Expression of human catalase in acatalasemic murine SV-B2 cells confers protection from oxidative damage

FreeRadicalBiology&Medicine, Vol. 15, pp. 581-588, 1993 Printed in the USA. All rights reserved.

0891-5849/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Original Contribution EXPRESSION OF HUMAN CATALASE SV-B2 C E L L S C O N F E R S P R O T E C T I O N

IN ACATALASEMIC MURINE FROM OXIDATIVE DAMAGE

BARBARA A. LINDAU-SHEPARD* and JACQUELIN B. SHAFFER ? *Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY, USA; and *The School of Public Health, State University of New York at Albany, Albany, NY, USA

(Received 15 January 1993; Revised 14 April 1993; Accepted 16 April 1993) Abstract--Reactive oxygen species have been implicated in aerobic organisms as causative agents in damage to DNA, proteins, and lipids. Catalase is a major enzyme in the defense against such oxidant damage. To determine whether increased catalase expression confers greater resistance to oxidant stress, a eukaryotic expression vector harboring a human catalase cDNA clone was constructed. Acatalasemic murine fibroblasts were then co-transfected with the catalase expression vector and pSV2-neo, and successfully transfected cells were identified by their ability to grow in the presence of geneticin. Clones that contained integrated copies of the catalase expression vector were identified by Polymerase Chain Reaction (PCR) analysis. Stably transfected geneticin-resistant cell lines that overexpressed catalase in potentially positive cell lines were confirmed by catalase enzyme assays. To examine the physiological relevance of catalase overexpression, cells were exposed to oxidant stresses (hydrogen peroxide and hyperoxia), and survival rates were determined. Results demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. These transfected cell lines will provide important models for further evaluation of the role of catalase in protecting cells against the toxic effects of oxygen-derived free radicals and their derivatives. Keywords--Catalase, Acatalasemic, Transfection, Hyperoxia, H202, Free radicals

mutases (SOD), catalase, and glutathione peroxidase (GPx). The interrelationship of the antioxidant enzyme activities in the defense against specific oxidant injuries has yet to be fully elucidated. Amstad et al. 6 suggested that in mouse epidermal cells, transfectants that overproduced CuZn SOD were hypersensitive to bursts of 02 plus H202 and that CuZn SOD overproduction had to be balanced with an increase of catalase expression to restore protection from oxidant damage. In contrast, overproducers of catalase alone showed increased protection from oxidant injuries relative to the parent cells. Spitz et al. 7,s have shown that Chinese hamster ovary cells became adapted to chronic oxidative stress mediated by H202 and that high levels of H202 tolerance are accompanied by increases in chromosome number. Hamster tracheal epithelial cells of the respiratory track respond to H202 by increasing their catalase expression in a dose-dependent manner. 9 Amstad et al. 6 have reported that catalase transfected mouse epidermal cells with a fourfold increase in catalase

INTRODUCTION

Reactive oxygen species, which include oxyradicals, peroxides, and hydroxyl radicals, have been implicated as causative agents in damage to DNA, proteins, and cell membranes) Such oxidant injuries can lead to carcinogenesis, inflammatory diseases, autoimmune conditions, and cell senescence,z3 The rate of production of reactive oxygen intermediates can be amplified by neutrophil activation, hyperoxia, metabolism of redox active drugs, radiation exposure, and ischemia: ,s Aerobic cells have an impressive system of protection against oxidative damage which is comprised of low-molecular-weight antioxidants and antioxidant enzymes. The major enzymatic defenses against reactive oxygen species are the superoxide dis-

•

Supported by NIH 815-3191E. Address correspondence to: Barbara A. Lindau-Shepard, Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA.

