oxidative stress induces differential gene expression in a ... m urea gel using the genomy lxr dna...

Oxidative Stress Induces Differential Gene Expressionin a Human Lens Epithelial Cell Line

Deborah A. Carper,1 Jennifer K. Sun,l Takeshi Iwata,l J. Samuel Zigler, Jr,1

Nobuhiro Jbaraki,2 Li-Ren Lin,5 and Venkat Reddy5

PURPOSE. TO identify differentially expressed genes in a human lens epithelial cell line exposed tooxidative stress.

METHODS. Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was usedto evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cellswere exposed for 3 hours to a single bolus of 200 JULM hydrogen peroxide. Differentially expressedgenes were identified through DNA sequencing and a nucleotide database search. Differentialexpression was confirmed by northern blot and RT-PCR analyses.

RESULTS. Using 18 primer sets, 28 RT-PCR products were differentially expressed between controland hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamideadenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and /3-hydroxyisobutyryl-coenzyme Ahydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold asearly as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b,cytochrome oxidase III, and 16S rRNA.

CONCLUSIONS. Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and pep tide process-ing. These early changes may reflect pathways involved in the defense, pathology, or both of thelens epithelium, which is exposed to oxidative stress throughout life. (Invest Ophthalmol Vis Sci.1999:40:400-406)

Oxidative stress is believed to be an important contrib-uting factor in maturity-onset cataract.1'2 Reactive ox-ygen species (ROS) such as hydrogen peroxide

CH2O2), superoxide anion, singlet oxygen, and the hydroxylradical are postulated to contribute to this process. High con-centrations of H2O2 have been reported in human cataractsand in the corresponding aqueous humor of the eye.2'3 In theorgan-cultured lens, the presence of ROS causes a number ofbiochemical changes, which lead to an increase in water-insoluble lens proteins and the appearance of lens opacity.4"8

The lens epithelium is especially vulnerable to oxidativestress.9"11 Damage to this single layer of cuboidal epithelialcells on the anterior surface of the lens can precede andcontribute to lens opacification. For example, generation ofROS in a cultured rat lens system led to irreversible damage inthe epithelial cell layer.9 Cellular and mitochondrial swelling,DNA fragmentation, and loss of cell viability were observed

From the 'Laboratory of Mechanisms of Ocular Diseases, NationalEye Institute, National Institutes of Health, Bethesda, Maryland; thedepartment of Ophthalmology, Nippon Medical School 1-1-5 Sendagi,Bunkyo-ku, Tokyo, Japan; and the 3Eye Research Institute, OaklandUniversity, Rochester, Michigan.

Submitted for publication June 24, 1998; revised September 11,1998; accepted October 1, 1998.

Proprietary interest category: N.Reprint requests: Deborah A. Carper, Building 6, Room 232, Na-

tional Eye Institute, National Institutes of Health, 9000 Rockville Pike,Bethesda, MD 20892.

before cataract formation. These data emphasize the fact thatthe lens epithelium plays a crucial role in lens transparency.

Generation of ROS can cause deleterious peroxidation oflipids, modification of proteins, and cleavage of DNA.12 How-ever, at low concentrations, ROS, which are generated asby-products of normal mitochondrial electron transport, canserve a valuable function in cell signaling.13"16 For example, anincrease in intracellular ROS via the increased expression ofthe GTP-binding protein racl leads to activation of the tran-scription factor nuclear factor-KB.15 In prokaryotes, H2O2 actsas a second messenger activating OxyR protein, a transcrip-tional activator of many antioxidant genes, including hydroper-oxidase I and glutathione reductase.17

The mRNA differential display (DD) technique developedby Liang and Pardee18 permits simultaneous identification ofupregulated and downregulated genes between two groups ofcells, tissues, or conditions. This comparative method usessmall amounts of RNA and is based on gel electrophoreticanalysis of subpopulations of cDNAs produced by reverse tran-scriptase-polymerase chain reaction (RT-PCR) using 3' an-chored oligo (dT) primers and short arbitrary 5' primers. Foreach primer set, cDNAs with differing signal intensities be-tween experimental conditions can be isolated and furthercharacterized. We have used this powerful method to comparethe changes in gene expression between control and oxida-tively stressed human lens epithelial cells. The goals of thisstudy were to identify early changes in gene expression thatare associated with oxidative damage in lens epithelial cells

