Department of Environmental Health Sciences, School of Public Health and Center for Free Radical Biology, University of Alabama, Birmingham, Alabama 35294-0022
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9. D3R cells were resistant to these agents without caspase activation. Antioxidant profiles revealed that D3R cells had significantly higher glutathione and glutathione reductase activity but markedly lower catalase, glutathione peroxidase, and aldehyde reductase activities than G4S cells. Thus A549 cells contain at least two distinct subpopulations with respect to predisposition to cell death and antioxidant profile. Because sensitivities to agents and the antioxidant profile were inconsistent, mechanisms independent of antioxidants, including the apparent inability to activate caspases in D3R cells, may play an important role. Regardless, the results suggest that antioxidant profiles of asymmetrical cell populations cannot predict sensitivity to oxidants and warn that the use of single subclones is advisable for mechanistic studies using A549 or other unstable cell lines.
oxidative stress; enzyme; caspase; apoptosis; genetic instability
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE A549 CELL LINE, ORIGINALLY established from a lung adenocarcinoma of a Caucasian male in 1972 (22), has been widely used as a model of lung type II epithelial cells, as is reflected by almost 2,000 published articles. However, like other carcinoma cell lines, A549 cells are a nonhomogenous cell population that consists of multiple clones with a modal chromosome number of 12 and counts of 64, 65, and 67 chromosomes occurring with high frequency (American Type Culture Collection, http://atcc.org/SearchCatalogs/longview.cfm?view=ce,323118,CCL-185&text=a549). In fact, several subpopulations with various characteristics have been isolated from the parental cell populations (14, 16). Nevertheless, the majority of research with A549 cells continues to be done using the heterogeneous population.
In our initial studies of apoptosis, we also found that A549
cells contain cells that are both susceptible and resistant to cytotoxicity by tumor necrosis factor- (TNF-
) plus actinomycin D. Therefore, several clones from the parental A549 cells were isolated by
limiting dilution, and TNF-
plus actinomycin D-susceptible (G4S) and
-resistant (D3R) clones were selected for additional studies.
Subsequent study revealed that the different sensitivity was dependent
on actinomycin D alone, and that the resistant/sensitive phenotype was
also indicative of the response to other macromolecule synthesis
inhibitors as well as to some oxidative insults.
Reactive oxygen species (ROS) are associated with the initiation of cell death by a variety of cytotoxic insults such as ultraviolet radiation and chemotherapeutic agents. Depending on the burden of ROS, the mode of cell death can be either apoptosis or necrosis or a combination of the two (12). Nonetheless, a network of antioxidants and antioxidant enzymes [superoxide dismutases (SOD) catalase, glutathione peroxidases (GPx), glutathione (GSH), NADPH generated by the pentose phosphate pathway (PPP), thioredoxin (Trx), peroxiredoxins, and glutathione S-transferases (GST)] protects cells against ROS and cytotoxic products of lipid peroxidation while maintaining cellular thiols in their reduced states.
Overexpression or deletion of various antioxidant enzymes can change the sensitivity to various cytotoxic insults (3, 6, 26, 27, 30, 33, 35, 45) as well as growth rates (5, 27), which substantiates the notion that ROS or their reactive derivatives are critical components of signal transduction pathways. Therefore, our simple original hypothesis was that the differences in the sensitivity of these clones to cytocidal agents, as well as other characteristics, would be related to differences in their cellular antioxidant levels. To address this possibility, the antioxidant profiles of the subclones were characterized. Although some antioxidants were higher in the resistant clone than in the susceptible clone, others including the frontline enzymes catalase and GPx were markedly lower. The results also showed that the two clones differ asymmetrically in factors other than antioxidant levels. Thus, even when oxidants are used to initiate cell death, antioxidant profiles alone cannot predict the sensitivity to cell death of asymmetrical clones. Furthermore, although subcloning appears to be advisable for any mechanistic study using the A549 cell line or other unstable cell lines, comparison among such clones is unreliable in determining mechanisms, as there are often unknown variables.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
Unless otherwise noted, all chemicals were from Sigma (St. Louis,
MO). Acetyl-Asp-Glu-Val-Asp-7-amino-4-(trifluoromethyl)-coumarine (Ac-DEVD-AFC) and
acetyl-Leu-Glu-His-Asp-7-amino-4-(trifluoromethyl)-coumarine (Ac-LEHD-AFC) were from Calbiochem (La Jolla, CA); 4-hydroxy-2-nonenal (HNE) was from Cayman Chemical (Ann Arbor, MI);
2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was from Oxis Research
(Portland, OR); glycylglycine was from ICN Biomedical (Aurora, OH); and
L--glutamyl 7-amino-4-methylcoumarin (AMC) was purchased
from Bachem Bioscience (King of Prussia, PA).
