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Carcinogenesis Advance Access originally published online on April 13, 2007
Carcinogenesis 2007 28(10):2166-2171; doi:10.1093/carcin/bgm093
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide

Andrew G. Cox1,2 and Mark B. Hampton1,*

1 Free Radical Research Group, Department of Pathology, Christchurch School of Medicine & Health Sciences, University of Otago, PO Box 4345, Christchurch, New Zealand
2 School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

* To whom correspondence should be addressed. Tel: +64 3 364 1524; Fax: +64 3 364 1083; Email: mark.hampton{at}chmeds.ac.nz


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 µM), but it did block cell death at apoptotic doses (<200 µM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells.

Abbreviations: AMC, 7-amino-4-methylcoumarin; AO, acridine orange; CBMN, cytokinesis-block micronuclei; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2O2, hydrogen peroxide; 5-IAF, 5-iodoacetamidofluorescein; PBS, phosphate-buffered saline


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Bcl-2 is an anti-apoptotic protein that was first identified as a proto-oncogene present at the breakpoint of a chromosomal rearrangement in B cell follicular lymphoma (1,2). Since the initial discovery, Bcl-2 has been found to be over-expressed in a variety of haematological malignancies and solid tumours (3). Previous studies suggest that Bcl-2 possesses antioxidant properties that protect cells against hydrogen peroxide (H2O2)-mediated cytotoxicity (4–7). However, it is not clear whether Bcl-2 protects the cells from H2O2 by enhancing the decomposition of H2O2 and the repair of oxidized cellular constituents or by preventing the cells from undergoing apoptosis.

Several studies suggest that Bcl-2 promotes a thiol-rich intracellular environment resistant to the cytotoxic actions of reactive oxidants such as H2O2 (6–9) and that the over-expression of Bcl-2 protects against oxidative damage (9). Hockenbery et al. (4) showed in their seminal study that Bcl-2 over-expression could block H2O2-mediated lipid peroxidation. Since then, other groups have demonstrated that the over-expression of Bcl-2 prevents oxidant-mediated lipid peroxidation (10–12). Bcl-2 over-expression can also lower protein carbonyl formation in response to oxidative stress (7), and Bcl-2-deficient mice show increased levels of protein carbonyls, indirectly supporting this proposal (13,14). However, several studies have produced findings contrary to the universal principle that Bcl-2 prevents oxidative damage in cells, with Bcl-2 over-expression exacerbating oxidative damage to both lipids and DNA in response to oxidative insult (15,16). Importantly, the process of apoptosis results in intracellular oxidation (17–19), and by blocking apoptosis and subsequent downstream oxidation, Bcl-2 could be mistaken as having antioxidant properties. It is important, therefore, to distinguish oxidation due to the initial stress from oxidation occurring as a consequence of apoptosis.

In this study, we investigated oxidative damage and the cytotoxicity of H2O2 in Jurkat T lymphoma cells over-expressing Bcl-2. We examined several markers of oxidative stress including oxidation of peroxiredoxins and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lipid peroxidation with the redox-sensitive lipid probe C-11 BODIPY 581/591 and protein carbonyl formation with a dinitrophenylhydrazine-based Enzyme-linked immunosorbent assay (ELISA). In addition, we performed the cytokinesis-block micronuclei (CBMN) assay to detect chromosome breakages in Jurkat T lymphoma cells exposed to genotoxic stress (20,21). Our results indicated that while Bcl-2 protected cells from H2O2 cytotoxicity by inhibiting apoptosis, over-expression did not protect cellular constituents from the initial oxidative stress itself. This phenotype appeared to promote genomic instability, and we propose it contributes to the oncogenic properties of Bcl-2.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Cell culture materials including RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin and geneticin were from Gibco BRL (Auckland, New Zealand). Hybond-polyvinylidene difluoride membrane and enhanced chemiluminescenceTM western blotting system were from Amersham Biosciences (Buckinghamshire, UK). C-11 BODIPY 581/591, 5-iodoacetamidofluorescein (5-IAF) was from Molecular Probes (San Diego, CA). The artificial caspase substrate Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (AMC) was from the Peptide Institute (Osaka, Japan). Mouse anti-Bcl-2 was from Zymed Laboratories (San Francisco, CA). The Carbonyl ELISA kit was from BioCell (Auckland, New Zealand). All other chemicals and reagents were from Sigma Chemical Co. (St Louis, MO) and BDH Laboratory Supplies (Poole, UK).

