JXB Advance Access originally published online on May 22, 2006
Journal of Experimental Botany 2006 57(8):1667-1676; doi:10.1093/jxb/erj194
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RESEARCH PAPER |
Reduced expression of top1ß gene induces programmed cell death and alters ascorbate metabolism in Daucus carota cultured cells
1Department of Plant Biology and Pathology, University of Bari, Via Orabona 4, I-70125, Bari, Italy
2Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, I-27100, Pavia, Italy
3Interdisciplinary Center for Biomedical Research (CIR) Università Campus Biomedico Via Longoni 83, I-00155 Roma, Italy
To whom correspondence should be addressed. E-mail: degara{at}botanica.uniba.it
Received 24 January 2006; Accepted 13 March 2006
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Abstract |
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Topoisomerase I (topo I) is a nuclear enzyme which plays a fundamental role in several pathways involving changes in DNA topology. The topo I-mediated reaction is accomplished by the transient covalent binding of the enzyme to DNA (topo I–DNA complex). Stabilization of the topo I–DNA complex, leading to irreversible double-strand breaks, has been reported to occur in animal cells under oxidative stress conditions and during apoptosis. In order to study the existence of a putative link between the topo I-mediated DNA damage and ascorbate (ASC) metabolism, also involved in the responses against oxidative stress and in the apoptotic process in plants, Daucus carota cells showing reduced expression of the top1ß gene encoding the topo Iß isoform were produced, using an antisense RNA strategy. Two independent transgenic lines (AT1-ß/22 and ß/36), characterized by a slow growth phenotype, resistance to camptothecin, a specific inhibitor of topo I, but sensitivity to etoposide, an inhibitor of topo II, were investigated in this study. In the absence of external stimuli, AT1-ß/22 and ß/36 cells underwent programmed cell death (PCD) in a precocious phase of the growth curve. ASC metabolism showed remarkable differences in AT1-ß/22 and ß/36 cells, compared with control, and the observed alterations were similar to those occurring in tobacco Yellow Bright-2 cells induced to enter PCD by exogenous stimuli. However, differently from other studied examples of PCD, overproduction of reactive oxygen species was not detected in AT1-ß/22 and ß/36 cells. The relevance of these findings in relation to the signalling pathways leading to PCD is discussed.
Key words: Antioxidants, ascorbate, Daucus carota, programmed cell death, topoisomerase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Eukaryotic DNA topoisomerase I (topo I, EC 5.99.1.2 [EC] ) plays an essential role in cellular processes, such as replication, transcription, and recombination, which require conformational changes in DNA topology. The eukaryotic topoisomerase I is a member of the type IB subfamily since it forms a covalent intermediate between a tyrosyl group of the protein and the 3'-end of the broken DNA strand (Wang, 1996). Stress accumulating within the topological domains of the DNA molecule can be dissipated by topo I, using the controlled breakage of a single DNA strand, passing DNA through the strand break and finally religating the cleaved DNA. Several reports indicate that, in multicellular eukaryotes, topo I is essential for correct development (Lee et al., 1993; Morham et al., 1996) whereas, in yeast mutants lacking topo I, the up-regulation of top2 gene encoding topoisomerase II is able to compensate for the lack of topo I (Thrash et al., 1984; Goto and Wang, 1985). An extensive description of the different roles related to DNA topoisomerases has recently been published (Wang, 2002).
For the first time in a eukaryotic organism, the presence of two functional top1 loci encoding two distinct forms of topo I (Topo I
and Topo Iß) has been reported in Daucus carota cells (Balestrazzi et al., 1996, 2000). This finding has been confirmed in the pufferfish Fugu rubripes (Smith et al., 2001) and in Arabidopsis thaliana (Takahashi et al., 2002). A functional analysis revealed that carrot top1ß could be considered analogous to one of the Arabidopsis top1 genes (Balestrazzi et al., 2003). Using the RNA interference approach, the Arabidopsis top1ß gene was disrupted and the resulting lines were crossed to top1 lines. The progeny was characterized by seedling lethality and the results suggested that, as previously reported for animal cells, topo I function is essential for plant survival (Takahashi et al., 2002).