A549 cells and isolation of the subclones. A549 cells were purchased from the American Type Culture Collection (ATCC). Parental cells and subclones were cultured in F-12K medium (GIBCO-BRL) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin within a humidified atmosphere containing 5% CO2 at 37°C.
Subcloning of A549 cells was done by limiting dilution using 96-well culture plates. Several wells that contained a single colony were randomly selected and aliquoted. An aliquot from each clone was treated with TNF-Cytotoxicity assay by crystal violet staining. Cells in 96-well plates (1.5 × 104 cells/well) were treated with various agents in the culture medium for 8 h. Cells were washed with PBS [that contained (in mM) 10 Na2HPO4, 1 KH2PO4, 137 NaCl, and 2.7 KCl, pH 7.4] and then fixed and stained with 100 µl of crystal violet solution [0.5% (wt/vol) crystal violet, 1.5% (vol/vol) formaldehyde, and 1% (vol/vol) ethanol] for 30 min. After the wells were washed with water, the stained cells were lysed with 1% (wt/vol) deoxycholate, and the absorbance at 550 nm was read in a microplate reader.
Caspase assays. Cells in 24-well plates (1.5 × 105 cells/well) were treated with various agents in medium for 2 or 4 h. After the treatment, both attached and detached cells were collected and combined. The cells were lysed in 250 µl of 0.1% Triton X-100/NaPi (0.1 M sodium phosphate buffer, pH 7.4) and centrifuged at 10,000 g for 10 min at 4°C to obtain the supernatant. Caspase assays were carried out in a 96-well assay plate. The final concentrations of each constituent in 200 µl were as follows: cell lysate equivalent to the original 6 × 104 cells, 10 mM dithiothreitol, 0.05% (vol/vol) Triton X-100, and 50 µM either Ac-DEVD-AFC (caspase 3 substrate) or Ac-LEHD-AFC (caspase 9 substrate) in NaPi. After the reaction progressed at 37°C for 60 min, the fluorescence intensity was measured in a fluorescence microplate reader (SpectraMax GeminiXS, Molecular Devices) with excitation and emission wavelengths of 400 and 500 nm, respectively. The values were converted to AFC concentrations using an external AFC standard.
Measurement of cellular antioxidant enzyme activities.
Unless otherwise indicated, the measurements were made using
aliquots of lysate prepared as follows: cells were cultured in 100-mm
culture dishes until attainment of ~70% confluence (D3R, ~3 × 106; G4S, ~1.5 × 106 cells/dish),
100% confluence (D3R, ~6 × 106; G4S, ~3 × 106 cells/dish), or overconfluence (24 h after confluence:
D3R, ~10 × 106; G4S, ~4.5 × 106
cells/dish). Cells were washed with PBS and lysed with 0.1% Triton X-100/NaPi. The lysates were centrifuged at 10,000 g for 15 min at 4°C, and the supernatants were collected. Lysates were
aliquoted and stored at 80°C, and each aliquot was used only once.
Protein concentrations were determined by the Bradford method (Bio-Rad) with BSA as a standard.
Antioxidant enzyme assays in microplate plate reader. Unless otherwise indicated, all antioxidant enzyme assays described were conducted at 22°C in a 96-well assay plate with a total volume of 200 µl. The reaction kinetics were measured with a SpectraMax Plus microplate reader (Molecular Devices) that was controlled by the dedicated software (SOFTmax Pro, Molecular Devices). Each sample was measured in either duplicate or triplicate.