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B.A. LINDAU-SHEPARDand J. B. SHAFFER

activity relative to the parent cells exhibited greater resistance to oxidant bursts generated by xanthine/ xanthine oxidase. The role of constitutively increased catalase activity in rescuing acatalasemic cells from oxidative damage induced by high levels of H202 has not yet been reported. Similarly, it has not been established whether upregulated catalase expression has any effect in protecting cells from the deleterious effects of elevated levels of oxygen. In this study we examined the role of catalase in mammalian cells exposed to oxidants generated extracellularly and intracellularly. We constructed a mammalian expression vector for the purpose of overproducing human catalase (hCAS) cDNA in acatalasemic murine SV-B2 cells by stable transfection. These cells were compared to the parent cell strain to evaluate the relative sensitivity to oxidative damage induced by H202 and by hyperoxia. Our results demonstrate that overproduction of catalase in SV-B2 cells enhances resistance to oxidative injuries mediated by H202 and hyperoxia. MATERIALS AND METHODS

Construction of expression vectors pBL-proA. A 1.4-Kbp PstI-PstI SV40 polyA fragment was isolated from pSV2-neo and cloned into the PstI site of pUC-18. A unique PstI site was created by deleting the PstI site at the 3' end of the polyA fragment through partial digestion with PstI, and blunt ends were produced by T4 DNA polymerase, prior to religation. A 619-bp fragment, consisting of the immediate early enhancer and promoter sequences of cytomegalovirus (CMV), was removed from pCMV-cat by restriction digestion with XbaI and PstI. This regulatory region was then ligated into the pUC- 18 multiple cloning site, 5' to the SV40 polyA sequences. The plasmid pCMV-cat was generously provided by Dr. H. Hofstetter. ~° Genes of interest were inserted into the PstI site, 3' to the promoter and 5' to the polyA signals. HindlII and XbaI cloning sites were created by first deleting the remaining multiple cloning sites of pUC-18 by restriction digestion and filling in the staggered ends with T4 DNA polymerase prior to ligation. PstI, XbaI, and HindIII adaptor sequences were designed and inserted into the PstI cloning site.

pBL-hcas2.3. A PstI-PstI 2.3-Kbp cDNA clone containing the entire coding region of human catalase, including its endogenous ATG nucleotides, 30-bp 5' noncoding sequences, and 650-bp 3' noncoding sequences containing the endogenous polyadenylation signal (AATAAA), was isolated from pCat 10.2,

which was provided by Dr. Robert Korneluk. ~~,~2This human catalase cDNA was then inserted into the Pstl site of pBL-proA. All enzymes were purchased from Boerhringer Mannheim.

Growth and transfection of cells SV-B2 murine fibroblasts deficient in catalase activity, obtained from Dr. William Lewis, ~3 were plated at a density of 3 × 10 6 cells/100 mm culture dish in Eagle's Special medium supplemented with 5% fetal bovine serum. After a 24-h incubation period, the cells were transfected by the calcium phosphate precipitate method of Graham and van der EbJ 4 The catalase expression vector pBL-hcas2.3 (15 ug) was co-transfected with 5 ~zg of pSV2-neo, which contains the neoR gene, providing a dominant selectable marker. Control clones were generated by transfecting the SV-B2 cells with pSV2-neo. Approximately 48 h after transfection, the cells were trypsinized and replated at a 1:5 dilution. The antibiotic Geneticin (G418) (Gibco, Baltimore, MD) was added (800 tsg/ml) the following day. G418-resistant colonies were isolated approximately 2 weeks posttransfection by trypsinization in cloning cylinders. Selected clones were grown to mass culture for further analysis.

Determination of catalase activity Catalase activity was determined according to the method of Aebi,t 5which involves the spectophotometric monitoring at 240 nm of the disappearance of t 0 mM H202 in the presence of cell homogenates. Cells were lysed in hypotonic buffer, sonicated for four 15-s bursts, and centrifuged (4°C) at 10,000 × g for 10 min. The supernatants were assayed for total protein concentration according to the bicinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL).

