©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Bcl-2 Oncoprotein Functions as a Pro-oxidant (*)

(Received for publication, December 9, 1994; and in revised form, December 26, 1994)

Howard M. Steinman (§)

From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanism by which the bcl-2 oncogene exerts its anti-apoptotic and antioxidant action is unknown. We found that expression of bcl-2 in superoxide dismutase-deficient (SOD) Escherichia coli resulted in increased transcription of the KatG catalase-peroxidase, a 13-fold increase in KatG activity and a 100-fold increase in resistance to hydrogen peroxide. In addition, mutation rate was increased 3-fold, and katG and oxyR, a transcriptional regulator of katG induction, were required for aerobic survival. These data indicate that Bcl-2 acts as a pro-oxidant in E. coli, i.e. Bcl-2 generates reactive oxygen intermediates. In support of a pro-oxidant mechanism in eukaryotic cells, we found a 73% increase in superoxide dismutase activity in a murine B-cell line overexpressing Bcl-2. Increases in reduced glutathione and in oxyradical damage to DNA, previously observed in other overexpressing cell lines, are additional evidence for a pro-oxidant mechanism. Thus, Bcl-2 does not appear to be an antioxidant. Instead, Bcl-2 appears to influence levels of reactive oxygen intermediates that induce endogenous cellular antioxidants. This activity of Bcl-2 may control entry into apoptosis.


INTRODUCTION

Eighty-five percent of human follicular B-cell lymphomas are associated with a translocation from chromosome 14 into the immunoglobulin heavy chain region of chromosome 18. This DNA rearrangement results in a chimeric mRNA and increased expression of a gene, bcl-2, shown by clonogenicity and tumorigenicity criteria to be an oncogene. In contrast to tumor suppressors and oncogenes that accelerate cell division, overexpression of bcl-2 delays programmed cell death. Intrinsic, non-translocated, bcl-2 appears to act similarly and delay apoptotic death during lymphocyte development(1, 2) .

How Bcl-2 produces this anti-apoptotic action is unknown. It is of interest that overexpression of bcl-2 in mammalian cell lines can decrease lipid peroxidation and can increase resistance to apoptotic killing by hydrogen peroxide, menadione, and depletion of glutathione. Moreover, introduction of bcl-2 in yeast mutants that lack cytosolic superoxide dismutase (SOD), (^1)mitochondrial SOD or both SODs partially corrects growth deficits seen under aerobic culture conditions(3, 4) . These observations on the antioxidant action of Bcl-2 are significant because reactive oxygen intermediates (ROI) can induce apoptotic death (1, 3) and act as second messengers by influencing transcription factors such as NF-kappaB and AP-1(5, 6, 7) .

The mechanism by which Bcl-2 produces this antioxidant effect is not understood. It may act directly as an antioxidant, chelate redox-active metals, or reduce ROI generation(3, 4) . The latter mechanism has been favored because Bcl-2 associates with membranes of mitochondria, nucleus, and endoplasmic reticulum and could influence transport of electrons to oxygen and ROI generation(1, 2, 3) . However, Bcl-2DeltaC, a recombinant form made by deletion of the C-terminal region, has 50-70% of the antioxidant and anti-apoptotic action of full-length Bcl-2 even though it is no longer membrane-associated(3) .

To investigate the mechanism by which Bcl-2 produces its antioxidant and anti-apoptotic activity, we introduced Bcl-2 in E. coli lacking SOD and observed an antioxidant effect similar to that seen in eukaryotic cells. Our data indicate that in E. coli Bcl-2 acts neither as an antioxidant nor to decrease ROI generation. Instead, Bcl-2 functions as a pro-oxidant, creating an oxidative stress that induces an antioxidant response and incidentally damages macromolecules sensitive to ROI. Evidence that this pro-oxidant mechanism is operative in mammalian cell lines comes from data of ours on induction of SOD in a B-cell line and prior data of others on glutathione levels and DNA damage (3, 4) that was not accountable by prevailing mechanisms of Bcl-2 action. The significance of a pro-oxidant mechanism is that it explains a large body of data on the antioxidant effect of Bcl-2 and suggests that Bcl-2 is a physiological agent that modulates ROI levels. This effect on ROI may influence the cellular commitment to apoptosis.