DNA is highly susceptible in vivo to reactive oxygen species (ROS) (i.e. hydrogen peroxide, hydroxyl radical, superoxide anion) responsible for a wide range of DNA lesions and, among these, 8-oxoguanine represents the most abundant base damage (Park et al., 1992). It has been shown that the stability of the topo I–DNA intermediate is increased in the presence of 8-oxoguanine (Pourquier et al., 1999). Some apoptotic agents which produce oxidative DNA lesions, such as staurosporine (Sordet et al., 2004), UV-light (Soe et al., 2004), and hydrogen peroxide (Daroui et al., 2004), were shown to be efficient inducers of topo I–DNA complexes in mammalian and yeast cells. One intriguing hypothesis, formulated by the authors, was that these intermediates could play an active role in sensing and/or amplifying the DNA damage, thus activating programmed cell death (PCD).
Recent results indicate that, in plants, a critical step in the signalling pathway leading to PCD is the decrease in the activity of the cytosolic ascorbate peroxidase (APX) and in the ascorbate level (De Gara, 2004). In fact, down-regulation of the APX activity and a decrease in the ASC content and redox state have been reported in the PCD activated by both NO and H2O2 as well as by heat shock in Nicotiana tabacum Bright Yellow-2 cells (de Pinto et al., 2002; Vacca et al., 2004). Studies in planta also confirmed a central role for ASC metabolism in PCD. The Arabidopsis mutants vtc1 and vtc2, having low levels of ascorbate, exhibit micro-necrotic areas and express pathogenesis-related genes in the absence of external stimuli, all symptoms induced during the hypersensitive PCD (Pavet et al., 2005). It is also known that the PCD induced by pathogen attack requires APX down-regulation (Mittler et al., 1998, 1999). Suppression of the ASC-dependent antioxidant system is probably required to permit the oxidative burst characterizing all kinds of PCD so far studied. However, the analysis of the changes in APX activity, protein amount, and gene expression suggests that different regulatory mechanisms are activated both upstream and downstream of the oxidative burst (Vacca et al., 2004; MC de Pinto, A Paradiso and L De Gara, unpublished results). This makes more complex the role of APX in the signalling pathway leading to PCD.
ASC metabolism has also been extensively studied for its relations with oxidative stress. It is part of the ascorbate–glutathione cycle, a network of reactions involved in ROS detoxification and present in several cellular compartments of both plant and animal cells (Noctor and Foyer, 1998; Asada, 1999; Karpinsky et al., 1999; Shigeoka et al., 2002).
In order to investigate the putative relationship existing between the topo I-mediated DNA damage and other intracellular pathways involved in defence responses, carrot cell lines showing a reduced expression of the top1ß gene have been obtained. The analysis of cellular viability, growth capability, ascorbate–glutathione cycle enzyme activities, as well as parameters indicative of the occurrence of oxidative stress, were performed in control and in top1ß down-regulated cells. Results suggest that the impairment in DNA metabolism caused by the decrease in top1ß gene expression also induces PCD in a precocious stage of cell suspension growth and in the absence of external stimuli. Moreover, top1ß down-regulation affects ASC metabolism, probably as a downstream step in the PCD activation.
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Materials and methods |
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Plant material
Carrot seeds (Daucus carota L. cv. Nantaise) were sterilized for 15 min in 70% (v/v) ethanol and subsequently for 40 min in 40% (v/v) sodium hypochloride. After several washes in sterile distilled water, seeds were induced to germinate on B5 medium (Gamborg et al., 1968) supplemented with 0.8% agar at 24 °C for 7 d. Hypocotyl segments (1 cm long) were used for Agrobacterium-mediated transformation.