Cell culture
Human Jurkat T lymphoma cell line was acquired from the American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Jurkat transfectants over-expressing Bcl-2 (pCI-neo-cDNA-bcl-2) and neo controls were generated as described previously (22) and grown in RPMI 1640 supplemented with 10% FBS and 315 µg/ml geneticin. Cells were maintained in a humidified incubator at 37°C and 5% CO2/air. For all experiments, exponentially growing cells were pelleted and re-suspended in complete medium at a concentration of 1 x 106 cells/ml. Cells were exposed to H2O2 in complete medium. When required, cells were incubated with zVAD.fmk (5 µM) or desferrioxamine (1 mM) for an hour before exposure to H2O2.

Cell viability
Cell viability was measured with the DNA-intercalating vital dye propidium iodide. Treated cells (5 x 105) were harvested by centrifugation and re-suspended in 1 ml of phosphate-buffered saline (PBS) containing 5 µg propidium iodide. The cell suspension was incubated in the dark for 10 min and then 10 000 cells were analysed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) to determine the percentage of propidium iodide-positive cells.

Caspase assay
Caspase activity within treated cells was determined fluorometrically by following the cleavage of Asp-Glu-Val-Asp-AMC. Treated cells (5 x 105) were collected by centrifugation and frozen at –80°C. Frozen pellets were re-suspended in 10 µl PBS and transferred to a 96-well plate. Ninety-five microlitres of caspase buffer (5 mM dithiothreitol, 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10% sucrose, 0.1% NP-40 and 0.1% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at pH 7.25) containing 50 µM Asp-Glu-Val-Asp-AMC was added to the sample and the rate of AMC production (excitation, 370 nm; emission, 445 nm) was followed at 37°C with a POLARstar Galaxy fluorescent plate reader (BMG Labtechnologies Pty Ltd., Mt Eliza, Australia).

Immunoblot detection of proteins
Protein extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane by western blotting. Polyvinylidene difluoride membranes were subsequently blocked and incubated with the appropriate primary antibody overnight at 4°C. Immunoreactivity was visualized by probing with an appropriate horseradish peroxidase-conjugated secondary antibody and detected using enhanced chemiluminescence (Amersham Biosciences).

IAF-labelling of oxidized thiols
Reversibly oxidized thiol-containing proteins were fluorescently labelled using a technique described by Baty et al. (23). Cell samples (5 x 106 cells) were harvested, washed in PBS and then re-suspended in 75 µl N-ethylmaleimide (NEM) buffer (10 mM NEM, 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycoltetraacetic acid (EGTA), CompleteTM protease inhibitors and pH 7.4) and incubated for 15 min. The cells were solubilized with 1% CHAPS for 15 min on ice and centrifuged at 16 000g for 4 min in order to pellet cell debris. The clarified extracts were desalted using Bio-Spin® 6 chromatography columns equilibrated with extract buffer. The resulting lysates were treated with 1 mM dithiothreitol for 10 min, followed by 200 µM 5-IAF for a further 15 min in the dark. 5-IAF-labelled samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then visualized using a Bio-Rad FX fluorescence scanner (excitation {lambda} = 488 nm and emission {lambda} = 530 nm, Bio-Rad Laboratories, Hercules, CA).

Lipid peroxidation assay
Lipid peroxidation was detected by utilizing the oxidation-sensitive lipophilic probe C-11 BODIPY 581/591, as described previously (24). Cells were incubated for 30 min in media containing 5 µM BODIPY C-11. After incubation, cells were washed in PBS and re-suspended in fresh RPMI 1640 supplemented with 10% FBS. Treated cells were analysed by a FACSCalibur flow cytometer (Becton Dickinson) to monitor the change in intensity of the BODIPY-green fluorescent signal.