EXPERIMENTAL PROCEDURES

Bacteriological Methods

We previously (8) constructed a mutant of E. coli K-12 with both SOD genes deleted and the sodB gene replaced by a kanamycin cassette (kan^r-for-sodB). A derivative of that strain in which kan^r is inactivated by insertion of Tn9 (Cm^r) was used in the current study. The kan^r-for-sodB was transduced with P1 phage (9) from strain LFH 1010 (8) into pdxH strain CGSC 4553. A kan^r pdxH transductant was transduced with a pool of random Tn9 insertions in strain LFH 1010 and pdxH transductants selected. By replica plating a Km^sCm^r strain was isolated. The kan::Tn9 mutation was transduced into DeltasodA strain OX2-1 (8) to obtain strain OX2-1.4A, the SOD host used in these studies. Activity staining following electrophoresis of cell extracts (8) demonstrated that strain OX2-1.4A was SOD. Bluescript vector or Bluescript plasmids containing cDNA of human Bcl-2 or Bcl-2DeltaC ((10) ; amber in place of the codon for Trp-214) were introduced by electroporation. OX2-1.4A is wild type for katE, katF, katG, oxyR, and lac. P1 transduction to appropriate drug resistance was used to introduce katE::Tn10, katF::Tn10, katG::Tn10(11) , oxyR::kan and Tn10 linked to Delta(argF-lac)169(12) . Standard methods (13) were used for UV induction of a lysogen harboring a katG::lacZ transcriptional fusion(14) , infection, and isolation of katG fusion lysogens of lac SOD strains. Standard growth conditions were 37 °C in Luria-Bertani broth (9) containing 100 µg/ml sodium ampicillin. BBL GasPak pouches were used for anaerobic growth on plates. Filter disc zones of inhibition was measured as described before (15) using 10 µl of 0.5-1 M solutions/filter. Cultures were grown to mid-log phase (OD = 0.3-0.6) unless indicated otherwise for filter disc tests and other procedures.

Biochemical Methods

beta-Galactosidase was assayed with o-nitrophenyl-beta-D-galactoside as substrate and activity expressed as Miller units(9) . Other enzyme activities were measured in sonic extracts. Continuous variablesPeroxidase activity was assayed by oxidation of dianisidine (16) and catalase and hydrogen peroxide concentration by the phenol-aminoantipyrine method(17) . One activity unit for peroxidase and catalase was defined as 1 µmol of hydrogen peroxide decomposed/min. Staining for catalase activity following non-denaturing gel electrophoresis was performed as described before(18) . SOD activity was measured by the pyrogallol method(15) ; 1 unit was defined as the amount for 50% inhibition of the non-enzymatic rate of autooxidation. Western blots following transfer of proteins from SDS-polyacrylamide gels (19) were probed with murine anti-Bcl-2 monoclonal antibody (DAKO), which also reacts with Bcl-2DeltaC, followed by anti-mouse immunoglobulin coupled to alkaline phosphatase. Protein concentration was determined by the BCA reagent ((15) ; Pierce).

Tissue Culture

NSO cells, a murine B-cell plasmacytoma line, wild type and stable bcl-2 transfectants, and their growth conditions have been described previously(20) .


RESULTS AND DISCUSSION

Demonstration of an Antioxidant Phenotype in E. coli

Bcl-2 has been expressed in E. coli(10) . A SOD host was chosen here because when bcl-2 was introduced in SOD mutants of yeast an antioxidant effect was seen(4) . Western blots of log phase extracts showed immunoreactive bands of the expected molecular weight and of equal intensity in SODE. coli containing bcl-2 and bcl-2DeltaC. No bands were seen from the control strain, SOD with Bluescript vector.

