; Muslimovi c et al., 2009). Many studies have shown that numerous inducers of cleavable complexes (including ETO) generate extensively fragmented chromosomes (Rello-Varona et al., 2006). For example, in cultured Chinese hamster lung fibroblasts (CHL cells), the stabilization of Topo II-DNA cleavable complexes caused disruption of chromosomal integrity and formation of chromosome-type aberrations (mainly breaks and exchanges, (Suzuki et al., 1995)). Earlier studies showed that ETO can also induce chromosomal aberrations in human fibroblasts (anaphase cells with entangled chromosomes; (Cimini et al., 1997)), female and male germ cells (Mailhes et al., 1996; Tateno and Kamiguchi, 2001), and in HeLaS3 cells (anaphase bridges; (Terasawa et al., 2014)). EPC (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is one of the naturally occurring alkaloids isolated from leaves of the evergreen tree Ochrosia elliptica Labill (Apocynaceae), found in Oceania. EPC and its derivatives exhibit significant activity against cancer cell lines (Fang et al., 2009; Kizek et al., 2012). In several animal tissues, expression levels of cytochromes P450 (CYP) and peroxidases that activate EPC are crucial for antitumor, cytostatic and genotoxic  et al., 2011). Although EPC does activities of this inhibitor (Stiborova not inhibit DNA religation mediated by Topo II, it induces DNA breakage enhancing the forward rate of cleavage (Kizek et al., 2012;  et al., 2006; Poljakova  et al., 2009). Hence, EPC-treated Stiborova Chinese hamster ovary (CHO) cells revealed chromosomal aberrations, including gaps and chromatid-type breaks (Takahashi-Hyodo et al., 1999). Despite no significant influence on mitotic activity, ETO and EPC (both applied at a dose of 200 mM) induce significant, yet transient,

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Fig. 3. Chromosomal aberrations during prophase, metaphase, anaphase, telophase, and post-telophase cells in Feulgen-stained root meristems following 8-h treatment with ETO, ETO þ MG132, EPC and EPC þ MG132 mixture; bar ¼ 10 mm.

chromosomal aberrations in root meristem cells of A. cepa. Following each treatment, the highest number of Feulgen-stained cells with disrupted chromosome morphology and/or organization was observed after 8-h incubation. Furthermore, ETO and EPC treatments resulted in an uneven segregation of genetic material into two daughter cells. As evidenced using immunocytochemical staining of b-tubulin, this result corresponds well with the dysfunction of the microtubule (MT) system and accumulation of large amounts of MTs at one of the cell poles. Noteworthy, a considerable reorganization of mitotic apparatus and formation of multipolar mitosis has been found by Balashova et al. (2008) in ETO-treated CHO-K1 cells. It cannot be excluded that, as in the case of HeLa cells (Rello-Varona et al., 2006), ETO- and EPC-induced chromosome breaks in A. cepa root meristem cells are correlated with the improper attachment of chromatids to kinetochore microtubules. Interactions of EPC with DNA, Topo II, and Topo II-DNA complexes were tested in different animal and human cell systems by monitoring the fluorescence intensity of the drug [e.g. (Wu et al., 2012)]. It was shown that, as a potent mutagen, EPC intercalates into DNA base pairs and inhibits Topo II activity with the

production of covalent DNA adducts. Observations in A. cepa root meristems revealed the appearance of nuclear green fluorescence as soon as after 2 h treatment and its steady increase throughout successive periods of incubation. Remarkably, Stiborova et al. (2014) demonstrated that the cytotoxicity of EPC corresponded to the levels of EPC-DNA adducts: the higher their level, the higher the cytotoxicity of EPC. In onion roots, however, the maximum number of aberrant cells preceded the time point at which the most massive EPC intercalation (stabilized later on) was observed by fluorescence microscopy. Such a time-effect, also noticed for the frequency of aberrant chromosome structures in a series of treatments with ETO, suggests some kind of adaptation response to the drug-induced cellular stress. In accord with this, only a relatively small number of ETO- and EPC-treated onion root cells (probably those with unrepaired DNA lesions) induced AL-PCD, as it was demonstrated earlier, in a considerably larger scale, after incubations with another _ anticancer drug, b-lapachone (Zabka et al., 2013). Contrasting with the effects of b-lapachone, however, an increase in the number of cell nuclei with evident signs of advanced degeneration, both after treatment with ETO and EPC, was accompanied by a gradual reduction in the number of cells with chromosomal aberrations.