Protein carbonyl ELISA
Protein carbonyls were detected using an ELISA kit as described previously (25). Briefly, the method involved derivatizing protein carbonyls from treated cell lysates with dinitrophenylhydrazine. Derivatized protein was then absorbed to a hi-bond microplate and detected by ELISA. The carbonyl content of each sample was determined by comparing to a standard curve.

Lysosomal destabilization assay
Lysosomal rupture was monitored using the acridine orange (AO) relocalization assay as described previously (26). Cells (1 x 106 cells/ml) were stained with AO (5 µg/ml) for 15 min before being pelleted and re-suspended in fresh media. AO-stained cells were exposed to H2O2 for various times before being analysed for increases in green fluorescence (lysosomal rupture) by a FACSCalibur flow cytometer (Becton Dickinson). All steps were carried out in the dark.

Cell growth
Cells were grown for a week and the number of viable cells was determined on a daily basis by staining cells with trypan blue under a hemocytometer. Cell culture medium was replaced when necessary by pelleting cells and re-suspending in fresh RPMI 1640 supplemented with 10% FBS to ensure optimal growth conditions.

CBMN assay
The CBMN assay was performed as described by Fenech et al. (21,27). Cells were treated with doses of H2O2 for 4 h before being cultured for a further 24 h in media containing 4.5 µg/ml cytochalasin B. The next day, cells were harvested by centrifugation and re-suspended in PBS. Fifty thousand cells were transferred to a glass slide by cytocentrifugation at 500 r.p.m. for 5 min. The slides were air-dried, fixed in absolute methanol for 10 min and stained using Diff-Quick. At least 1000 binucleate cells were counted on each slide. Micronuclei were scored using the criteria described previously (27).

Statistics
Values are shown as the mean ± SE of at least three individual experiments. All statistical analyses were performed with the software package SigmaStat (Systat, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Bcl-2 blocks apoptosis but not H2O2-mediated necrosis
Parental Jurkat cells and the B9 line stably transfected with a Bcl-2 expression vector were assessed for their response to treatment with H2O2. B9 cells displayed a marked increase (~60-fold) in the level of Bcl-2 protein expression when compared with the parental Jurkat cell line (Figure 1A). The Jurkat cells were vulnerable to H2O2, with 50% cell death (LD50) at a dose of 50 µM (Figure 1B). The B9 cells showed increased resistance to H2O2 with an LD50 of ~200 µM. The rate of H2O2 clearance was very similar (results not shown), with both the Jurkat and B9 cells consuming >80% of a 50 µM bolus within 30 min.


Figure 1
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  		Fig. 1. Bcl-2 prevents H2O2-induced apoptosis in Jurkat T lymphocytes. (A) Bcl-2 expression levels in Jurkat and B9 cells. Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted for Bcl-2. As a loading control, all samples were further blotted for GAPDH. (B) Cell death in Jurkat and B9 cells challenged with a range of H2O2 doses and incubated for 24 h. Cells were subsequently stained with propidium iodide and subjected to FACS analysis to differentiate the proportion of propidium iodide-positive cells in the population. (C) Caspase activity in Jurkat and B9 cells were exposed to H2O2 for 6 h. Cell samples were subsequently removed and assayed for caspase-3 activation by monitoring the cleavage of Asp-Glu-Val-Asp-AMC. Values represent the mean ± SE of three independent experiments. *indicates a significant difference (P < 0.05) between Jurkat and B9 cells at the same dose as determined by a two-way repeated measures analysis of variance with Bonferroni multiple comparison (SyStat).

 
A morphological assessment of treated Jurkat cells revealed that at low concentrations of H2O2 cells appeared to shrink and show signs of membrane blebbing, consistent with the induction of apoptosis. However, at higher concentrations of H2O2 the Jurkat cells became swollen and rounded, consistent with necrotic cell death. In comparison, the B9 clones treated with H2O2 displayed no morphological signs of apoptosis, but did appear necrotic at higher doses of H2O2. To quantify this effect, caspase activity was assessed in H2O2-treated Jurkat and B9 cells. The Jurkat cells exhibited a sharp increase in caspase activity at the lower doses of H2O2, peaking with a 12-fold increase in activity at 50 µM H2O2 (Figure 1C). Higher doses of H2O2 (>400 µM) did not activate caspases, indeed, caspase activity dropped below that seen in untreated cells. There was no caspase activation in the B9 clones at any dose, and they also showed decreased caspase activation at higher concentrations. Caspase assays were performed at later time-points to check if Bcl-2 was delaying apoptosis at low doses of H2O2, however, no activity was detected (data not shown).