Total SOD activity was evaluated by the cytochrome c/xanthine/xanthine oxidase system according to the methods of McCord and Fridovich (32) with modifications. The final concentrations of each constituent were as follows: 100 µg/ml cell lysate, 10 µM ferricytochrome c, 3.32 mU/ml xanthine oxidase, 50 µM xanthine, 10 µg/ml catalase, and 100 µM diethylenetriaminepentaacetic acid (DETAPAC) in 0.05% (vol/vol) Trition X-100/NaPi. The rate of ferricytochrome c reduction was monitored at 550 nm. The addition of known amounts of exogenous SOD into the lysate produced the expected increases in SOD activity that indicated the absence of interference from either oxidases (i.e., cytochrome c oxidase) or reductases in the lysate (data not shown). One unit was defined as 50% inhibition of the cytochrome c reduction, and the activity was calculated from the following equation: activity (in U/ml) = Vcontrol/VlysateOther antioxidants: enzymes. MnSOD activity was measured in the in-gel nitroblue tetrazolium assay (7). Confluent cells in 100-mm dishes were homogenized in 200 µl of PBS by sonication on ice. After centrifugation at 10,000 g for 15 min at 4°C, the supernatant was collected. The protein (50 µg) was resolved by 10% native PAGE at 4°C, and MnSOD activity was measured. The intensity of the MnSOD activity was within a linear range, which was confirmed using serially diluted authentic Cu,ZnSOD.
Intracellular GSH content was determined by HPLC according to the methods of Fariss and Reed (17), with modifications as reported previously (29), using cells cultured in six-well plates. Trx content was determined by an ELISA kit (Wakan, Kyoto, Japan) using detergent-free cell lysate (detailed in Antioxidant enzyme assays in microplate plate reader) according to the manufacturer's instructions. Protease inhibitors [final concentrations of 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.4 µM aprotinin, 11 µM leupeptin, 20 µM bestatin, 7.5 µM pepstatin A, and 7 µM E64] were added to the lysate before the assay. Catalase activity was measured with a spectrophotometer at 25°C from the rate of H2O2 decomposition using an extinction coefficient ofMeasurement of the steady-state H2O2
concentration.
Intracellular H2O2 concentration was estimated
from the rate of endogenous catalase inactivation by
3-amino-1,2,4-triazole according to the methods of Royall and
colleagues (37) with slight modifications. Cells in
six-well plates (4.5 × 105 cells/well) were incubated
with 25 mM aminotriazole (ATZ) in medium for various times. At the end
of the incubation, cells were washed with PBS and immediately lysed
with 0.3 ml of 0.1% Triton X-100/NaPi on ice, and lysate was prepared
as described (see Measurement of cellular antioxidant enzyme
activities). Catalase activities were measured as described
previously, and the rate constant (kcatalase)
for the pseudo-first-order inactivation kinetics was determined. The
steady-state H2O2 concentration
([H2O2]) was calculated from the following
relationship: [H2O2] = kcatalase/k1, where k1 is the second-order rate constant of
compound I (catalase + H2O2 compound I + H2O) formation from catalase and
H2O2 (1.7 × 107
M
1 · s
1).
Data and statistics. All values expressed are means ± SD. Statistical analysis was carried out with Student's t-test or one-way ANOVA followed by Student-Newman-Keuls test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of A549 subclones.
Our initial studies were designed to investigate the mechanism of cell
death caused by the combination of TNF- and actinomycin D using A549
cells as a model. However, the studies revealed that although some
cells (<10%) died in several hours, the rest were resistant to the
treatment even for 24 h (data not shown). Therefore, several
clones from the parental A549 cells were isolated. On the basis of the
sensitivity to actinomycin D plus TNF-
treatment, a highly
susceptible clone and a highly resistant clone were selected for
detailed study. Subsequently, it was found that the susceptibility of
the clone to actinomycin D plus TNF-
treatment was almost completely
dependent on actinomycin D alone (data not shown). The growth rate of
the actinomycin D-resistant clone was faster than that of the
susceptible clone (~1.5 times; see Fig. 2E, control). The
susceptible clone was named G4S for its relative susceptibility and
slower growth rate and the resistant clone D3R for its relative resistance and rapid growth rate.
|
Sensitivity of clones to cytotoxic agents.