H202 treatment

H202 concentration was determined as described by Spitz et al. 7 Cells were plated at a concentration of 2 × l03 cells/cm 2. When the cell density had doubled 24 h later, the cells were treated with H202 ( 10 umol/l X l07 cells, in medium without phenol red) for 1 h. Following this treatment, cells were washed three times with Dulbecco's phosphate-buffered saline, trypsinized, counted by Coulter counter, and plated for colony formation at a density of I × l03 cells per T-75 flask. After 8 to 9 d colonies were fixed, stained

Human catalase in acatalasemic cells

with crystal violet, and counted using an automated colony counter (New Brunswick Scientific Co. Inc.).

Hyperoxia treatment Cells were plated at a concentration of 2 x l 0 3 cells/cm 2 into 60-mm culture dishes. Two days later, at cell concentrations of 1.6 x l05 cells/plate, cells were exposed to 95% 02/5% CO2 in an oxygen chamber (Bellco Glass, Inc., Vineland, N J) for 24 h. Cells were trypsinized, counted by Coulter counter, and plated for colony formation at a density of 250 to 500 cells per 60-mm plate. After 8 to 9 d colonies were fixed, stained with crystal violet, and counted using an automated colony counter (New Brunswick Scientific Co. Inc.).

RNA isolation and northern blots Total RNA was isolated from cells using the procedure of Chomczynski and Sacchi.16 A total of 30 #g of total RNA was electrophoresed on a 1.2% agarose, formaldehyde-formamide gel with a 20-mM MOPS running buffer. Gels were stained with ethidium bromide and photographed for reference to determine relative RNA sizes by comparing to the 28S and 18S rRNA bands. RNA was transferred to nitrocellulose according to Maniatis et al. ~7 Purified 3' noncoding human catalase cDNA was labeled with [a32P] dATP by using a random primed DNA labeling kit (Boehringer Mannheim). Hybridization was performed according to Maniatis et al. 17 Autoradiography was performed by exposing the hybridized nitrocellulose filter to Kodak XAR-2 film using intensifying screens at - 7 0 ° C for 3 to 6 d.

Polymerase chain reaction and southern blot Genomic DNA was extracted from cells by the method of Reymond. Is Oligonucleotides for PCR were generated by an Applied Biosystems (Foster City, OH) Model 381A DNA synthesizer. To generate DNA fragments that covered the entire 2.3-Kbp hCAS cDNA in pBL-hcas2.3, 5' oligonucleotide primer sequences were based on CMV promoter sequences and 3' oligonucleotide primer sequences were based on SV polyA sequences. DNA amplification was performed according to instructions provided by Perkin Elmer-Cetus GeneAmp DNA Amplification Reagent Kit (Perkin Elmer Corp., Norwalk, CT) with

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30 ~g ofgenomic DNA per 100 ul reaction mix, in a Perkin Elmer-Cetus DNA Thermal Cycler instrument under the following conditions: denature 94°C --1 min; anneal 45°C--2 min; extend 72°Cm2 rain (25 cycles total). A total of 10 #1 of PCR-generated DNA was electrophoresed on a 1% agarose gel with TBE running buffer. DNA was transferred from gels to nitrocellulose according to Maniatis et a1.17Hybridizations and subsequent autoradiographies were performed with 3' noncoding human catalase cDNA probes as described previously for Northern blots, with the exception that films were developed after an exposure time of 6 t o 18h.

Analysis of CuZn SOD, Mn SOD, and GPx activity Cells were harvested and lysates prepared as described above. Samples were electrophoresed in 10% polyacrylamide. Sodium decyl sulfate (SDS) was omitted from the gel and running buffer, and the samples were not boiled prior to electrophoresis. Staining for SOD enzymatic activity was performed according to Beauchamp and Fridovich. 19The gel was soaked in 2.5 mM nitroblue tetrazolium for 20 min and then in a solution of 45 mM potassium phosphate (pH 7.0)/ 0.028 mM riboflavin/30 mM TEMED for 20 min. The gel was illuminated until it became blue, except at the achromatic positions of SOD activity, and was then photographed. GPx was assayed according to the procedure described by Gunzler and Flohr. 2° RESULTS