Protein and lipid oxidation still occurs in cells over-expressing Bcl-2
Oxidation of the thiol proteins GAPDH and peroxiredoxin 2 are sensitive markers of exposure to H2O2 (28). Thiol oxidation in GAPDH is visualized by first blocking free cysteine residues, and then reducing any oxidized cysteines and labelling them with 5-IAF. The IAF-labelled band corresponding to GAPDH is easy to identify by sodium dodecyl sulfate–polyacrylamide gel electrophoresis due to its high abundance and sensitivity to oxidation. We compared GAPDH oxidation in Jurkat and B9 cells 10 min after treatment with H2O2 and observed that oxidation was virtually identical between the two cell types at each H2O2 concentration (Figure 2A). Furthermore, Jurkat and B9 cells treated with a bolus of 100 µM H2O2 experienced a similar rate of GAPDH oxidation over time (Figure 2B).


Figure 2
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  		Fig. 2. GAPDH and peroxiredoxin 2 oxidation in Jurkat and B9 cells exposed to H2O2. (A) GAPDH thiol oxidation was monitored, by 5-IAF labelling, in Jurkat and B9 cells exposed to various concentrations of H2O2 for 10 min. (B) Jurkat and B9 cells were treated with 100 µM H2O2 for various times before samples were harvested and labelled with 5-IAF. (C) Peroxiredoxin 2 over-oxidation as monitored by non-reducing western blotting, in Jurkat and B9 cells exposed to various concentrations of H2O2 for 10 min. (D) Jurkat and B9 cells were treated with 100 µM H2O2 for various times before samples were harvested and peroxiredoxin 2 assessed as in (C). Images are illustrative of at least three independent experiments.

 
In contrast to GAPDH, peroxiredoxin 2 is over-oxidized in Jurkat cells treated with H2O2 (28). On a non-reducing gel, peroxiredoxin 2 runs as a dimer because it is immediately oxidized following cell lysis (29). Dimer formation is prevented if peroxiredoxin 2 is oxidized to the sulphinic acid (over-oxidized). The majority of peroxiredoxin 2 was over-oxidized at 10 µM H2O2 and within 5 min in both the Jurkat and B9 cells (Figure 2C and D).

To assess damage to lipids, cells were loaded with the oxidation-sensitive lipophilic probe C-11 BODIPY 581/591, and then treated with H2O2. One hour after challenge with 100 µM H2O2, there was an indistinguishable increase in lipid peroxidation between both cell lines (Figure 3).


Figure 3
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  		Fig. 3. Lipid peroxidation in Jurkat and B9 cells treated with H2O2. (A) Jurkat and B9 cells were loaded with C11-BODIPY581/591 and exposed to 100 µM H2O2 for 1 h before being harvested and subjected to flow cytometry analysis for BODIPY-green fluorescence. (B) Values represent the mean ± SE of at least four independent experiments.

 
Protein carbonyl formation was also assessed in Jurkat and B9 cells exposed to 100 µM H2O2. The initial rate of protein carbonyl formation following H2O2 exposure was similar in both cell lines (Figure 4A). However, carbonyl formation continued to increase in the Jurkat cells whereas remaining at a constant level in the B9 cells (Figure 4A). There are two potential explanations for this phenomenon. The first is that downstream apoptotic events lead to carbonyl formation, however, significant apoptosis is not evident for 6 h in this model (30), and the general caspase inhibitor zVAD.fmk did not block carbonyl formation (Figure 4B). The iron chelator desferrioxamine was able to inhibit peroxide-mediated carbonyl production (Figure 4B), consistent with the work of Brunk et al. implicating lysosomal disruption (26,31). We used the lysomotropic dye AO to monitor lysosomal destabilization in cells exposed to 100 µM H2O2. The AO-relocalization assay measures increases in cytosolic green fluorescence as the dye is released from burst lysosomes. Jurkat cells exposed to H2O2 displayed an early increase in lysosomal rupture that peaked an hour after exposure, whereas Bcl-2 over-expression provided some protection from lysosomal rupture (Figure 4C).