400Investigative Ophthalmology & Visual Science, February 1999, Vol. 40, No. 2Copyright © Association for Research in Vision and Ophthalmology

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IOVS, February 1999, Vol. 40, No. 2 Differential Gene Expression in a Human Lens Cell line 401

and also to evaluate functional systems that may be involved incell signaling and defense. We have identified several geneswith altered expression in stressed cells that are involved insignaling and posttranslational processing. In addition, we ob-served decreased expression of genes for specific mitochon-drial enzymes involved in electron transport.

METHODS

Cell Culture and Isolation of RNACells from the SRA 01-04 human lens epithelial cell line19 weregrown in Dulbecco's modified Eagle's medium (DMEM; GIB-CO-BRL Life Technologies, Gaithersburg, MD) supplementedwith 20% fetal bovine serum (GIBCO-BRL) and 0.05 mg/mlgentamicin (GIBCO-BRL) in a 5% CO2 37°C incubator. Thesecells attach to the surface of the plastic flask and appearcuboidal in shape when confluent. The viable cells werecounted using trypan blue stain (GIBCO-BRL), and equal ali-quots were placed into 75-cm2 flasks. Once the cells reachedapproximately 95% confluence (2 X 106 cells/75-cm2 flask),the cells were washed with DMEM that did not contain sup-plements. A 10-ml volume of DMEM, containing either water or200 fxM H2O2 (Mallinckrodt, Paris, KY), and not containing anyother supplements, was then added to each 75-cm2 flask. Thecells were incubated for an additional 3 hours for the DD or for

0 to 24 hours for the time course experiments. Flasks werethen washed twice with 1X PBS (GIBCO-BRL), and the cellswere harvested by lysis with 4 ml RNA denaturing solution(guanidinium thiocyanate; Stratagene, La Jolla, CA). The num-ber of attached viable control or H2O2-treated cells did notchange (2 X 106 cells/75-cm2 flask by trypan blue exclusion)after 3 hours of treatment but was reduced to 85% of theoriginal cell number by 24 hours. The RNA was processedusing the Stratagene RNA Isolation Kit (Stratagene) followed byRNase-free DNasel treatment (Boehringer-Mannheim, India-napolis, IN). The quality of the RNA was evaluated by agarosegel electrophoresis.

Differential Display AnalysisRNA populations from control and H2O2-treated cells werecompared using the Differential Display Hieroglyph mRNAProfile Kit (Genomyx, Foster City, CA). Eighteen primer setswere used, which represents approximately 10% to 20% of themore than 10,000 genes expressed in a cell. Anchor primers 1and 2 were combined with arbitrary primers 1, 2, and 4.Anchor primers 8, 9, and 10 were used with arbitrary primers1 through 4. First-strand synthesis of cDNA was performedwith 200 ng RNA, 0.2 ju,M anchor primer (Genomyx), 20 URNasin (Promega, Madison, WI), dNTP mix (25 JLIM each,Genomyx), 10 mM dithiothreitol, 40 U Superscript II RT En-zyme, and Superscript II RT Buffer (the last three were fromGIBCO-BRL). After first-strand synthesis, PCR was performedin duplicate for each condition using the Hieroglyph kit andprotocol (Genomyx), the AmpliTaq enzyme (Perkin-Elmer,Foster City, CA), and [a-33P]dATP (DuPont-NEN, Wilmington,DE) in a Perkin-Elmer GeneAmp 9600 thermocycler (Perkin-Elmer) as described previously.20 Radiolabeled PCR productswere electrophoretically separated on a 4.5% polyacrylamide 8M urea gel using the Genomyx LR DNA Sequencer, which canseparate products up to 2 kb in length. Differential display-

PCR gel bands were visualized using X-Omat film (Kodak,Rochester, NY).

Reamplification, Cloning, Sequencing, andIdentification of DD Bands

Differentially expressed DD bands were excised from the poly-acrylamide gel and reamplified using the same primers andprotocol (Hieroglyph kit; Genomyx) as in the original PCR, butwithout radiolabeled dATP, as previously described.20 To ver-ify DNA amplification, the PCR products were purified byWizard PCR preparation (Promega, Madison, WI) and analyzedby agarose gel electrophoresis.