In analyzing E. coli for an antioxidant phenotype, H(2)O(2) was the principal focus, because hydrogen peroxide is implicated in the antioxidant action of bcl-2 overexpression in murine cell lines. Bcl-2 cell lines show increased resistance to killing by H(2)O(2) and buthionine sulfoximine, an inhibitor of glutathione synthesis, and display decreased H(2)O(2) production following treatment with diethyl maleate, which reacts with free thiol groups(3, 4) . In a filter disc method, SOD strains expressing Bcl-2DeltaC or Bcl-2 showed increased resistance to hydrogen peroxide compared to the control strain (25 ± 0.5, 37 ± 3 versus 42 ± 0.4 mm zones, respectively). Resistance to copper sulfate and paraquat were not increased in the Bcl-2DeltaC or Bcl-2 strains compared to the control strain.

Bcl-2DeltaC Protects against Hydrogen Peroxide Killing by Decomposition of Peroxide

To analyze the mechanism of the antioxidant activity in SODE. coli, the Bcl-2DeltaC strain was studied, because its resistance to H(2)O(2) was greater than that of the Bcl-2 strain. Log phase cultures were exposed to a range of H(2)O(2) concentrations and then plated. The results (Fig. 1) showed that H(2)O(2) was acting as a bacteriocidal not a bacteriostatic agent. At 25 mM H(2)O(2), survival of the Bcl-2DeltaC strain was 2 orders of magnitude greater than the Bluescript control strain.


Figure 1: Resistance to hydrogen peroxide. Log phase cultures grown aerobically in shaker flasks were treated for 15 min at 37 °C with the indicated concentration of hydrogen peroxide, then diluted and plated for colony formation. Similar results were obtained if bovine catalase was added to 2 µg/ml after the 15-min incubation.



Although resistance to H(2)O(2) has been amply documented in mammalian cell lines overexpressing bcl-2, the mechanism of this resistance has not been characterized(1, 2, 3, 4) . Decomposition of peroxide by cellular catalases/peroxidases is one possibility. To determine if resistance to H(2)O(2) correlated with peroxide destruction in E. coli, the concentration of H(2)O(2) was measured during the course of a killing experiment. Hydrogen peroxide is structurally similar to water. Cellular and external pools rapidly equilibrate, and the concentration in the medium reflects that in the cytoplasm(21) . Hydrogen peroxide in culture supernatants was quantified in a 15 min killing experiment with 20 mM H(2)O(2). (^2)For the control strain, survival was 3% and the H(2)O(2) concentration after 15 min was reduced to 1.6 mM. For the Bcl-2DeltaC strain, survival was 62% and the final [H(2)O(2)] was 80-fold less: 0.02 mM. Clearly, differences in survival were attributable to differences in the decomposition of H(2)O(2).

KatG Is Induced Constitutively in E. coli with Bcl-2DeltaC

The preceding data suggested that catalase levels might be higher in the Bcl-2DeltaC strain. E. coli contains two catalases, encoded by katE and katG, and does not contain glutathione peroxidase. KatG and KatE are readily separable by electrophoresis under non-denaturing conditions and can be visualized by in situ staining of the gel (Fig. 2). KatG catalase activity was significantly increased in the Bcl-2DeltaC strain compared to the control strain. Levels of KatE are comparable in the two strains. While KatE is cytoplasmic, KatG is found predominantly in the periplasm(22) . High levels of the KatG periplasmic catalase activity in the Bcl-2DeltaC strain are consistent with its resistance to peroxide killing; H(2)O(2) is decomposed in the periplasmic space, preventing damage to the inner membrane and cytosol and leading to increased survival.


Figure 2: Catalase zymogram. Lanes1 and 2 are sonic extracts from Bluescript control and Bcl-2DeltaC strains (21 µg of protein/lane). Lane3 is 0.2 µg of bovine catalase.