Figure 2 shows the difference in the
sensitivity of D3R and G4S clones to various agents evaluated by
crystal violet staining. Cell death by actinomycin D was preferentially
induced in G4S cells (Fig. 2A). For example, at an
actinomycin D concentration of 0.5 µg/ml, the number of G4S cells
declined to 40% of the control at 8 h while 90% of D3R cells
remained viable. The apparent slight decrease in the number of D3R
cells at the high actinomycin D concentration was due not to cell death
but to growth arrest as revealed by microscopic observation (data not
shown). The more evident contrast could be seen at 24 h. Figure
2E shows the number of cells present after 24 h of
exposure compared with the initial number of cells before exposure. It
also shows the relative detachment of cells at 24 h (Figure
2E, inset). Thus both cell death and growth arrest were
examined. The number of D3R cells remained the same as the initial
value, but no cell death was microscopically observed (Fig.
2E), indicating the cells committed to growth arrest. In
contrast, the number of G4S cells evaluated as dead by crystal violet
staining was reduced to 15% of the original value, although this value
represented some debris from dead cells, and, microscopically, almost
all cells were observed as shrunken dead cells (Fig. 2E). Thus G4S cells were susceptible, whereas D3R cells were almost completely resistant to the cytotoxic effects of actinomycin D under
our experimental conditions.
|
Identification of mode of cell death.
Apoptotic cell death, which is characterized as the activation of
caspases, is associated with cell death by various cytocidal agents
(15, 23). Therefore, using the activities of caspase 3 and
caspase 9 as indexes of apoptosis, the mode of cell death by
the cytocidal agents was investigated. Both caspase 3 (Fig. 3A) and caspase 9 (Fig.
3B) were activated in G4S cells but not in D3R cells after
treatment with actinomycin D, anisomycin, HNE, and DMNQ at doses lethal
to G4S, which is consistent with the sensitivity of each clone to these
agents as measured by crystal violet staining (see Fig. 2). Thus, in
G4S cells, cell death by the cytocidal agents was at least partially
due to apoptosis and involved the mitochondrial/caspase 9 cascade (23). In contrast, the limited cell death in D3R
cells, which occurred with HNE and DMNQ but not the macromolecule
synthesis inhibitors, appeared to be via some other mechanism.
|
Antioxidant profile of clones.
The difference between the clones in the sensitivity to oxidants
suggested that differences in the cellular antioxidant systems might be
responsible. Because antioxidant enzyme activity can be affected by
various culture conditions including cell confluency (8,
9), antioxidant enzyme profiles were investigated at three
different cell densities (Table 1):
subconfluent (~70%), confluent (~100%), and overconfluent (24 h
after confluence).
|
|
Steady-state H2O2 concentration in clones.
A possible consequence of the difference in catalase and GPx activities
would be a difference in the intracellular hydroperoxide steady-state
concentration, especially H2O2. That is, lower
peroxidase activity in D3R cells could allow the
H2O2 steady-state concentration to be
relatively higher under physiological conditions, whereas the
H2O2 steady-state concentration could be
relatively lower for the higher peroxidase-expressing G4S cells. In
cells, H2O2 freely diffuses into the
peroxisomes where almost all catalase is sequestered. Thus the
cytosolic steady-state level of H2O2 can be
estimated from the rate of inactivation of endogenous catalase by ATZ
(37), although it actually reflects the steady-state concentration in the peroxisomes. Catalase inactivation by ATZ was 2.5 times faster in D3R than G4S cells, with half-life values of 208 and
527 s, respectively (Fig. 5). The
calculated steady-state H2O2 concentrations of
D3R and G4S cells were 196 and 77 pM, respectively. Thus the result was
consistent with a presumable consequence of the difference in
peroxidase activities between the clones, thereby substantiating the
different peroxidase activities in vivo.
|
Relative resistance to chemical oxidation.
High concentrations of exogenous hydroperoxides have often been used to
study cell death, even though the physiological relevance is clearly
questionable. Nonetheless, as the differences in the enzymes that
directly interact with H2O2 were strikingly
different, we tested the relative resistance to what may better be
considered as chemical oxidation. In marked contrast to the DMNQ and
HNE results, when cells were exposed to bolus
H2O2 or tert-butyl hydroperoxide (t-BOOH) for 8 h, G4S cells showed relative resistance
to both hydroperoxides (Fig. 6). At the
nonphysiological concentration of 2 mM H2O2,
the number of viable cells in the D3R cell culture was reduced to 41%
of the control, whereas that of G4S cells was 68% of the control (Fig.