Construction of a binary plasmid containing the antisense top1ß cDNA and carrot transformation
PCR was used to amplify a cDNA fragment required to produce the antisense construct. The 588 bp fragment spanning the 5'-UTR and 467 bp from the coding region of top1ß cDNA was obtained with the following oligonucleotide primers: beta-1 (5'-CGAGCTCGCTGCTCGTAACGTCCAT-3') and beta-2 (5'-TCCCCCGGGGGATCCTCCAACCGGGAGTC-3'). The SacI and SmaI sites are underlined. PCR reactions were performed at standard conditions: clone pTop11, containing the full-length top1ß cDNA (Balestrazzi et al., 2000), was utilized in the reactions performed with beta-1/beta-2. Aliquots containing 5–10 ng of template were used in 30 µl of reaction mixture containing 10 mM TRIS–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, dNTPs (200 µM each), oligonucleotide primers (100 ng each), and 0.5 U Taq polymerase (Perkin Elmer, Cetus). Amplification was carried out at 94 °C for 50 s, 48 °C for 50 s, and 72 °C for 1 min for 35 cycles using a DNA Thermal Cycler 480 (Perkin Elmer, Cetus). The PCR product was analysed on a 1.2% agarose gel and recovered using the Qiaex II gel extraction kit (Qiagen). The PCR product, digested with SacI/SmaI, was subsequently cloned in the antisense orientation into the pBI121 binary vector, previously digested with SacI/SmaI (Jefferson, 1987). The antisense insert was under the control of the CaMV 35S promoter and the NOS-ter region. Sequence analysis confirmed the presence of the top1 antisense cDNA fragment in the recombinant binary vector. The construct, named AT1-ß, was introduced into Agrobacterium GV2260 strain by electroporation. The binary vector pBI121 was also transferred into the same strain as the control. Agrobacterium-mediated transformation of carrot hypocotyl fragments was performed as described by Hardegger and Sturm (1998). Hypocotyl fragments from 7-d-old seedlings were transferred into an overnight-grown Agrobacterium culture for 3–5 min and then co-cultivated on semi-solid B5 medium supplemented with 1 mg l–1 NAA and 0.5 mg l–1 BAP (Duchefa Biochemicals). After 3 d, explants were washed with liquid B5 medium and transferred on semi-solid B5 medium containing 1 mg l–1 NAA, 0.5 mg l–1 BAP, 200 mg l–1 cefotaxime, and 200 mg l–1 vancomycin. Two weeks later the hypocotyl segments were transferred on the same medium containing 1 mg l–1 NAA, 0.5 mg l–1 BAP, 200 mg l–1 cefotaxime, 200 mg l–1 vancomycin, and 10 mg l–1 geneticin (Duchefa Biochemicals). After 6 weeks, calli were transferred on fresh medium. Transformation efficiency was defined as the number of geneticin-resistant calli obtained compared with the total number of co-cultivated explants. Fifty hypocotyl explants were used for the transformation experiment. After 4–6 weeks, calli growing on explants were split and transferred onto fresh solid medium. Subsequently, carrot cell suspension cultures were obtained for each antisense top1 line and for the control line carrying the empty pBI121 vector. For this purpose, aliquots (0.2–0.5 g) of calli were transferred into 50 ml flasks containing 10–20 ml liquid B5 medium supplemented with 0.5 mg l–1 2,4-D and 10 mg l–1 G-418 disulphate (Duchefa Biochemicals).
Cell culture
Daucus carota cell lines were routinely propagated in B5 medium supplemented with 30 g l–1 (w/v) sucrose, 10 mg l–1 G-418 disulphate, and 0.5 mg l–1 2,4-D. Cells were subcultured to fresh culture medium every 10 d by transferring 5 ml to 45 ml of fresh medium and incubated at 80 rpm at 27 °C in darkness. The Package Cell Volume (PCV), corresponding to the ratio between the cell volume and the total suspension volume, was measured collecting cell suspension aliquots (10 ml) by centrifugation at 250 g for 6 min at 4 °C.
Northern blot analysis
Poly(A)+ RNA was extracted from actively proliferating carrot cell suspension cultures by affinity chromatography on oligo (dT)-cellulose (Sigma Aldrich) as previously described (Balestrazzi et al., 1996). For northern blot analysis, poly(A)+ RNAs were run on 0.6% agarose denaturing formaldehyde gels and subsequently blotted to nylon membranes (HybondTM-N+, Amersham Biosciences) according to the manufacturer's instructions. Hybridization conditions were as follows: 50% (v/v) formamide, 5x SSC, 0.5% SDS, 5x Denhardt's solution, 100 µg ml–1 salmon sperm DNA at 42 °C for 18 h. The 588 bp fragment spanning the 5'-UTR and 467 bp from the coding region of top1ß cDNA and the 519 bp fragment containing the 5'-UTR and 250 bp from the coding region of top1 cDNA (Balestrazzi et al., 1996) were used as probes to detect the top1ß and the top1 mRNAs, respectively. The 1.8 kb cDNA fragment spanning the carrot 18S rDNA region was used as constitutive control (A Balestrazzi et al., unpublished data). Probes were [32P]-dCTP labelled using the Hexa Label PlusTM DNA Labelling Kit (Fermentas). Filters were washed with 1x SSC/0.1% SDS at 65 °C for 10 min. Densitometric analysis was performed using a Biostep GmbH apparatus with the argus X1 3.3.0 software.