Enzyme assays were performed to quantitate KatG. KatG can be assayed in the presence of KatE because KatG has peroxidase activity while KatE is exclusively a catalase(16) . Using a peroxidase assay, we found that KatG activity was increased 13-fold when Bcl-2DeltaC is present (0.12 versus .0095 dianisidine units/mg protein). Since katG expression is regulated transcriptionally(23, 24) , an increased rate of katG transcription was expected in the Bcl-2DeltaC strain. The rate of transcription of a katG::lacZ transcriptional fusion was in fact 2-4-fold greater in the Bcl-2DeltaC strain than in the control strain (Fig. 3). These data indicate that the increased resistance to H(2)O(2) in Bcl-2DeltaC E. coli is not attributable to an antioxidant activity of Bcl-2 itself. Instead, resistance is due to an increased expression of katG that occurs throughout growth, in the absence of H(2)O(2).


Figure 3: Rates of katG transcription. beta-Galactosidase activity from a katG::lacZ transcriptional fusion was measured during growth of lac derivatives of Bcl-2DeltaC and control strains. Introduction of the lac mutation did not change the relative resistance of the strains to H(2)O(2) (filter disc method). The doubling times for Bluescript control and Bcl-2DeltaC strains were 48 and 42 min, respectively.



Evidence That the Bcl-2DeltaC Strain Experiences a Constitutive Oxidative Stress

KatG activity is ordinarily low during exponential growth of E. coli (24). Why is the KatG level increased 13-fold in the Bcl-2DeltaC strain? KatG is inducible(23, 24) . Exposure of E. coli to µM concentrations of H(2)O(2) induces katG and renders E. coli resistant to mM H(2)O(2)(25) . This induction is transcriptionally controlled by the OxyR regulator(23, 24) . However, in our studies, KatG levels were elevated constitutively in the Bcl-2DeltaC strain without prior exposure to hydrogen peroxide. This constitutively high level of KatG suggested that during ordinary aerobic culturing the Bcl-2DeltaC strain experienced an oxidative stress sufficient to induce an antioxidant response, i.e. induce katG. Two studies were performed to evaluate this hypothesis.

First, we measured the rate of mutation to rifampicin resistance(15) . Reactive oxygen species can be mutagenic and cause oxidative damage to DNA(26, 27) . Increases in mutation rate, DNA fragmentation, and formation of 8-hydroxy deoxyguanosine are accepted biomarkers of increased oxidative stress. In the absence of peroxide and any other added oxidant, the mutation rate to rifampicin resistance was 3-fold higher in the Bcl-2DeltaC strain compared to the control strain (Table 1). This result is consistent with the presence of a constitutive oxidative stress in the Bcl-2DeltaC strain in ordinary aerobic cultures. After an H(2)O(2) challenge, the mutation rate in the control strain was increased 4-fold, as expected from the known mutagenic effects of hydrogen peroxide. After the same challenge, mutation rate of the Bcl-2DeltaC strain was increased only 20%. This was consistent with the 13-fold increase in KatG activity that was expected to decompose H(2)O(2) and prevent its mutagenic action.



Additional evidence for a constitutive oxidative stress came from construction of isogenic strains containing insertionally inactivated alleles of katE, katF, and katG by transduction. Introduction of katE::Tn10 or katF::Tn10 mutations in the Bcl-2DeltaC strain did not attenuate resistance to H(2)O(2) compared to the corresponding mutations in the control strain (filter disc method; data not shown). These results were expected, because KatE levels are comparable in the two strains and KatE is not implicated in H(2)O(2) resistance of the Bcl-2DeltaC strain. KatF is , involved in induction of KatE during stationary phase(28) .