Transfection of mouse acatalasemic fibroblast cells SV-B2 with hCAS A cDNA clone encoding human catalase was used to construct a mammalian expression vector, pBLhcas2.3, with control elements consisting of the immediate early CMV promoter region and a 1.4-Kbp SV40 polyA fragment, shown schematically in Fig. I. SV-B2 cells were co-transfected with pBL-hcas2.3 and pSV2-neo by the calcium phosphate precipitation method. Transfectants were selected by their ability to survive and form colonies in the presence of the antibiotic G418. Twenty-five colonies were selected, grown in mass culture, and examined for catalase activity and for the presence of integrated intact hCAS cDNA. For this purpose, specific primers to pBL-hcas2.3, 5' and 3' to the hCAS cDNA, corresponding to se-

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Fig. 1. Construction of the generic mammalian expression vector pBL-proA and the human catalase expression vector pBLhcas2.3. A 1.4-Kbp Pstl to Pstl SV40 polyA fragment was isolated from pSV2-neo and cloned into the Pstl site ofpUC-18. A unique Pstl site was created by deleting the Pstl at the 3' end of the polyA fragment through partial digestion with Pstl, and blunt ends were produced by T4 DNA polymerase, prior to ligation. A 619-bp fragment consisting of the immediate early promoter sequences of cytomegalovirus (CMV) was removed by digestion with Xbal and Pstl from pCMV-cat. This regulatory region was then ligated into the pUC- 18 multiple cloning site, 5' to the SV40 polyA sequence. In addition to the Pstl cloning site, HindIII and Xbal sites were created by first deleting the remaining multiple cloning sites of pUC-l 8 and then inserting adaptor nucleotides containing Xbal and HindIli into the PstI site. Construction of the pBL-hcas2.3 human catalase expression vector: A Pstl to Pstl 2.3-Kbp cDNA clone containing the entire coding region of human catalase--including its endogenous ATG, 30-bp 5' noncoding sequence, and 650-bp 3' noncoding sequences containing the endogenous polyA signal--was isolated from pCAT 10.2. This human catalase cDNA was then inserted into the Pstl site of pBL-proA.

Expression of hCAS in the transJected SV-B2 derived cell strains

q u e n c e s in the C M V p r o m o t e r a n d SV40 p o l y A region, were synthesized. T h e s e p r i m e r s were used to a m p l i f y the h C A S c l o n e f r o m the g e n o m i c D N A o f a selected n u m b e r o f t r a n s f e c t e d cell strains b y t h e p o l y m e r a s e c h a i n reaction. T h e entire 2 . 3 - K b p h C A S c D N A was a m p l i f i e d f r o m the m u r i n e b a c k g r o u n d , which confirmed the integration of the intact hCAS c D N A i n t o the cell s t r a i n ' s g e n o m e (Fig. 2). R e p r e s e n tative t r a n s f e c t e d strains were c h o s e n for m o l e c u l a r a n d p h y s i o l o g i c a l analysis. C o n t r o l cell strain B l - n e o was o b t a i n e d b y t r a n s f e c t i n g SV-B2 cells with pSV2-neo.

T o t a l c e l l u l a r R N A was isolated f r o m several t r a n s fectants. E x p r e s s i o n o f r e c o m b i n a n t h C A S in SV-B2 t r a n s f e c t a n t s was c o n f i r m e d b y N o r t h e r n b l o t a n a l y sis. T o t a l c e l l u l a r R N A was i s o l a t e d f r o m several transfectants. T w o different p r o b e s were used to detect catalase R N A : a full-length m o u s e catalase c D N A , a n d a 6 5 0 - b p 3' u n t r a n s l a t e d region isolated f r o m h C A S c D N A . T h e r e is a great a m o u n t o f h o m o l ogy b e t w e e n t h e c o d i n g region o f m o u s e a n d h u m a n co

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Fig. 2. Southern blot analysis of amplified hCAS cDNA from transfected cells. Cell strains were examined for the presence of integrated hCAS cDNA. Lanes 4, 5, and 6: negative controls. Lane l 1: catalase expression vector as positive control. Lanes l, 2, 3, 7, 8, 9, and 10: strains co-transfected wi_thpSV2-neo and pBL-hcas2.3, selected on the basis of their resistance the G418.