Cell death and oxygen consumption
Carrot cell suspension cultures (AT1 transgenic and control lines) were stained with the Evan's blue dye and cell death was determined by spectrophotometric analysis according to Carimi et al. (2003). For each line, two independent experiments were performed with each assay done in triplicate. Dyed cells were also microscopically analysed according to de Pinto et al. (1999). For each cell suspension line oxygen consumption was measured using a Gilson 5/6 oxygraph (Gilson Medical Electronics Inc., Middletown, WI). The rate of oxygen consumption was obtained as a tangent to the initial part of the progress curve and expressed as nanoatoms of O2 min–1 mg–1 of protein.
DNA laddering
Twenty-day-old cells (1 g) were collected and homogenized in liquid N2. DNA was extracted using the CTAB method according to Murray and Thompson (1980). DNA samples were digested with 100 µg ml–1 DNase-free RNase for 1 h at 37 °C, electrophoresed on a 1.5% (w/v) agarose gel containing 1x TAE (40 mM TRIS-acetate, and 1 mM EDTA pH 8.0), and stained with ethidium bromide.
Extraction and analysis of ascorbate pool
Cells were collected by filtration on Whatman 3MM paper in a Buchner funnel connected to a conical flask and a vacuum pump and weighed (fresh weight). Cells (0.2 g) were homogenized with four volumes of cold 5% (w/v) meta-phosphoric acid at 4 °C in a porcelain mortar. The homogenate was centrifuged at 20 000 g for 15 min at 4 °C, and the supernatant was collected for the analysis of ascorbate. ASC content and redox state were measured as described by de Pinto et al. (1999).
Enzyme assays
Aliquots (0.3 g) from each cell suspension line were homogenized in liquid N2 with a mortar and pestle. Four volumes of a buffer containing 50 mM TRIS–HCl (pH 7.8), 0.05% (w/v) cysteine, and 0.1% (w/v) BSA, were added just as the last trace of liquid N2 disappeared. The thawed mixture was then ground and centrifuged at 20 000 g for 15 min. The supernatant was used for spectrophotometric analysis.
DHA reductase (glutathione: dehydroascorbate oxidoreductase, EC 1.8.5.1 [EC] ), AFR reductase (NADH: ascorbate free radical oxidoreductase, EC 1.6.5.4 [EC] ) cytosolic ascorbate peroxidase (L-ascorbate: hydrogen peroxide oxidoreductase, EC 1.11.1.11 [EC] ) were assayed according to de Pinto et al. (2003). Protein measurement was performed according to Bradford (1976) using bovine serum albumin as a standard.
Measurement of H2O2
The release of H2O2 in the cell suspension medium was measured according to Bellincampi et al. (2000). In brief, 1 ml of cell cultures was harvested by centrifugation (10 000 g, 20 s, 25 °C), and H2O2 concentration was measured in the supernatant. An aliquot of supernatant (500 µl) was added to 500 µl of assay reagent (500 µM ferrous ammonium sulphate, 50 mM H2SO4, 200 µM xylenol orange, 200 mM sorbitol). After 45 min of incubation the peroxide-mediated oxidation of Fe2+ to Fe3+ was determined by measuring the A560 of the Fe3+-xylenol orange complex.
Lipid peroxidation
The level of lipid peroxidation was measured in terms of malondialdehyde content determined by the thiobarbituric acid reaction as described by Zhang and Kirkham (1996). Cells (0.4 g) were homogenized in 4 ml of 0.1% (w/v) trichloroacetic acid. The homogenate was centrifuged at 10 000 g for 10 min. One millilitre of the supernatant was diluted 1:5 (v/v) with 20% (w/v) trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The mixture was heated at 95 °C for 30 min, cooled on ice, and centrifuged at 10 000 g for 10 min. The absorbance (532 nm) of the supernatant was measured. Values corresponding to non-specific absorption at 600 nm were subtracted from the values obtained at 532 nm. The concentration of malondialdehyde was calculated using the extinction coefficient of 155 mM cm–1.