Figure 4
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  		Fig. 4. Protein carbonyl formation and lysosomal destabilization in Jurkat and B9 cells treated with H2O2. (A) Jurkat and B9 cells treated with 100 µM H2O2 were harvested periodically over time and kept at –80°C. Protein carbonyls were detected by ELISA using a streptavidin-conjugated horseradish peroxidase detection system. (B) Cells were incubated with zVAD (5 µM) or desferrioxamine (DFO) (1 mM) for an hour, before being exposed to 100 µM H2O2 for 3 h. (C) Jurkat and B9 cells were pre-stained with AO (5 µg/ml) and then exposed to 100 µM H2O2 for various times before being harvested and subjected to flow cytometry analysis for green fluorescence. Values represent the mean ± SE of at least three independent experiments. *indicates a significant difference (P < 0.05) between Jurkat and B9 cells at the same time (A and C), or the effect of inhibitors on cells treated with H2O2 (B), as determined by a two-way repeated measures analysis of variance with Bonferroni multiple comparison (SyStat).

 
Oxidatively damaged cells over-expressing Bcl-2 continued to proliferate and show signs of genomic instability
The adverse effects of oxidative stress have been shown to affect the proliferative capacity of cells (32). When Jurkat cells were treated with 100 µM H2O2, there was progressive cell death with 10% survival after 3 days (Figure 5). After 8 days, Surviving Jurkat cells showed signs of proliferation. In contrast, when the B9 cells were treated with H2O2, they went into a temporary growth arrest for a 2- to 3-day period without significant cell loss, consistent with the viability studies. The growth-arrested cells remained metabolically active, requiring regular media change. At 3 to 4 days, the cells began to proliferate again.


Figure 5
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  		Fig. 5. Monitoring growth of Jurkat and B9 cells treated with H2O2 over time. The growth of treated Jurkat and B9 cultures treated with 100 µM H2O2 was monitored daily by counting cells with a hemocytometer using the trypan blue exclusion assay. The culture medium was replaced daily to avoid overgrowth. Values represent the mean ± SE of at least four independent experiments.

 
To assess genomic instability, Jurkat and B9 cells were exposed to H2O2 for 4 h before being treated with the microfilament assembly inhibitor cytochalasin B and cultured for a further 20 h. The resultant binucleate cells were examined microscopically to detect chromosomal breakages (micronuclei formation) (Figure 6A). The CBMN data revealed that Jurkat cells exhibited a very low basal level of micronucleated cells in control conditions; however, the number substantially increased after treatment with 50 µM H2O2 (Figure 6B). Jurkat cells exposed to 100 µM H2O2 became apoptotic or necrotic, making it impossible to count micronucleated cells. B9 cells showed significantly more micronucleated cells in both basal conditions and following treatment with H2O2. B9 cells exposed to 100 µM H2O2 expressed an extremely high level of micronucleated cells (35 per 1000).


Figure 6
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  		Fig. 6. Micronuclei formation in Jurkat and B9 cells treated with H2O2. (A) Illustrative photomicrograph of a binucleate cell containing two micronuclei. (B) Micronuclei formation in Jurkat and B9 cells treated with 50 and 100 µM H2O2 after 1 day. Treated cells were harvested and transferred to a glass slide by cytocentrifugation. Values represent the mean ± SE of at least three independent experiments. *indicates a significant difference (P < 0.05) between Jurkat and B9 cells at the same dose as determined by a two-way repeated measures analysis of variance with Bonferroni multiple comparison (SyStat).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Previous studies have reported that Bcl-2 can protect cells from damage following an oxidative insult (9). In many of these investigations, the authors proposed that Bcl-2 possessed antioxidant properties that prevent oxidative damage. We now show that Bcl-2 does not provide protection against the initial oxidative damage mediated by H2O2, but by acting as an anti-apoptotic protein, it does reduce the associated cytotoxicity. The net outcome of this action is damage without death, as illustrated by elevated micronuclei formation. We propose that this contributes to the tumorigenesis associated with Bcl-2 expression.