6A), and at 500 µM t-BOOH, the number of viable
D3R cells was reduced to 9% of the control, while that for G4S cells
was still 66% of the control (Fig. 6B). Thus, although G4S
cells were more susceptible to DMNQ (see Fig. 2C), in which addition of catalase extracellularly protected cells by >50% (see Fig. 2F), the cells were relatively resistant to chemical
oxidation by bolus hydroperoxide addition. Conversely, although D3R
cells showed relative resistance to DMNQ, the cells were more
susceptible to the bolus hydroperoxide treatments. Although 2 mM
H2O2 caused more extensive cell death in D3R
than G4S cells, neither caspase 3 nor caspase 9 activities could be
detected in the D3R cells or in the G4S cells dying from the exposure
even up to 4 mM H2O2 [a dose lethal to both
clones (data not shown)], which demonstrates that the mode of cell
death caused by this nonphysiological concentration of
H2O2 was via a caspase-independent mechanism.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines of tumor cell origin are well known to readily undergo chromosomal rearrangement, multiplication, and spontaneous mutation (21). Reflecting this fact, several subclones and subpopulations that bear different morphologies as well as different sensitivities to some xenobiotics have been previously isolated from the parental population of A549 cells (14, 16). In this study, we isolated two clones (D3R and G4S cells) from the A549 cell line that differed markedly in terms of morphology (see Fig. 1), growth rate (see Fig. 2E), caspase activation (see Fig. 3), cell death (see Figs. 2 and 6), and antioxidant profile (see Fig. 4 and Table 1).
In any single cell type, higher GR activity or GSH content renders cells resistant to cell death (13, 34). Compared with G4S cells, D3R cells had higher GSH content (1.6 times) and GR (3.1 times), GST (1.6 times), and 6PGD (1.8 times) activities and had comparable activities (<1.5 times) in Cu,ZnSOD, MnSOD, NQO, GGT, Trx, TrxR, and G6PD (see Fig. 4 and Table 1). Thus one may suspect that the difference in the activity of the GSH system (GSH, GR) may be in part responsible for the different sensitivity of the clones. However, G4S cells also had higher activities of GPx (5.5 times), catalase (3 times), and AR (4 times), which suggests that attributing the difference in sensitivity of the clones to the difference in the GSH system is incompatible with the total picture. Similarly, whereas the much-higher catalase and GPx activities in G4S cells correlate with the relative susceptibility to bolus addition of nonphysiological concentrations of hydroperoxides (H2O2 and t-BOOH; Fig. 6), that sensitivity is inconsistent with the GR- and GSH-profile differences. Thus the relationship between the antioxidant profiles and the the relative susceptibility to the macromolecular synthesis inhibitors DMNQ and HNE or to bolus hydroperoxide is not straightforward. As such, we suggest that the prediction of relative susceptibility or the drawing of mechanistic conclusions from comparisons of antioxidant defenses between the two clones is confounded by many variables. Although we cautiously consider in the following text some of those variables, the potential remains for many other factors having nothing to do with oxidant-antioxidant balance to contribute to differences in susceptibility.
Defective caspase activation program in D3R cells is likely a contributing mechanism for different predisposition of clones to cell death. Apoptosis is a likely component of cell death in G4S cells induced by all four agents (actinomycin D, anisomycin, DMNQ, and HNE) because all of these activated caspase (see Fig. 3). Nevertheless, none of the agents tested induced any measurable increase in the activity of either caspase 9 or caspase 3 in D3R cells (see Fig. 3). Thus D3R cells appear to have a defect in the caspase-activation program itself whereby the cells show absolute resistance to the macromolecule synthesis inhibitors, whose toxicological mechanism is apparently more apoptotic than necrotic. On the other hand, the sensitivity of D3R to DMNQ and HNE toxicity is likely due to the induction of necrotic cell death by these agents. Although DMNQ and HNE appear to induce both apoptosis and necrosis, only the G4S cells were susceptible to both cell death mechanisms.