Human catalase in acatalasemic cells

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Fig. 3. Northern blot analysis of total RNA extracts from stable transfectants. The locations of the 18s and 28s ribosomal RNAs are shown in the figure. (A) RNAs were probed with a 32p-labelledfull-length mouse catalase cDNA. Lanes: 1, 2, 3, 6, and 7:30 /~gtotal RNA from transfectants. Lane 4:30/Lg total RNA from B l-neo control. Lane 5:60 #g total RNA from B 1-neo control. (B) RNAs were probed with a 32p-labelled650-bp 3' noncoding hCAS cDNA fragment. Lanes: 1,2, 5, 6, and 7:30/zg total RNA from transfectants. Lane 3:30 #g total RNA from Bl-neo control. Lane 4:60 ug total RNA from Bl-neo control. catalase; however, the 3' n o n c o d i n g region o f these species have n o homology. S a m p l e s o f a p p r o x i m a t e l y 30 #g total cellular R N A f r o m several transfectants, a n d 30 #g a n d 60 #g samples from the c o n t r o l B 1-neo, were electrophoresed o n f o r m a l d e h y d e - f o r m a m i d e gels a n d transferred to nitrocellulose. T h e h u m a n catalase R N A from the transfectants h y b r i d i z e d with the full-length m o u s e catalase c D N A probe as well as with the m o u s e catalase R N A o f the c o n t r o l B 1-neo. However, the m o u s e catalase R N A signal was very low; at 30 #g o f R N A n o catalase R N A signal was visible a n d 60 #g R N A was necessary to detect the catalase R N A signal (Fig. 3A). T h e p r o b e consisting o f the 3' u n translated hCAS c D N A did n o t a p p e a r to hybridize with m o u s e R N A o f the B l - n e o control. E v e n at the

Table 1. Catalase Activity in Transfected Cell Strains

c o n c e n t r a t i o n o f 60 #g R N A n o signal was observed. However, this p r o b e hybridized efficiently with the catalase R N A o f the transfectants, i n d i c a t i n g that the i d e n t i t y o f this catalase R N A is a n h C A S c D N A t r a n script (Fig. 3B).

Increased catalase activity associated with recombinant hCAS expression T a b l e 1 s u m m a r i z e s the e n z y m a t i c data o b t a i n e d o n catalase activity for selected transfectants. C o m pared to the c o n t r o l strain B l - n e o , the catalase activity o f the five e x p e r i m e n t a l strains was a p p r o x i m a t e l y 2.8- to 4-fold higher. T h e r e were n o detectable changes in C u Z n S O D a n d M n S O D activities i n a n y o f the selected cell strains. T h e activity o f g l u t a t h i o n e peroxidase also r e m a i n e d u n c h a n g e d for all the cell strains tested (data n o t shown).

Catalase Activity Cell Strain

Units/mg Proteinb

Relative Increase in Activity

n=

Bl-neo BLS 3 BLS 6 BLS 7 BLS 15 BLS 19

0.46 _+0.12 1.63 + 0.46 1.62 _+0.23 1.32 + 0.15 1.80 _+0.19 1.90 _+0.16

1.00c* 3.54 3.52 2.87 3.91 4.13

6 4 4 4 4 4

Cells were grown in monolayer cultures and protein was extracted when density was approximately at 80% confluency. The protein concentration of the cell extracts was measured and appropriate volumes of extracts were added at room temperature to an assay mixture of phosphate buffer (pH 6.8) containing l0 mM H202. The rate of decrease in absorbance at 240 nm was measured in the first minute of incubation. The units are defined as the number of micromoles of H202 consumed per minute per mg protein. a n = number of independent assays. b Mean + standard error of the mean of n samples each measured 4 times. ¢The control cell (Bl-neo) activity is arbitrarily selected to be equal to 1.00. * All experiments are statistically different from control.