Reamplified bands were directly sequenced using the Per-kin-Elmer 310 fluorescent sequencer and the DNA FS sequenc-ing kit and protocol (Perkin-Elmer). The Ml3 universal reverseprimer provided by the Genomyx system was used to initiatesequencing. Bands that contained more than one PCR product,as evidenced by overlapping DNA sequences, were cloned intothe TOPO TA cloning vector Gnvitrogen, Carlsbad, CA). Plas-mid preparations (Wizard Plasmid Preparation; Promega) ofthe cloned DNA were then sequenced using a primer, synthe-sized on a DNA synthesizer (Applied Biosystems, Foster City,CA), specific for the Sp6 site in the cloning vector. Homologysearches of nucleotide databases were performed through theNational Center for Biotechnology Information using theBLAST network service (National Library of Medicine, Be-thesda, MD).

Confirmation of Expressed Gene ChangesNorthern blot analysis and RT-PCR were used to verify dieexpressed gene changes observed with the DD gels. For north-ern blot analysis, 3 ju,g RNA from the original DD experiment or5 jag or 10 jitg from two subsequent experiments was run in astandard formaldehyde agarose gel.21 After the gel was blottedwith Biotrans nitrocellulose according to protocol GCN, CostaMesa, CA), the blot was probed with 32P-radiolabeled directDD bands or subcloned DNA. To generate a probe from thesubcloned DNA, primers specific for each target gene weresynthesized (Applied Biosystems) and used to produce specificPCR products (260 bp to 430 bp) using AmpliTaq enzyme(Perkin-Elmer). These products were subsequently separatedby agarose gel electrophoresis, extracted from the gels (QiagenGel Extraction Kit; Qiagen, Chatsworth, CA), and sequenced toverify the correct target gene. An 18S rRNA probe (Ambion,Austin, TX) was used to normalize for variations in RNA load-ing. Radioactive labeling was carried out using a randomprimer kit (GIBCO-BRL) and [a32P]dCTP (DuPont). After stan-dard hybridization and wash protocols at 42°C GCN), the blotwas exposed to X-Omat film (Kodak) and also to fluorescentdetection and quantitation using Storm and ImageQuaNT soft-ware (Molecular Dynamics, Sunnyvale, CA). To evaluate ex-pressed gene changes by RT-PCR, target genes were comparedbetween the two cell conditions using 300 ng RNA pretreatedwith DNasel (Boehringer-Mannheim), 200 nM specific geneprimers generated on a DNA synthesizer (Applied Biosystems),and the One Step RT-PCR system and protocol (GIBCO-BRL).GAPDH was used as a housekeeping enzyme normalizationcontrol. Controls lacking reverse transcriptase (heat-inactivat-ed) were performed under identical conditions. PCR was car-ried out with a single set of specific gene primers or in com-bination with GAPDH. PCR products were verified by size


402 Carper et al. IOVS, February 1999, Vol. 40, No. 2

Control Hydrogen Peroxide

FIGURE 1. Micrographs of human lens epithelial cell line SRA 01-04. Cells were incubated for 3 hours inDMEM without (Control) or with 200 ;u,M H2O2 (Hydrogen Peroxide).

determination on agarose gels and by nucleotide sequencing.For comparative analysis, these products were run on a 1.0%agarose gel, visualized by ethidium bromide-staining, andquantified using the Kodak digital camera and BioMax IDImage Analysis Software (Kodak). One Step RT-PCR (GIBCO-BRL) was also used to generate the cytochrome oxidase III PCRproduct used as a probe on northern blot analysis. The probewas verified by DNA sequencing.

RESULTS AND DISCUSSION

Human lens epithelial cells from the immortalized SRA 01-04cell line were incubated in DMEM without bovine serum albu-min or antibiotics for 3 hours in the presence or absence of asingle bolus of 200 juM H2O2. These conditions were chosenon the basis of previous findings that these parameters elicitmorphologic and biochemical changes in cultured human lensepithelial cells.22 In addition, it has been shown that H2O2

levels can be quite high, varying from 10 ixM to 660 /xM in theaqueous humor of cataract patients.23 However, with othermethods of measurement, H2O2 levels have been reported tobe less than 10 p,M.2? For this reason, a recent study hasattempted to address this issue by investigating the ability ofaqueous humor (bovine) to generate high concentrations ofH2O2.

