(Received for publication, December 9, 1994; and in revised form, December 26, 1994)
From the
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.
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), ()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-
B 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-2C, 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.
In
analyzing E. coli for an antioxidant phenotype,
HO
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
O
and
buthionine sulfoximine, an inhibitor of glutathione synthesis, and
display decreased H
O
production following
treatment with diethyl maleate, which reacts with free thiol
groups(3, 4) . In a filter disc method,
SOD
strains expressing Bcl-2
C 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-2
C or Bcl-2 strains compared to the
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 HO
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
O
correlated with peroxide destruction in E. coli, the
concentration of H
O
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
O
. (
)For the control strain,
survival was 3% and the H
O
concentration after
15 min was reduced to 1.6 mM. For the Bcl-2
C strain,
survival was 62% and the final [H
O
]
was 80-fold less: 0.02 mM. Clearly, differences in survival
were attributable to differences in the decomposition of
H
O
.
Figure 2:
Catalase zymogram. Lanes1 and 2 are sonic extracts from Bluescript control and
Bcl-2C 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-2C 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-2
C strain. The
rate of transcription of a katG::lacZ transcriptional
fusion was in fact 2-4-fold greater in the Bcl-2
C strain
than in the control strain (Fig. 3). These data indicate that
the increased resistance to H
O
in Bcl-2
C 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
O
.
Figure 3:
Rates of katG transcription.
-Galactosidase activity from a katG::lacZ transcriptional fusion was measured during growth of lac
derivatives of Bcl-2
C and control
strains. Introduction of the lac
mutation
did not change the relative resistance of the strains to
H
O
(filter disc method). The doubling times for
Bluescript control and Bcl-2
C strains were 48 and 42 min,
respectively.
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-2C
strain compared to the control strain (Table 1). This result is
consistent with the presence of a constitutive oxidative stress in the
Bcl-2
C strain in ordinary aerobic cultures. After an
H
O
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-2
C strain was increased only 20%. This was consistent
with the 13-fold increase in KatG activity that was expected to
decompose H
O
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-2C strain did
not attenuate resistance to H
O
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
O
resistance of the Bcl-2
C 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-2C katG::Tn10 mutant could not be similarly constructed. The following three
observations suggested that Bcl-2
C 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
O
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-2
C 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-2
C 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-2C strain to
H
O
, KatG was essential for survival during
ordinary aerobic growth. These data and the increased mutation rate are
convincing evidence that the Bcl-2
C SOD
strain
experiences a constitutive oxidative stress. The only difference
between control and Bcl-2
C strains is expression of bcl-2
C. Therefore, our data indicate that Bcl-2
C
creates an oxidative stress, i.e. Bcl-2
C 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
O
or redox cycling
compounds imposed experimentally.
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-
B and AP-1
proposed as a redox-sensitive transcription factors (5, 6, 7) from treatment of cell cultures
with exogenous agents, e.g. H
O
,
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.