D3R cells are susceptible to bolus addition of very high concentrations of H2O2 and t-BOOH (Fig. 6), although caspases were not activated (data not shown). In this case, the defective caspase-activation program in D3R cells no longer confers a protective role because the mode of death is probably necrosis. The results here appear to parallel what has been observed in the Jurkat T-cell line in which a caspase activation-deficient clone has been shown to be defective in the expression of the proapoptotic protein Bak (44).Potential mechanism for paradoxes in H2O2 toxicity and peroxidase activity. G4S cells were more susceptible to DMNQ than were D3R cells in a catalase-inhibitable manner (see Fig. 2, C and F) but showed relative resistance to bolus nonphysiological concentrations of hydroperoxides (Fig. 6). This apparent paradoxical contrast in susceptibility to hydroperoxide toxicity may be explained by differences in the site of H2O2 attack in DMNQ toxicity and in bolus hydroperoxide toxicity. Although it is probable that DMNQ could be reduced in the cell by oxidoreductases, it may actually be reduced at the outer surface of the plasma membrane by a transmembrane oxidoreductase that can reduce quinones (41). Because H2O2 is highly diffusible through membranes, it is therefore conceivable that DMNQ derived H2O2 signals for cell death by affecting a target at or near the plasma membrane. In this scenario, the level of activities of intracellular GPx and catalase would be far less relevant in preventing cytotoxicity than when hydroperoxide directly damages intracellular targets. In fact, complete inactivation of endogenous catalase by ATZ pretreatment had no effect on DMNQ sensitivity (data not shown), which suggests that the site of redox cycling of DMNQ as well as the target of H2O2 are unlikely to be cytosolic. In contrast, although the physiological relevance of bolus addition of high concentrations of H2O2 is questionable, high H2O2 concentrations throughout the cell are quickly reached, and differences in cytosolic H2O2 scavenging ability may then account for the differences in resistance to cell death.
Caveat for studies using cell lines. Although the A549 cell line has been widely used, it is difficult to elicit consistent cellular responses in this cell line (personal communication from three other laboratories and our experience). It is likely that the ratio of clones such as those described here, which can vary with culture conditions, would contribute to inconsistencies. As with other cell lines from which subclones have been shown to vary in response, so should future studies with A549 cells. In this regard, the D3R and G4S cells, which are now extensively characterized, would be more useful individually in future investigations than would the parental A549 cell line. Nonetheless, although efforts at subcloning are expected to produce a population with genetic homogeneity that will be preserved over certain generations, due to the inherent genetic instability of cancer cells, it is probably best to use them for only several passages.
Although characterization of antioxidants and antioxidant enzyme profiles of two A549 cell subclones have revealed paradoxical inconsistencies between antioxidant enzyme activities and sensitivities to the corresponding oxidants used to induce cell death, the antioxidant enzyme profile alone cannot predict propensity to apoptosis, especially when cells are compared that are dissimilar in so many respects. Thus, although the individual clones may be useful for future studies, comparisons between them are fraught with hazards. ![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Dr. Mutay Aslan for comments on the H2O2 measurement by aminotriazole. We also thank Dr. Victor Darley-Usmar for valuable discussions.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants HL-37556 and ES-05511.
Address for reprint requests and other correspondence: H. J. Forman, Dept. of Environmental Health Sciences, School of Public Health, Univ. of Alabama at Birmingham, RPHB-317, 1530 3rd Ave. South, Birmingham, AL 35294-0022 (E-mail: hforman{at}uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 24, 2002;10.1152/ajplung.00025.2002
Received 18 January 2002; accepted in final form 27 April 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aebi, H.
Catalase in vitro.
Methods Enzymol
105:
121-126,
1984[ISI][Medline].
2.
Alin, P,
Danielson UH,
and
Mannervik B.
4-Hydroxyalk-2-enals are substrates for glutathione transferase.
FEBS Lett
179:
267-270,
1985[ISI][Medline].
3.
Arai, M,
Imai H,
Koumura T,
Yoshida M,
Emoto K,
Umeda M,
Chiba N,
and
Nakagawa Y.
Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells.
J Biol Chem
274:
4924-4933,
1999
4.
Arner, ES,
Zhong L,
and
Holmgren A.
Preparation and assay of mammalian thioredoxin and thioredoxin reductase.
Methods Enzymol
300:
226-239,
1999[ISI][Medline].
5.
Arnold, RS,
Shi J,
Murad E,
Whalen AM,
Sun CQ,
Polavarapu R,
Parthasarathy S,
Petros JA,
and
Lambeth JD.
Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1.