2ii Levels of H2O2 were found to be in the range (^lOO/xM) that causes cataracts in organ culture.

By light microscopy, the morphology of the H2O2-ex-posed cells appeared unchanged during the 3-hour treatmentperiod used for DD analysis (Fig. 1). No change in the numberof attached viable cells, as determined by trypan blue exclu-sion, was observed at this time point, and the yield of RNAfrom the control and H2O2 conditions was approximatelyequal, being approximately 25 t̂g total RNA per flask.

Differential display analysis indicated a number of differ-ences in gene expression between control and H2O2-treated

cells. Using 18 primer sets, 28 DD bands, from 500 bp to 1.4 kbin length, were observed to be either upregulated (n = 10) ordownregulated in - 18) in the H2O2 treatment group (Fig. 2).Fourteen of die 28 differentially expressed bands, those show-ing the strongest intensities and best resolutions, were ream-plified and sequenced. Analysis using nucleotide databasesearch identified 10 of the 14 genes. For example, the banddesignated by the top arrow in Figure 2 was glutamine cyclo-transferase by BLAST search. Of the genes identified by directsequencing (>96% match), 2 were mitochondrial cytochromeb, 3 were mitochondrial 16S rRNA, and 2 were cytoplasmicglycoprotein 130, a receptor component for interleukin-6. Thisemphasizes the fact that arbitrary primers, being only 10 bp inlength, can prime at several sites within a given RNA species,resulting in redundancy of cDNA products. In addition, rRNAs,which lack long polyA tails, nevertheless can be reverse-tran-scribed, as has been reported in previous DD studies.12 Otherbands identified by direct sequencing were ribosomal proteinS10 and mitochondrial nicotinamide adenine dinucleotide(NADH) dehydrogenase subunit 4. Four of the DD bands gavemultiple signals on nucleotide sequencing, suggesting that twoor more expressed genes were contained in the excised DDbands. Thus, the remaining 4 unidentified bands were sub-cloned to obtain pure populations of expressed genes. Twentyclones were picked from each subcloned DD band and se-quenced. From 2 to 4 expressed genes were identified for eachsubclone, some of which were identical with the directlysequenced DD bands. The lower band (bottom arrow) inFigure 2 turned out to be a mixed population consisting ofmitochondrial NADH dehydrogenase subunit 5 and cyto-chrome b. Table 1 shows a list of the differentially expressedgenes identified by direct or subcloned DNA sequencing.

Northern blot analyses were run to confirm the expressedgene changes detected by DD (Fig. 3). Glutamine cyclotrans-ferase (QC) changed the most, with a 10-fold decrease in


IOVS, February 1999, Vol. 40, No. 2 Differential Gene Expression in a Human Lens Cell line 403

FIGURE 2. Autoradiograph of a DD gel. RT-PCR products from control(—) and H2O2-treated (+) cells in duplicate were compared. Arrowsindicate downregulation of expressed genes. Top arrow, glutaininecyclotransferase; bottom arrow, a mixture of mitochondrial NADHdehydrogenase subunit 5 and cytochrome b. This automdiogmph rep-resents approximately 25% of the length of the gel for anchor primer8/arbitrary primer 1, Expressed gene changes were not observed withthis primer set in other areas of the gel.

expression in the H2O2-treated cells, similar to that observed inthe original DD gel (Fig. 2, top arrow). This protein is respon-sible for posttranslational modification of N-terminal pyroglu-tamyl residues of neuropeptide precursors.25'26 The cyclizationof the N-terminal glutaminyl residue converts these precursorsinto biologically active hormones. QC has been identified in avariety of tissues, including pituitary, brain, B-lymphocytes,and retina and also in plants. The peptide precursors for QC inthe human lens epithelial cells are unknown; however, the

presence of QC mRNA indicates a distinct mechanism of pep-tide processing in lens cells. The dramatically lowered level ofQC mRNA in oxidatively stressed cells may indicate a reduceddemand for processing enzymes either due to lower peptideconcentrations or turnover.