Results with the katG::Tn10 allele were quite different. The mutant of the control strain was readily constructed. However, the Bcl-2DeltaC katG::Tn10 mutant could not be similarly constructed. The following three observations suggested that Bcl-2DeltaC katG::Tn10 mutant had not been isolated because it was sensitive to the oxidative stress of ordinary aerobic culturing. (a) Construction of the katG::Tn10 mutant was possible if transductants were selected anaerobically. (b) Transductants could be isolated aerobically in plates containing 1-6 µg ml bovine catalase. Since intracellular H(2)O(2) is in rapid equilibrium with the medium, an external catalase can compensate for absence of KatG in the cell. (c) The saturation density of the Bcl-2DeltaC katG::Tn10 mutant in aerobic LB broth was proportional to the amount of bovine catalase added to the medium. For 0, 0.025, 0.05, and 0.1 µg of bovine catalase ml, the OD at saturation was 0.18, 0.87, 1.2, and 1.5. In contrast, aerobic growth of the control strain with the katG::Tn10 mutation was not influenced by bovine catalase. Similar results were obtained with a oxyR::kan mutation. Catalase was required for introduction of oxyR::kan in the Bcl-2DeltaC strain but not for the control strain.

Results with katG and oxyR null mutations indicate that KatG was not merely important in the tolerance of the Bcl-2DeltaC strain to H(2)O(2), KatG was essential for survival during ordinary aerobic growth. These data and the increased mutation rate are convincing evidence that the Bcl-2DeltaC SOD strain experiences a constitutive oxidative stress. The only difference between control and Bcl-2DeltaC strains is expression of bcl-2DeltaC. Therefore, our data indicate that Bcl-2DeltaC creates an oxidative stress, i.e. Bcl-2DeltaC acts as a pro-oxidant and increases ROI levels in the E. coli cell. A pro-oxidant mechanism proposes that Bcl-2 elevates ROI, which induce the endogenous antioxidants responsible for the antioxidant phenotype. Incidental to induction of an antioxidant response is damage to cellular targets that are sensitive to oxyradicals, e.g. DNA. The increase in ROI associated with Bcl-2 is presumed adequate to signal the induction of antioxidants but small compared to the potentially lethal doses of H(2)O(2) or redox cycling compounds imposed experimentally.

Does Bcl-2 Act as a Pro-oxidant in Mammalian Cell Lines?

Overexpression of bcl-2 in mammalian cell line reduces killing by H(2)O(2) and other oxidants (1, 2, 3, 4) . How bcl-2 expression leads to such antioxidant activity is not known. Our characterization of Bcl-2DeltaC in E. coli indicates that it acts as a pro-oxidant. Data of ours and others show that when bcl-2 is overexpressed in mammalian cell lines, two critical components of the pro-oxidant mechanism are observed: induction of endogenous antioxidants and incidental damage to DNA. We analyzed superoxide dismutase in NSO cells, a murine B-cell line, stably transfected with bcl-2. The cells were grown under ordinary aerobic conditions without any imposed stress. We found that SOD activity in wild type and Bcl-2 NSO cells is predominantly CuZnSOD with little MnSOD (95% versus 5% of the total activity, respectively). In Bcl-2 NSO cells, total SOD activity was increased by 73% compared to wild type NSO cells (260 ± 3 versus 150 ± 14 units/mg cell protein). SOD is a major antioxidant enzyme and can be induced in response to oxidative stress. Its increase in the absence of an imposed oxidative stress suggests that Bcl-2 NSO cells experience an oxidant stress in normal aerobic culturing. In addition, our data suggested that the level of CuZnSOD was critical in the cellular decision to enter apoptosis. Data of others support this suggestion. Down-regulation of CuZnSOD caused apoptotic death in PC12 neuronal cells(29) , while overexpression of MnSOD had no effect on apoptosis in FL5.12 B-cells(3) .