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Results |
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Production of transgenic carrot cell lines expressing the antisense top1ß construct
Carrot cell transformation has been performed using an Agrobacterium tumefaciens strain engineered with a pBI121 binary vector containing, in antisense orientation, the 588 bp fragment spanning both the 5'-UTR and a sequence of 467 bp from the coding region of top1ß cDNA. The generated construct was named AT1-ß. This sequence was selected from the 5' region of the top1ß cDNA to obtain the specific and independent down-regulation of the top1ß gene. Previous observations showed that the less conserved domain between the two carrot proteins (32% similarity) and generally among all eukaryotic topoisomerases I, is the N-terminal region, while similarity rises to 89% in the C-terminal region of the two enzymes (Balestrazzi et al., 2000). A schematic representation of the top1ß antisense construct is shown in Fig. 1. The AT1-ß construct was then transferred into Daucus carota explants by A. tumefaciens-mediated transformation, with the following efficiencies: 3.2% (AT1-ß) and 29.68% (pBI121 control line).
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Molecular analyses
In order to confirm the presence of the top1ß antisense cDNA insert in the carrot genome, 17 AT1-ß cell lines were tested by Southern blot analysis using a gene-specific probe corresponding to the same top1 antisense region (data not shown). To this aim, carrot genomic DNA was digested with a restriction enzyme with no target site inside the antisense construct to discriminate between endogenous and transgene sequences.
Results from Southern analyses showed that among the 17 carrot lines, 10 were found to be single-copy, three contained two copies, and two lines had three and four copies of the antisense insert, respectively. A high mortality rate among the selected antisense top1 lines during the in vitro culture of both cell suspensions and calli was observed and most of the transgenic lines did not survive. This result was not unexpected due to the essential function for cell viability carried out by top1 genes.
Two independent transgenic cell lines (AT1-ß/36 and AT1-ß/22) showing a slow growth phenotype and carrying one and two transgene copies, respectively, were selected for further analysis.
To verify the steady-state levels of both top1 and top1ß transcripts in the selected AT1 lines and in the control line, carrying only the empty vector pBI121 (Control), northern blot hybridization was performed (Fig. 2A, C). To identify both top1 and top1ß mRNAs, gene-specific probes were used. Fluctuations in the steady-state levels of top1 and top1ß mRNAs, due to differential RNA loading, were normalized using a 18S rDNA probe previously isolated in this laboratory (A Balestrazzi et al., unpublished data). The normalized values of top1 and top1ß mRNAs are reported in Fig. 2B and D.
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Northern analysis showed that in both AT1-ß/22 and ß/36 lines top1ß expression was down-regulated. The antisense-mediated reduction of top1ß mRNA was about 46% in AT1-ß/22 and about 65% in AT1-ß/36, respectively. This depletion was associated with a less strong but evident reduction of top1
transcript level (about 20%) in AT1-ß/22 cells, whereas in AT1-ß/36 cells an enhanced top1 mRNA accumulation (at least 50%) was observed. This up-regulation of top1 transcript was confirmed by results from relative quantitative RT-PCR using 18S rRNA as internal control (data not shown).
Sensitivity to topoisomerase inhibitors
The anticancer drug camptothecin (CPT) is known as a specific inhibitor of topo I and an inducer of mammalian apoptosis (Kaufmann, 1998). When the carrot AT1-ß/22 and ß/36 cell lines were evaluated for vitality by the Evan's Blue spectrophotometric assay in the presence of CPT (0.25 µM), a pronounced decrease in the percent of dead cells was observed in both antisense lines, compared with the control (Fig. 3). By contrast, these cells showed sensitivity to etoposide, a topoisomerase II specific drug (Andoh and Ishida, 1998), similar to the control cell line (Fig. 3), thus suggesting that no significant differences in topo II levels occurred in both AT1-ß/22 and ß/36 cells.
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Growth curve and cell viability
The growth patterns of control, AT1-ß/22 and ß/36 lines are shown in Fig. 4.