Proc Natl Acad Sci USA
98:
5550-5555,
2001
6.
Bai, J,
Rodriguez AM,
Melendez JA,
and
Cederbaum AI.
Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.
J Biol Chem
274:
26217-26224,
1999
7.
Beauchamp, C,
and
Fridovich I.
Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.
Anal Biochem
44:
276-287,
1971[ISI][Medline].
8.
Bello, RI,
Gomez-Diaz C,
Navarro F,
Alcain FJ,
and
Villalba JM.
Expression of NAD(P)H:quinone oxidoreductase 1 in HeLa cells: role of hydrogen peroxide and growth phase.
J Biol Chem
276:
44379-44384,
2001
9.
Bishop, CT,
Mirza Z,
Crapo JD,
and
Freeman BA.
Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions.
In Vitro Cell Dev Biol
21:
229-236,
1985[ISI][Medline].
10.
Cadenas, E.
Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism.
Biochem Pharmacol
49:
127-140,
1995[ISI][Medline].
11.
Carlberg, I,
and
Mannervik B.
Glutathione reductase.
Methods Enzymol
113:
484-490,
1985[ISI][Medline].
12.
Chandra, J,
Samali A,
and
Orrenius S.
Triggering and modulation of apoptosis by oxidative stress.
Free Radic Biol Med
29:
323-333,
2000[ISI][Medline].
13.
Cotgreave, IA,
and
Gerdes RG.
Recent trends in glutathione biochemistryglutathione-protein interactions: a molecular link between oxidative stress and cell proliferation?
Biochem Biophys Res Commun
242:
1-9,
1998[ISI][Medline].
14.
Croce, MV,
Colussi AG,
Price MR,
and
Segal-Eiras A.
Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549).
Pathol Oncol Res
5:
197-204,
1999[Medline].
15.
Davis, W, Jr,
Ronai Z,
and
Tew KD.
Cellular thiols and reactive oxygen species in drug-induced apoptosis.
J Pharmacol Exp Ther
296:
1-6,
2001
16.
Enger, MD,
Tesmer JG,
Travis GL,
and
Barham SS.
Clonal variation of cadmium response in human tumor cell lines.
Am J Physiol Cell Physiol
250:
C256-C263,
1986
17.
Fariss, MW,
and
Reed DJ.
High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives.
Methods Enzymol
143:
101-109,
1987[ISI][Medline].
18.
Flitter, WD,
and
Mason RP.
The enzymatic reduction of actinomycin D to a free radical species.
Arch Biochem Biophys
267:
632-639,
1988[ISI][Medline].
19.
Flohe, L,
and
Gunzler WA.
Assays of glutathione peroxidase.
Methods Enzymol
105:
114-121,
1984[ISI][Medline].
20.
Forman, HJ,
Shi MM,
Iwamoto T,
Liu RM,
and
Robison TW.
Measurement of -glutamyl transpeptidase and
-glutamylcysteine synthetase activities in cells.
Methods Enzymol
252:
66-71,
1995[ISI][Medline].
21.
Freshney, RI.
Transformation in culture of animal cells. In: Culture of Animal Cells (4th). New York: Wiley-Liss, 2000, p. 269-283.
22.
Giard, DJ,
Aaronson SA,
Todaro GJ,
Arnstein P,
Kersey JH,
Dosik H,
and
Parks WP.
In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.
J Natl Cancer Inst
51:
1417-1423,
1973[ISI][Medline].
23.
Green, DR,
and
Reed JC.
Mitochondria and apoptosis.
Science
281:
1309-1312,
1998
24.
Ikeda, K,
Kajiwara K,
Tanabe E,
Tokumaru S,
Kishida E,
Masuzawa Y,
and
Kojo S.
Involvement of hydrogen peroxide and hydroxyl radical in chemically induced apoptosis of HL-60 cells.
Biochem Pharmacol
57:
1361-1365,
1999[ISI][Medline].
25.
Karp, DR,
Shimooku K,
and
Lipsky PE.
Expression of -glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death.
J Biol Chem
276:
3798-3804,
2001
26.
Kuninaka, S,
Ichinose Y,
Koja K,
and
Toh Y.
Suppression of manganese superoxide dismutase augments sensitivity to radiation, hyperthermia and doxorubicin in colon cancer cell lines by inducing apoptosis.