Previous data of others, not consonant with an antioxidant mechanism, supports a pro-oxidant mechanism for Bcl-2. The effect of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, was studied in a murine hypothalamic neural cell line overexpressing bcl-2. The Bcl-2 cell line was more resistant to BSO killing than the control. However, prior to any BSO treatment, glutathione concentration was more than 2-fold increased in the Bcl-2 cell line (4) . This enigmatic increase in glutathione, a major thiol antioxidant, is understandable by a pro-oxidant mechanism in which bcl-2 overexpression creates an oxidative stress that induces cellular antioxidants and leads to resistance against subsequent, potentially lethal oxidative stresses. Damage to DNA was observed in a study of a T-cell hybridoma. DNA fragmentation was increased in a bcl-2 transfectant (0.9 versus 0%) in the absence of an external apoptotic stress(4) . This is consistent with overexpression of bcl-2 leading to increased ROI, evidenced by damage to DNA.

The effect of Bcl-2 on ROI generation has been considered by others (1, 2, 3, 4) . Decreased generation was reported in a neuronal cell line, while no change in ROI production was reported for a pro-B cell line expressing bcl-2. Those results neither conflict with nor preclude a pro-oxidant mechanism. In the pro-oxidant mechanism, ROI are increased prior to entering an apoptotic pathway. The focus in the preceding studies was the effect of Bcl-2 after oxidative or other stresses produce apoptosis.

A pro-oxidant mechanism for Bcl-2 has three significant implications. First, it accounts for data unexplained by prevailing theories of Bcl-2 antioxidant action. How could overexpression of Bcl-2 increase resistance to such a wide range of agents, including -irradiation, glutathione inhibitors, and redox cycling agents? If Bcl-2 acts as a pro-oxidant and induces a cellular antioxidant response, resistance to such a panel of reagents is not unexpected. Second, it implies that oxidative stress and ROI are integral control elements in the cellular decision to enter apoptosis, not merely experimental tools to initiate apoptosis in cell lines. Finally, the pro-oxidant mechanism is significant in implying that Bcl-2 is a physiological agent that modulates cellular ROI levels. ROI have been proposed as second messengers, and NF-kappaB and AP-1 proposed as a redox-sensitive transcription factors (5, 6, 7) from treatment of cell cultures with exogenous agents, e.g. H(2)O(2), menadione, metal chelators, or N-acetylcysteine. It is assumed that these agents reflect processes occurring in vivo. However, there is little quantitative information on physiological fluctuations in ROI, e.g. from respiratory burst activity during inflammation or from increased leakage of electrons from damage to mitochondria. A pro-oxidant function for Bcl-2 would provide a benchmark for what constitutes a physiological oxidative stress and a means of determining if this stress has a regulatory role. How Bcl-2 influences ROI levels is not clear. Analysis of oxyradical production by in vivo spin trapping may give insights.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM 40468, National Science Foundation Grant MCB 9220055, and a grant from The Amyotrophic Lateral Sclerosis Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3010; Fax: 718-892-0703; steinman{at}aecom.yu.edu.

(^1)
The abbreviations used are: SOD, superoxide dismutase; ROI, reactive oxygen intermediate(s); BSO, buthionine sulfoximine.

(^2)
Hydrogen peroxide concentration was not diminished in medium without E. coli.


ACKNOWLEDGEMENTS

I thank Dr. Michael Cleary, Stanford University, for Bluescript plasmids containing Bcl-2 and Bcl-2DeltaC; Dr. Barbara Bachmann, Yale University, for E. coli strains CGSC 4553 (pdxH) and CGSC 7096 (Tn10 near the Delta(argF-lac)169); Dr. Peter Loewen, University of Manitoba, for strains UM 120, 122, and 202 containing Tn10 insertions in katE, katF, and katG; Dr. Gisela Storz, National Institutes of Health, for strain GS09 (oxyR::kan) and for MC 4100 with a katG::lacZ transcriptional fusion; and the following colleagues at Albert Einstein College of Medicine: Dr. Betty Diamond and Subhransu Ray for wild type and Bcl-2-expressing NSO cell lines, Xiaoyan Zhu for construction of E. coli strain OX2-1.4A, and Laurie Weinstein and Gregory St. John for technical assistance.


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