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The package cell volume (PCV), measured every 4 d during the progression of the control cell suspension culture, revealed an active growth pattern since a 5-fold increase in PCV values was recorded at 4 d. A further increase in the PCV values was observed at 8 d while the suspension culture entered the stationary phase at 12–14 d and no significant fluctuations were present until the end of the experiment (20 d). On the contrary, both AT1-ß/22 and ß/36 lines were characterized by a slow growth phenotype. As shown in Fig. 4, only a 2-fold increase in PCV values was evident at 4 d and moderate growth, particularly for AT1-ß/36 cells, was subsequently observed. In the case of AT1 lines, the overall PCV values were significantly reduced, compared with control cells, thus indicating a general decrease in cellular proliferation.
In order to characterize the general metabolic state of the carrot cell suspension lines, their capability to consume oxygen was determined. The oxygen consumption, evaluated in 8-d-old cultures, was similar in all the cell lines tested and corresponded to 60±6, 55±7, and 40±9 nanoatoms O2 consumed min–1 mg–1 protein in control, AT1-ß/36 and AT1-ß/22 cells, respectively.
Cell death was analysed by staining with Evan's Blue, a dye able to enter dying cells. In AT1 cell suspension cultures, the presence of non-viable cells was clearly shown after 4 d of growth and cell death increased with time; in the control line, the number of dead cells visualized by Evan's Blue was lower during the entire growth period, reaching values of 19±1, 32±2, and 28±3% in the control, AT1-ß/36, and AT1-ß/22 cells, respectively, in the stationary phase. However, in the case of both AT1 lines, dying cells showed cytoplasmic shrinkage, a hallmark of programmed cell death (PCD) in plants (de Pinto et al., 2002; Vacca et al., 2004), whereas this feature was absent in the stained control cells (Fig. 5). The occurrence of PCD in AT1-ß/22 and ß/36 lines was further substantiated by the analysis of DNA laddering, performed in cells collected during the stationary phase (Fig. 6).
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Ascorbate metabolism
It has recently been reported that an impairment in ascorbate (ASC) metabolism occurs as an early event in the PCD signalling pathway in tobacco Bright Yellow-2 cells (de Pinto et al., 2002; Vacca et al., 2004). In order to verify whether the cellular processes involving ACS might be affected as a consequence of top1ß down-regulation, the levels and redox state of ASC as well as the activity of the ascorbate–glutathione cycle enzymes were analysed in the carrot AT1-ß/22 and AT1-ß/36 cells.
In the control cells, ASC content was in the range of 3000–4000 nmol g–1 FW. It showed a transient increase during the growth curve, reaching its maximum level in the exponential phase (at about 10 d of growth) (Fig. 7). This behaviour was in agreement with that reported for ASC in other plant cell-suspension cultures (de Pinto et al., 1999). The ASC redox state (i.e. the ratio between the reduced form and the reduced plus oxidized forms) was maintained at a value of 0.8–0.9 in the period ranging from 4–14 d of growth, whereas it decreased at value of about 0.5 by 19 d. In the antisense AT1 cells the ASC level (around 1000 nmol g–1 FW), was remarkably lower than in control cells. Moreover, the antisense cell lines did not show the transient increase in ASC content previously described for the control cells and almost similar ASC values were maintained during the whole growth period (Fig. 7). The ASC redox state did not differ significantly between control and AT1 cells during the first 14 d of growth. However, when cells entered the stationary phase, a more remarkable decrease was observed in the top1ß-depleted carrot cells, reaching values of 0.14 and 0.26 for AT1-ß/22 and ß/36 cells, respectively, at 19 d of culture.
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In order to verify whether the decrease in the ASC content of the AT1 cells was due to an impairment of the ASC biosynthetic pathway, the activity of the enzyme catalysing the L-galactono-
-lactone (GL) oxidation to ASC was analysed in one of the antisense lines (AT1-ß/22). To this end, different concentrations of GL were added to the culture medium of 10-d-old carrot cells, and changes in ASC content were measured after 1 h of incubation with GL. Both control and AT1 cells were able to convert GL to ASC, but different efficiencies were recorded. In fact, in AT1-ß/22 cells the increase in ASC content, expressed in percentage terms, was much lower than in control cells, in particular, when lower GL concentrations were used (Fig. 8). After 2 h of incubation with GL, AT1-ß/22 cells showed a reduced ASC biosynthetic capability, whereas, this difference became irrelevant after 24 h (data not shown).