Table 2. H202and Hyperoxia Resistance in hCAS Transfected Cell Strains Oxidant Challenges Relative Colony-Forming Ability~ Cell Strain

Hyperoxia

H202

Bl-neo BLS 3 BLS 6 BLS 7 BLS 15 BLS 19

1.00 2.40 + 0.15b 4.00 _+0.53 1.50 _+0.34 4.30 + 0.96 4.30 + 0.75

5.10 _+ 1.37c 4.20 _+ 1.13 2.20 _+0.35 5.00 + 0.98 5.60 + 0.88

1.00

=Relative to the number of colonies in the control B l.neo arbitrarily assigned the number 1.00. b Mean + standard error of the mean of the ratio of each cell strain to the control B l-neo for 4 independent assays each done in triplicate. c Mean + standard error of the mean of the ratio of each cell strain to the control B l-neo for 5 independent assays each done in duplicate.

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B . A . L1NDAU-SHEPARD a n d J. B. SHAFFER

Effects of hCAS expression on cell survival and growth properties in response to oxidative stresses Cell doubling time for the five experimental strains and the control strain B l-neo was calculated prior to oxidant treatment and found to be approximately 17 h for all strains except for strain BLS3, for which the doubling time was 19 h. Table 2 shows the results of clonogenic survival experiments in which five strains overexpressing catalase were exposed to 10 umol H202/107 cells for 1 h at 37 °C. Comparing each strain with the control strain B l-neo, elonogenic survival ranges from about 2.2-fold to 5.6-fold greater for the catalase transfectants. These data were collected by counting all visible colonies, ranging from 1 mm to 4 mm in diameter. However, there was a notable difference in the density of the colonies: B 1-neo and BLS7 cell strains had colonies ranging from 1 mm to 2 mm in diameter with 50 to 150 cells per colony; whereas the majority ofBLS3, BLS6, BLS15, and BLSI9 colonies were approximately 3 mm in diameter and formed very dark crystal violet strains. These colonies, when viewed under the microscope, were composed of densely packed cells too numerous to count. These results indicate that a quantitative, as well as qualitative, increase in colony-forming ability correlated with increased catalase activity in catalase transfectants. The five experimental cell strains and the control strain Bl-neo were exposed to 95% oxygen for 24 h before colony plating. Table 2 shows the clonogenic survival results. The differences in survival ability between the experimental strains are signifcant (P < .05). BLS7 had a 1.5-fold greater colony forming ability than B 1-neo, the lowest among the five cell strains tested. BLS7 also exhibited the lowest level of catalase activity. The data were collected by counting visible colonies. There was a noticeable difference in size and density of colonies between the cell strains, with strains B l-neo and BLS7 having significantly smaller and less dense colonies than strains BLS3, BLS6, BLS 15, and BLS 19. When the transfectants were challenged with H202, a relationship between clonogenic survival and catalase activity was detected (Fig. 4). A similar relationship was noted when these cells were challenged with 95% 02 (Fig. 5). When compared to SV-B2 and B l-neo, the mouse fibroblasts with normal catalase activity showed a 4.0-fold higher resistance to H202 and a 3.6-fold higher resistance to hyperoxia. DISCUSSION

Catalase and glutathione peroxidase are the major enzymes responsible for the enzymatic decomposi-

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tion of H202. Both enzymes have been shown to be equally involved in the detoxification of H2O2 in human erythrocytes, although catalase accounts for more than half of the removal of H20 2 when H202 is generated at high rates? 1 Studies described in this communication have focused on the protective effects of catalase against oxidant damage in SV-B2, a fibroblast cell line established from the homozygous C3H/Cs b acatalasemic mouse 22 by W. Lewis.13 When compared to the wild type cells, these cells displayed approximately 70% less catalase activity and were markedly more sensitive to the toxicity of H202. The catalase gene mutation of the C3H/Cs b mouse strain was shown to involve a single nucleotide transversion, from G -~ T, located in the third position of amino acid 11 in the catalase monomer, changing the codon for glutamine to histidine.23 Transfection of SV-B2 cells with the catalase expression vector pBL-hcas2.3 resulted in the isolation of several clonally derived cell strains which expressed elevated levels of enzymatically active catalase. This catalase activity was approximately 2.8- to 4.0-fold