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Fig. 4. Frequency distribution (%) of nuclear Feulgen-DNA contents in the onion root meristem cells treated with ETO (A) and EPC (B) (dark gray diagrams; arbitrary units [a.u.]), in comparison to the control (black histogram profiles). The tables on the right side of each histogram represent the estimated frequencies (%) of G1, S, and G2 cells in the control, ETO- and EPC-treated root meristems.

During mitosis and meiosis in plants, histone H3 is highly phosphorylated at Ser10 in the N-terminal tail of H3 histones, predominantly in pericentromeric regions of chromosomes (Schroeder-Reiter et al., 2003). Hans and Dimitrov's (2001) hypothesis, termed the 'ready production label' model, assigns a specific function to this modification as a marker for cellular competence to anaphase chromosome segregation after passing earlier phase to phase transitions and cell cycle checkpoints. Our previous study using continuous incubations of A. cepa root meristems with 0.75 mM hydroxyurea (HU) provided evidence that long-term exposure to replication stress results in an early H3S10 phosphorylation (extending from G2 phase to late telophase) in cells which had entered premature chromosome condensation (PCC, (Lyu et al., 2007)). Current experiments show that onion root meristem cells incubated with ETO and EPC reveal strong H3S10Ph immunofluorescence localized to the chromosome arms far beyond their pericentromeric regions. Thus, it seems possible that, irrespective of the cause of the loss or damage of the DNA, additional regions of H3S10 phosphorylation are induced under stress conditions at new chromosomal sites (possibly those bearing lesions). Similar experiments carried out by Liu et al. (2012) revealed that due to defective G2/M checkpoint activation, S4A/S8A mutant cells were progressing into mitosis with unresolved DNA damage and pan-nuclear H3S10 phosphorylation. Incubations of onion seedlings with ETO and EPC revealed slightly increased numbers of G1-and S-phase cells and decreased frequency of G2-phase cells, compared to the control. These results _ correspond to some extent to our previous observations (Zabka et al., 2014) showing that the prolonged treatment with other

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topoisomerase II inhibitors, ICRF-193 and doxorubicin, brings about almost complete decline in the G2-phase population and a significant accumulation of G1 cells. However, our present data clearly differ from those in animals, where ETO and EPC arrested cells in G2/M phase (Mak et al., 2005; Rello-Varona et al., 2006; Chiu et al., 2005; Litwiniec et al., 2013; Poljakov a et al., 2013). Thus, it cannot be excluded that the cell cycle checkpoints in plants are considerably less sensitive or much more refractory to DNA lesions and allow cells (after some adaptation time) to continue interphase and to enter mitotic division regardless of the persistence of DNA breaks _ or other types of topological abnormalities (Zabka et al., 2012; Cools and De Veylder, 2009). Despite various mechanisms of action, the influences of either ETO or EPC on root meristem cells in A. cepa are hard to be distinguished, since they result in similar chromosomal lesions and almost the same physiological outcome. The only important difference inferred from the comparative analysis between the effects of the two inhibitors concerns their catalytic activities in the presence of MG132 and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. It should be noted in this context that Topo II cleavable complexes induced by ETO are ubiquitinated and degraded by 26S proteasomes (Mao et al., 2001). This degradation is associated with the conversion of Topo II cleavable complexes into protein-free DSBs (true DSBs). Tammaro et al. (2013) showed two cellular mechanisms by which ETO induced DSBs: one dependent on transcription and associated with proteasomal degradation of Topo II and the other dependent on DNA replication but not on proteolysis. At low ETO concentrations, replication-dependent mechanism dominated while at its high doses both mechanisms were active. Gradual decrease in cells with chromosomal aberrations observed after longer incubations (>10 h) of onion root meristems treated with ETO might thus be explained by the elimination of Topo II cleavable complexes via the ubiquitin/26S proteasome pathway, followed by non-homologous end joining (NHEJ) repair of DSBs, as was the case in human K562 and HeLa cells (Kantidze et al., 2006). Using neutral comet assay, Zhang et al. (2006) demonstrated that ETO-induced g-H2AX signals in mouse embryonic fibroblasts (MEFs) were attenuated (40% reduction) upon proteasome inhibition with MG132. These results confirmed that Topo II-DNA covalent complexes are rapidly degraded by proteasomes, which is followed by the repair of the Topo II-concealed DSBs (Mao et al., 2001). In contrast to this, simultaneous incubation of onion seedlings with ETO þ MG123 mixture resulted in an accelerated onset of chromosomal instability. It seems possible then that MG132 inhibited degradation of not only Topo II but also of mitotic cyclins (Skoufias et al., 2007), which might be the main cause of acceleration of mitotic aberrations, as compared with the plants treated with only ETO. Such an interpretation of the results can find some confirmation in a slight increase in MI values noticed during the first incubation periods of onion seedlings with MG132 and EPC þ MG132. At the same time, however, considerable attenuation and prolongation of the onset of aberrations has been observed following joint incubations of onion roots in the mixture of EPC þ MG132. In view of that, despite similar effects induced by ETO and EPC, cells exposed to their mixtures with MG132 reveal contradictory responses. This, in turn, implies that not only constituents of the cell cycle control system, but also some other molecular factors need to be considered and more effort must be directed toward the question of mechanisms which contribute to diversity of physiological effects exerted by Topo II (or its inhibitors) and proteasome-dependent pathways. Till now, the effects of proteasome inhibitors on plant cell response to DNA damage is not fully understood.