Cytokine-inducible nuclear protein, which is involved insignal transduction and is considered to be a member of theprimary response gene family,27 increased twofold in stressedcells. Cytokine-inducible nuclear protein was first identifiedfrom a cDNA library prepared from interleukin-1 and tumornecrosis factor-a-stimulated human dermal microvascular en-dothelial cells. Its location in the nucleus, the presence of anankyrin-like repeat structure, and its similarity to IxB-like pro-teins suggest that this protein plays a role in the regulation ofgene expression.

By northern blot analysis, glycoprotein 130 decreased1.5-fold, alternative splicing factor (ASF) increased 4-fold (datanot shown because of faintness of the band), and ribosomalprotein S10 mRNA did not change between the two condi-tions. The latter finding emphasizes one of the inherent prob-lems of DD, in which low annealing temperatures that arenecessary to obtain PCR products can produce false positives("=10% in our study).

NADH dehydrogenase subunits 4 and 5 and cytochrome bof the mitochondrial electron transport system decreased four-to sixfold in stressed cells. The mitochondrial gene product16S rRNA was only slightly decreased. This specific effect onthe electron transport system may be the result of a negativefeedback mechanism, because mitochondria produce H2O2 asone of the by-products of cellular respiration. For the mito-chondrial and cytoplasmic RNAs, no degraded transcripts wereobserved on northern blot analysis (data not shown).

RT-PCR was used to confirm differential gene expressionfor probes with low or undetectable signals on northern blotanalysis (Fig. 4). ASF 2 increased fourfold in H2O2-treated cells.ASF is a serine/arginine-rich protein that activates alternativemRNA splicing and influences 5' splice-site selection based onits concentration in the cell.28'29 This represents an importantstep in the expression of many proteins and serves as a quan-titative and qualitative control on gene expression. For exam-ple, homologues of ASF in Drosophila control sex determina-tion; in vertebrates, ASF controls the expression of isoforms ofj3-tropomyosin and cardiac troponin T gene.29'31 Recently,ASF was reported to be upregulated in mouse malignant skintumors, a pathology that is associated with alternative splicingof integrins in the early stages of carcinogenesis.32 It is reason-

TABLE 1. Expressed Genes Identified by DifferentialDisplay

Mitochondrial Cytoplasmic

NADH dehydrogenasesubunit 4

NADH dehydrogenasesubunit 5

Cytochrome b16S rRNA

Glutamine cyclotransferaseCytokine-inducible nuclear

proteinGlycoprotein 130Ribosomal protein S10Alternative splicing factorj3-hydroxyisobutyryl-coenzyme

A hydrolaseCathepsin



Cont.H202

Glutamine cyclotransferase

Cytokine inducible protein

GIycoproteinl30

Ribosomal protein S10

Mitochondrial NADH dehydrogenase 4

Mitochondrial NADH dehydrogenase 5

Mitochondrial cytochrome b

Mitochondrial 16S rRNA

Cytoplasmic 18S rRNA

Cytoplasmic 18S rRNA

0.1 X

2X

0.67 X

IX

0.25 X

0.15 X

0.25 X

0.8 X

Blot A

BlotB

FIGURE 3- Composite of northern blot analysis using DD bands asprobes. All RNAs were analyzed on blot A (3 fig total RNA/lane) exceptcytokine-inducible protein, which was analyzed on blot B (10 p,g totalRNA/lane) from a separate experiment. X represents the relative in-crease or decrease in specific RNAs in the H2O2-treated cells whencompared with control cells after normalization to 18S rRNA usingimageQuaNT Software. No degraded RNA bands were observed. AllRNA hybridizations were at the expected size for the full-length cDNAsreported in the nucleotide database and original publications.

able to assume that splice-site selection via the upregulation ofASF is one of the mechanisms important in the rapid recruit-ment of functional proteins during stress conditions, such asseen in the human lens epithelial cells in our study.