To assess the impact of H2O2 on cells, we monitored the oxidation of two thiol proteins, GAPDH and peroxiredoxin 2, which are very sensitive to low concentrations of H2O2, along with the more general markers of oxidative damage, lipid peroxidation and protein carbonyl formation. There are several conflicting reports on the ability of Bcl-2 over-expression to prevent lipid peroxidation. This includes the original study by Hockenbery et al., where they detected differences at later time-points (4,11). This has to be interpreted with caution, however, as the induction and execution of apoptosis disrupts the redox environment of cells (19,33,34). For example, Cai et al. (17,35) have shown that cytochrome c released from the mitochondria of apoptotic cells increases superoxide production. In the same investigation, Bcl-2 over-expression prevented superoxide production by maintaining cytochrome c in the mitochondria.

The difference in protein carbonyl formation that we observed between Jurkat and B9 cells is another example of an apparent antioxidant activity of Bcl-2 that can be linked to a secondary effect. Our results support those of Zhao et al. (36), who reported secondary lysosomal rupture in cells treated with H2O2, which was inhibited by Bcl-2. The release of redox-active iron from these vesicles could promote carbonyl formation that continued well after the initial H2O2 was consumed. Also, protein carbonyls formed by H2O2 exposure are efficiently removed by the proteasome (37), and Lee et al. (7) have demonstrated that cells over-expressing Bcl-2 have a higher proteasomal activity. While these activities assist a cell in coping with an oxidative stress, Bcl-2 can not be described as having direct antioxidant properties.

H2O2 has a clear biphasic response in the Jurkat cell line, inducing apoptosis at lower doses before switching to necrosis at higher doses (30). The apoptotic range is defined by caspase activation, whereas at necrotic doses there is no caspase activation; indeed, there is inhibition of basal caspase activity consistent with oxidative inactivation of these cysteine-dependent enzymes (38). While Bcl-2 was able to protect against apoptotic doses of H2O2, it had no effect on cytotoxicity at necrotic doses and it did not prevent caspase inactivation. These observations strengthen the conclusion that Bcl-2 was acting as an anti-apoptotic factor but not as an antioxidant.

Over-expression of Bcl-2 enhanced the proportion of micronucleated cells seen in Jurkat cells following H2O2 exposure. Taga et al. (39) have demonstrated that cells over-expressing Bcl-2 have a higher frequency of micronuclei formation following X-irradiation. The doses of H2O2 that we found to be mutagenic (50–100 µM H2O2) are in line with previous work by Cherbonnel-Lasserre et al. (40). Our findings are also in accordance with the results of several other groups who have demonstrated that Bcl-2 over-expression enhances DNA and chromosomal damage, promoting mutagenesis in cells exposed to an oxidative insult (15,40–43). These investigations used a variety of mutagenic stimuli including ionizing radiation, benzene metabolites and H2O2 to show that Bcl-2 enhances mutagenesis. The data therefore demonstrate that by overriding apoptosis, Bcl-2 enhances genomic instability and renders cells more susceptible to tumourigenesis.

In summary, the results of this study have clearly demonstrated that Jurkat cells over-expressing Bcl-2 exhibit the same initial level of oxidative damage to cellular constituents as the parental Jurkat cells in response to H2O2 exposure. Furthermore, H2O2-treated Jurkat cells over-expressing Bcl-2 continued to proliferate, despite damage to cellular constituents. The inability of Jurkat cells over-expressing Bcl-2 to undergo apoptosis increased their susceptible to genomic instability as a result of H2O2 exposure.


   Acknowledgments
 
This work is supported by a Sir Charles Hercus Research Fellowship from the Health Research Council of New Zealand to M.B.H.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 21, 2006; revised April 3, 2007; accepted April 4, 2007.


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