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Fig. 5. Immunolocalization of b-tubulin in root meristem cells of A. cepa during metaphase, anaphase, and telophase from the control, ETO- and EPC-treated seedlings (8-h incubation). Immunolabeled b-tubulin and the same cells counterstained with propidium iodide (PI) are shown in every second row; bar ¼ 10 mm.

4. Materials and methods 4.1. Plant material and treatments Seeds of A. cepa var. Dawidowska (obtained from Vegetable  w) were sown on filter paper moistAgricultural Farm in Lubiczo ened with distilled water and germinated for 4 days at room temperature. Selected seedlings (1.5 ± 0.2 cm) were transferred to Petri dishes filled with distilled water/0.075% DMSO (SigmaeAldrich) mixture (control conditions), 100 mM MG132 (SigmaeAldrich), 200 mM etoposide (ETO; SigmaeAldrich), 200 mM ellipticine (EPC; SigmaeAldrich) or the mixtures of either ETO or EPC with 100 mM MG132. All these chemicals were dissolved in DMSO (final concentration < 0.1%) to prepare stock solutions. The final concentration of MG132 was based on the data from

experiments on Arabidopsis thaliana (Wang et al., 2011; Lachaud et al., 2013); the working concentrations of ETO and EPC were determined experimentally. All treatments were made at 20  C, in the dark.

4.2. Feulgen staining and cytophotometry Root tips of A. cepa were excised and fixed in Carnoy's mixture (absolute ethanol and glacial acetic acid; 3:1, v/v) for 1 h at room temperature. The material was washed 3 times with 96% ethanol and stored in 70% alcohol. Before staining, roots were rinsed three times with distilled water and hydrolyzed for 1 h in 4 M HCl. After that, roots were stained with Schiff's reagent (pararosaniline; SigmaeAldrich) for 1 h, rinsed in SO2-water (3 times; 3 min each) and distilled water. Apical fragments of roots (2-mm long) were

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Fig. 6. Immunofluorescence detection of phosphorylated histone H3S10 in root meristem cells of A. cepa during prophase, metaphase, anaphase, and telophase from the control, ETO- and EPC-treated seedlings (8-h incubation). DAPI-stained nuclear DNA of the same cells are shown in every second row; bar ¼ 10 mm.

€ser, Braunschweig, squashed onto microscope slides (Menzel-Gla Germany) in a drop of acetic acid (45%) and placed on dry ice until frozen. The slides were rinsed in 70% ethanol and embedded in Canada balsam. To evaluate mean values of mitotic indices and to quantify the occurrence of nuclei with chromosomal aberrations and with AL-PCD symptoms, 20 000 cells taken from 10 root meristems per each data point (for each experimental series) were analyzed using E100 microscope (Nikon). The cells were photographed under Optiphot-2 microscope equipped with DXM 1200 CCD camera (Nikon). Nuclear DNA content was measured using Jenamed 2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided Cytophotometer v1.2 (Forel, Lodz, Poland) for image analysis. In each experimental series about 8000 cell nuclei were analyzed. The extinction was measured at 565 nm and calibrated in arbitrary units (a.u.), with the 2C and 4C reference values recorded from half-telophase and prophase cells, respectively.