Br J Cancer
83:
928-934,
2000[ISI][Medline].
27.
Li, N,
Oberley TD,
Oberley LW,
and
Zhong W.
Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: mechanistic studies.
J Cell Physiol
175:
359-369,
1998[ISI][Medline].
28.
Lind, C,
Cadenas E,
Hochstein P,
and
Ernster L.
DT-diaphorase: purification, properties, and function.
Methods Enzymol
186:
287-301,
1990[Medline].
29.
Liu, RM,
Gao L,
Choi J,
and
Forman HJ.
-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal.
Am J Physiol Lung Cell Mol Physiol
275:
L861-L869,
1998
30.
Manna, SK,
Zhang HJ,
Yan T,
Oberley LW,
and
Aggarwal BB.
Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-B and activated protein-1.
J Biol Chem
273:
13245-13254,
1998
31.
Mannervik, B,
and
Guthenberg C.
Glutathione transferase (human placenta).
Methods Enzymol
77:
231-235,
1981[Medline].
32.
McCord, JM,
and
Fridovich I.
Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein).
J Biol Chem
244:
6049-6055,
1969
33.
Nomura, K,
Imai H,
Koumura T,
Arai M,
and
Nakagawa Y.
Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.
J Biol Chem
274:
29294-29302,
1999
34.
O'Donovan, DJ,
Katkin JP,
Tamura T,
Husser R,
Xu X,
Smith CV,
and
Welty SE.
Gene transfer of mitochondrially targeted glutathione reductase protects H441 cells from t-butyl hydroperoxide-induced oxidant stresses.
Am J Respir Cell Mol Biol
20:
256-263,
1999
35.
Pani, G,
Bedogni B,
Anzevino R,
Colavitti R,
Palazzotti B,
Borrello S,
and
Galeotti T.
Deregulated manganese superoxide dismutase expression and resistance to oxidative injury in p53-deficient cells.
Cancer Res
60:
4654-4660,
2000
36.
Poli, G,
and
Schaur RJ.
4-Hydroxynonenal in the pathomechanisms of oxidative stress.
IUBMB Life
50:
315-321,
2000[ISI][Medline].
37.
Royall, JA,
Gwin PD,
Parks DA,
and
Freeman BA.
Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-.
Arch Biochem Biophys
294:
686-694,
1992[ISI][Medline].
39.
Shi, M,
Gozal E,
Choy HA,
and
Forman HJ.
Extracellular glutathione and -glutamyl transpeptidase prevent H2O2-induced injury by 2,3-dimethoxy-1,4-naphthoquinone.
Free Radic Biol Med
15:
57-67,
1993[ISI][Medline].
40.
Srivastava, S,
Chandra A,
Bhatnagar A,
Srivastava SK,
and
Ansari NH.
Lipid peroxidation product, 4-hydroxynonenal and its conjugate with GSH are excellent substrates of bovine lens aldose reductase.
Biochem Biophys Res Commun
217:
741-746,
1995[ISI][Medline].
41.
Sun, IL,
Sun EE,
Crane FL,
Morre DJ,
Lindgren A,
and
Low H.
Requirement for coenzyme Q in plasma membrane electron transport.
Proc Natl Acad Sci USA
89:
11126-11130,
1992[Abstract].
42.
Tian, WN,
Braunstein LD,
Pang J,
Stuhlmeier KM,
Xi QC,
Tian X,
and
Stanton RC.
Importance of glucose-6-phosphate dehydrogenase activity for cell growth.
J Biol Chem
273:
10609-10617,
1998
43.
Verhaegen, S,
McGowan AJ,
Brophy AR,
Fernandes RS,
and
Cotter TG.
Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line.
Biochem Pharmacol
50:
1021-1029,
1995[ISI][Medline].
44.
Wang, GQ,
Gastman BR,
Wieckowski E,
Goldstein LA,
Gambotto A,
Kim TH,
Fang B,
Rabinovitz A,
Yin XM,
and
Rabinowich H.
A role for mitochondrial Bak in apoptotic response to anticancer drugs.
J Biol Chem
276:
34307-34317,
2001
45.
Zhang, P,
Liu B,
Kang SW,
Seo MS,
Rhee SG,
and
Obeid LM.
Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2.
J Biol Chem
272:
30615-30618,
1997