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greater than the parent cells' or the control transfectants' activity. Southern blot analysis of PCR-amplifled hCAS cDNA from the genomic DNA extracted from selected transfectants confirmed the integration of the full-length hCAS clone. In addition, BLS2, a G418-resistant strain with catalase activity comparable to that of the control cells, revealed no evidence of hCAS cDNA in its genome. From Northern blot analysis it is evident that the hCAS cDNA is transcribed in the transfected strains. The stable integration ofhCAS cDNA and the transcription of its message correlate with increased enzymatically active catalase in the transfected cell strains. The physiological relevance ofupregulation ofcatalase in SV-B2 derived cell strains has been evaluated by subjecting five transfectants with increased catalase activity to cytotoxic levels of extracellularly applied (H202) and intracellular (95% 02) generators of oxidative stress, n202 crosses biological membranes easily; previous data suggest that H 2 0 2 is a major oxidant leading to damage to macromolecules, including DNA single-strand breakage. 6 We subjected five catalase overexpressing transfectants to cytotoxic levels of H 2 0 2 and evaluated cell viability by clonogenic sur-

587

vival. The determination of the proliferative capacity of cells, as defined by their colony-forming ability, is an accurate cell survival test, because in this assay cells are scored as viable if they divide to form macroscopic colonies. The results of this experiment demonstrated a significant resistance to HEO2-induced oxidant stress in cells overexpressing catalase. BLS7, which had the lowest catalase activity of the five transfectants, exhibited a lower survival rate when compared to the other transfectants. Hyperoxia increases significantly the rate of intracellular generation of H202 .24,25 The consequences of upregulation of a specific antioxidant enzyme in cells exposed to hyperoxia have been examined. We subjected the five catalase transfectants to 95% O2. The results of these experiments indicated that cell strains exhibiting higher levels of catalase activity were more competent to survive and form colonies than strains with lower catalase activity. The lowest catalase overexpressing strain (BLS7) also had the lowest colonyforming ability. The results ofH202 and hyperoxia challenges demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. Additionally, these results suggest a correlation between degrees of resistance to H202-induced injuries and catalase activity. However, resistance to H202 and hyperoxia do not correlate completely. Different mechanisms are involved in the oxidant stresses produced by H202 and hyperoxia. The mitochondrion is a major target for hyperoxia-mediated toxicity. 26 When H 2 0 2 is administered directly, it diffuses readily across cell membranes thus damaging numerous cellular components. Judging from SOD activity gels, no significant change in SOD activity was observed between the control strain and the catalase transfected strains. Although the quantitative accuracy of activity gels is not as precise as more sensitive methods such as spectrophotometry, significant increases in activity can be visualized. Similarly, enzymatic activity of GPx was identical in the control strain and all catalase transfected strains. This strongly suggests that, under our experimental conditions, catalase is the major enzyme involved in the rescue of the transfected cells from oxidant damage. In our study we found that moderate increases of catalase activity confer protection from oxidant stress induced by hyperoxia and H 2 0 2 , which is an indication that these treatments resulted in high levels of intracellular H 2 0 2 . Our results suggest that catalase detoxifies both H 2 0 2 of extracellular and intracellular origin in the SV-B2 derived cell strains. The expression of human catalase in mouse cells afforded us

588

B.A. LINDAU-SHEPARDand J. B. SHAFFER

with the advantage to detect the cross-species catalase message by using a human catalase specific probe. Acatalasemic cells were effectively rescued from oxidant damage by restoring their catalase activity. REFERENCES

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