4.3. Immunocytochemical staining of microtubules (b-tubulin) Apical parts excised from the control roots and from seedlings treated with 200 mM ETO and 200 mM EPC were fixed for 40 min in MTSB-buffered (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, pH 7.0; pH 6.9) 3.7% paraformaldehyde. After washing with MTSB and maceration for 15 min (37  C) in a citric acid-buffered digestion solution (pH 5.0) containing 2.5% pectinase (Fluka, Germany), 2.5% cellulase (Onozuka R-10; Serva, Heidelberg, Germany) and 2.5% pectolyase (ICN, Costa Mesa, CA, USA), the roots were rinsed with MTSB and squashed onto slides. After drying, slides were pretreated with MTSB-buffered 8% bovine serum albumin (BSA; SigmaeAldrich) and 0.1% Triton X-100 (50 min, 20  C) and incubated with mouse monoclonal anti-b-tubulin antibody (1:750; SigmaeAldrich), dissolved in MTSB with 1% BSA. After 18-h incubation at 4  C, the slides were washed with MTSB and incubated (20  C) for 1.5 h with goat anti-rabbit Alexa Fluor 488® secondary antibody

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Fig. 7. Nuclear Feulgen-staining of A. cepa root meristem cells. Interphase nucleus from the control root (A) and the nuclei showing AL-PCD symptoms after 18-h treatment with ETO (B), ETO þ MG132 (C), EPC (D), and ETO þ MG132 (E); bar ¼ 10 mm. The frequency of cells with AL-PCD symptoms (%) from onion root meristems after incubations with: ETO, ETO þ MG132 (F), EPC, and EPC þ MG132 (G). Student's t-test p-values in the graph field: A e p < 0.001, B e p < 0.005, C e p < 0.02, D e p < 0.05.

(Cell Signaling), dissolved in MTSB (1:500). After rinsing with MTSB and counterstaining with propidium iodide (PI; 0.3 mg/ml), slides were embedded using a PBS/glycerol mixture (9:1) with 2.3% diazabicyclo[2.2.2]octane (DABCO; SigmaeAldrich). Immunofluorescence of b-tubulin-labeled cells was observed under Eclipse E-600 epifluorescence microscope (Nikon) and photographed with DS-Fi1 CCD camera (Nikon). B2 filter (blue light l ¼ 465e496 nm) was used for Alexa Fluor 488® and G2 filter (green light; l ¼ 540/25 nm) for PI. 4.4. Immunocytochemical detection of phosphorylated H3 (Ser10) histones Excised 1.5 mm long root tips from the control and ETO- and EPC-treated onion seedlings were fixed in 3.7% paraformaldehyde in PBS buffer for 40 min, at 4  C, and macerated for 15 min in a pectinase/cellulase/pectolyase mixture (37  C) and squashed, as before. Then the slides were pretreated with PBS-buffered 8% BSA and 4% Triton X-100 for 50 min. Rabbit polyclonal antibodies raised against human H3 histones phosphorylated at Ser10 (SigmaeAldrich) dissolved in PBS buffer (1:500) were added to the slides (4  C, dark wet chamber). After the overnight incubation, slides were washed three times in PBS buffer and incubated for 1.5 h with the goat anti-mouse Alexa Fluor 555® secondary antibody (1:400; Cell Signaling) dissolved in PBS buffer and counterstained with 40 ,6diamidino-2-phenylindole (DAPI, SIGMA, 0.4 mg/mL1), washed in PBS buffer and mounted in DABCO. Observations were made using Eclipse E-600 microscope equipped with G2 filter (green light;

l ¼ 540/25 nm) and U2 filter (UVB light; l ¼ 340e380 nm). All experiments performed using immunofluorescence methods were repeated 2e3 times. 4.5. Microfluorimetric analysis of EPC-DNA complexes Onion seedlings treated with 200 mM EPC for 0.5, 2, 4, 6, 8, 10, 12, 14, 16, 18 h were fixed in 3.7% paraformaldehyde in PBS buffer for 40 min, at 4  C. After enzymatic maceration, root tips were squashed onto glass slides. Following drying, slides were mounted in DABCO. Nuclear fluorescence intensity was measured by Eclipse E-600 miscroscope using blue light (l ¼ 465e496 nm). The images were recorded at exactly the same time of integration using DS-Fi1 CCD camera. Quantitative measurements of immunofluorescence of EPC-DNA complexes were made after converting color images into gray scale and expressed in arbitrary units as mean pixel values (pv) spanning the range from 0 (dark) to 255 (white). The total number of analyzed cells for one set of data was always more than 1000, if not indicated otherwise. Obtained data were expressed as the mean values ± standard deviation of the mean (S.D.), and Student's t tests for paired data were used to compare individual variables. Author contribution statement _ is the author of the conception, the design of the study, the A.Z. English version of the text, and contributed to acquisition of the results and performed most of the analyses. Both K.W. and J.T.P

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