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The activity of the cytosolic ascorbate peroxidase (APX) was markedly altered in the top1ß-depleted cells (Fig. 9A). In control cells, APX activity transiently increased during the growth period, reaching its maximum value during the exponential phase, whereas in AT1 cells the activity of the enzyme was much lower and remained almost constant for the whole of the tested period. On the other hand, the enzymes involved in ASC recycling, responsible for the reduction of the ASC oxidized forms, showed higher activities in AT1 cells compared with the control line (Fig. 9B, C). Concerning the glutathione reductase (GR) activity, no significant differences were evident in control and antisense cells (data not shown).
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The release of H2O2 in the culture medium was also measured. As shown in Table 1, both in control and antisense cells the H2O2 content increased during the growth period, however, AT1-ß/22 and ß/36 cells had lower levels of H2O2 than control cells. The levels of lipid peroxidation was consistently slightly lower in the antisense cells than in control ones (22.6±7.3, 2.4±1.0, 3.75±1.25 nmol g–1 FW for control, AT1-ß/22 and ß/36 cells at 14 d of culture, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study reports on the isolation and characterization of two independent transgenic cell lines of Daucus carota (AT1-ß/22 and ß/36) showing antisense-mediated depletion of the top1ß gene expression. These cell lines, characterized by a strong down-regulation of the top1ß gene expression, offer an important tool to analyse the consequences of topoisomerase Iß depletion toward a wide variety of fundamental processes in plants.
Due to the central role of topo I in DNA metabolism, deficiency in top1 gene expression might negatively influence cell culture growth and vitality. In Drosophila and mammals, topo I activity seems to be essential for the correct development, in particular for embryo development during specific stages characterized by cell proliferation (Lee et al., 1993; Morham et al., 1996). In yeasts, topo I deletion mutants showed reduced growth rates. However, the effects resulting from low levels of top1 gene expression are much less marked in yeasts than in animals, due to the up-regulation of the top2 gene expression coding for DNA topoisomerase II (Goto and Wang, 1985). Both AT1-ß/22 and ß/36 cell lines were characterized by a slow growth phenotype and by precocious cell death. In the AT1-ß/22 cell line, the reduction in top1ß gene expression was not compensated by an increased accumulation of top1 mRNA. By contrast, northern analysis indicated that top1 was also down-regulated in AT1-ß/22 cells. This might suggest that, even with the antisense top1ß insert, cross-hybridization between the AT1-ß antisense RNA and the endogenous top1 transcript had occurred.
A different response was observed in AT1-ß/36 cells, where the reduction of the ß transcript level was more pronounced than in AT1-ß/22 line. As shown by molecular analyses, in AT1-ß/36 cells an up-regulation of top1 mRNA level seems to be present. This situation resembles the top2-mediated compensation mechanism observed in top1-deficient yeast strains. Similarly to yeast, AT1-ß/36 cells maintained a slow growth phenotype.
Pharmacological studies demonstrate that cell sensitivity to specific topoisomerase drugs correlates with the enzyme level (Kaufmann, 1998). No significant differences in CPT sensitivity between the two AT1 lines were evident. Since no information concerning the individual sensitivity to this drug of carrot topo I and ß isoforms are currently available, it is not possible to assess the contribution of each enzyme to this cellular response.
The analysis carried out on AT1-ß/22 and ß/36 cell suspension cultures exposed to etoposide, a specific topo II inhibitor, suggests that the level of this enzyme was not affected. Consequently, no functional compensation involving topo II seems to occur in response to top1ß gene down-regulation in both antisense lines. These data are consistent with the observations reported in animal cells (Morham et al., 1996).
It has recently been reported using microarray analysis that the expression of several genes involved in cellular proliferation is down-regulated in mammalian topo I-deficient cells. As suggested by the authors, the reduced growth rate observed in mammalian cell lines could represent an attempt of the cell to maintain DNA and RNA integrity in a condition of low levels of topo I (Soret et al., 2003). A similar mechanism for preserving genome integrity would be also activated in carrot AT1-ß/22 and ß/36 cells. The reduced growth rates observed for other carrot AT1-ß lines, recovered during this study, further support the correlation between top1ß gene expression levels and cell viability/proliferation (A Balestrazzi, unpublished results).
In addition, a high mortality rate was recorded among the selected antisense top1 carrot lines during in vitro experiments and, although possible reasons that caused cell mortality and consequently loss of the AT1 antisense lines can only be discussed, it is tempting to speculate that a critical threshold of topoisomerase I activity needs to be present in the cell. To support this hypothesis, further investigations on AT1-ß/36 line showing a strong reduction of top1ß transcript level accompanied by up-regulation of the mRNA, will be carried out in future.
Both AT1 lines underwent PCD in the absence of exogenous stimuli starting from the 4th day of culture. Several reports, in which inhibition of topo I activity was pharmacologically obtained by camptothecin treatment (Kaufmann, 1998; Hoeberichts et al., 2001), highlight the correlation between the reduction in topo I activity and apoptosis both in animal and plant cells. These results indicate that the reduction in topo I levels induced in carrot cells by antisense technology also triggers PCD. Interestingly, cells undergoing PCD also showed a remarkable decrease in APX activity and ASC content, events which also characterize PCD in tobacco-cultured cells (de Pinto et al., 2002; Vacca et al., 2004). As far as the ASC content is concerned, an impairment in ASC biosynthesis also seems to occur in AT1-ß/22 and ß/36 cells. These data seem to suggest that the biosynthetic capacity was much lower in the antisense carrot cells than in the control line, even if the experimental approach does not allow it to be excluded that differences in GL uptake between control and AT1 cell lines might also affect their capability to convert GL into ASC. The reduction in APX activity and ASC content cannot be ascribed to the random insertion of the antisense construct into a specific region of the carrot genome carrying ASC-related genes with the consequent disruption of these essential functions, since the two independent transgenic lines are characterized by similar alterations in both ASC content and APX activity. The increase in the ASC recycling enzymes AFRR and DHAR observed in AT1 cells might represent a homeostatic response aimed at overcoming, at least in part, the ASC shortage. A decrease in cellular antioxidant properties has been shown to be a component of the complex strategies carried out by cells undergoing PCD, in order to generate the oxidative burst occurring during this process (de Pinto et al., 2002; Vacca et al., 2004; Henmi et al., 2005; Pavet et al., 2005). Surprisingly, the AT1 cells did not show an increase in ROS production or lipid peroxidation. Even if further studies are required to verify this hypothesis, it can be speculated that the impairment of ASC metabolism occurring in AT1 cells might be activated downstream of the DNA damage caused by the low expression level of top1ß gene, as part of the normal signalling pathways leading to PCD. This idea is supported by the fact that at least APX undergoes different regulatory mechanisms during PCD (Mittler et al., 1998; Vacca et al., 2004). Some events could be controlled by ROS production, while some others could occur upstream of the activation of the ROS-generating system, and could contribute to the oxidative burst required for PCD activation. Apparently, topo I is able to trigger PCD, entering a step downstream of the ROS overproduction in the signalling pathway leading to cell death.
In conclusion, these results suggest that different and tightly correlated pathways responsible for successful PCD can be independently switched on in carrot cells. Interestingly, the reported data seem to indicate the presence of an apoptotic pathway activated in the absence of ROS overproduction. ROS-independent mechanisms of apoptosis activation have been reported in animal cells (Hug et al., 1994), even if they do not seem to occur frequently. Due to the novelty of this finding, a more extensive investigation will be carried out in future to dissect the complex molecular and cellular mechanisms underlying ROS-independent PCD induction.
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Acknowledgements |
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This work was supported by a grant of the ‘Ministero dell'Università e della Ricerca Scientifica’ (PRIN 2004, n. 2004052535), Italy.
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Footnotes |
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* These authors contributed equally to the present work.
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Abbreviations |
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topo, topoisomerase; top1, topoisomerase I gene; ROS, reactive oxygen species; PCD, programmed cell death; APX, ascorbate peroxidase; ASC, ascorbate; PCV, package cell volume; AFRR, ascorbic free radical reductase; DHAR, dehydroascorbate reductase; AT1-ß, antisense top1ß cDNA insert; CPT, camptothecin; GL, L-galactono-
-lactone; GR, glutathione reductase. |
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