jS-Hydroxyisobutyryl-coenzyme A (CoA) hydrolase (E.C.3-1.2.4; /3-H) increased 10-fold in H2O2-treated cells, whereascathepsin increased slightly (1.4-fold after normalization toGAPDH; Fig. 4). In light of our observations on the differentialexpression of mitochondrial gene products, it is interesting tonote that j3-H, although derived from the nuclear not themitochondrial genome has a mitochondrial leader sequenceand is located in the mitochondrial matrix space.33 This en-zyme catalyzes the hydrolysis of S-hydroxyisobutyryl-CoA, anintermediate in the valine catabolic pathway.33 It has beenpostulated that 0-H plays a role in cellular defense, because itprotects cells from the effects of the highly toxic metabolitemethacrylyl-CoA, which is another intermediate in the catab-

oiism of valine upstream of S-hydroxyisobutyryl-CoA.Methacrylyl-CoA is a thiol-reactive molecule that could inacti-vate numerous enzymes in the absence of a mechanism de-signed to minimize its intramitochondrial concentration.34 Ithas been reported that an infant born with a deficiency of j3-Hexhibited large amounts of cysteine/cysteamine conjugates ofmethacrylic acid, indicating that conjugation betweenmethacrylyl-CoA and glutathione had occurred.34 Such a loss ofglutathione could be expected to create an imbalance in theredox state of the mitochondria if not the entire cell. Theincrease in J3-H transcripts could reflect an increase in valinecataboiism and could also include an enhanced metabolism ofmethacrylyl-CoA. The association between the increase in j3-Htranscripts and the decrease in mitochondrial gene products isnot clear; however, it is plausible that, although mitochondria]electron transport function is down regulated, defense mecha-nisms are being induced to protect mitochondrial integrity.

Because mitochondrial transcripts seemed disproportion-ately affected by oxidative stress, the rate of differential geneexpression of several mitochondrial gene products was evalu-ated in cells at 1 to 24 hours after a single bolus of 200 JJLMH2O2 (Fig. 5). NADH dehydrogenase subunit 4 decreased two-fold as early as 1 hour after cells were treated with H202. Thelevel of this transcript declined to 30% of control at 3 hours and20% of control at 5 hours. The level rose to 33% of control at24 hours. In contrast, cytochrome b transcripts showed asteady decrease, from 92% of control at 1 hour, to 42% at 3hours and 37% at 5 hours, and ending at 20% of control at 24hours. Cytochrome oxidase III, although not originally de-tected by DD, was also evaluated in the time course experi-ments. It followed a pattern similar to that of cytochrome b.Transcript levels of cytochrome oxidase III from H2O2-treatedcells were 95% of control at 1 hour, 55% at 3 hours, 50% at 5hours, and 20% of control at 24 hours. As observed in theoriginal DD experiment, 16S rRNA transcripts were generallyless affected by oxidative stress than the other mitochondrialgene products. The level of 16S rRNA was 92% at 1 hour, 75%at 3 hours, 65% at 5 hours, and 31% of control at 24 hours.

Mitochondria are sensitive to oxidative stress. In the pres-ence of ROS, mitochondria undergo morphologic and bio-chemical changes, including swelling,9 loss of electron trans-port capacity,35 and decreased transcription of geneproducts.12'36 Our findings agree with a recent DD study onhamster fibroblasts, in which oxidative stress (4 /j,M to 10 /xM

Cathespin ASF p-H

GAPDH

FIGURE 4. Ethidium bromide-stained agarose gel of RT-PCR differen-tially expressed genes. Expression levels of cathepsin, ASF 2, and/3-hydroxy isobutyryl- coenzyme A hydrolase (j3-H) were evaluated incontrol (—) and H2O2-treated (+) cells. Each reaction contains addi-tional primers for GAPDH as an internal standard. The same resultswere obtained for cathepsin, ASF, and )3-H using their respectiveprimers alone. The PCR products were quantified and normalized toGAPDH using Kodak BioMax ID Image Analysis Software.


IOVS, February 1999, Vol. 40, No. 2 Pifferential Gene Expression in a Human Lens Cell line 405

c/3

E

lhr 3hr 5hr 24hr

E8

NADH dehydrogenase

Cytochrome b

§ 9 * 9 Cytochrome oxidase III

O^A^MMMMMf £ 16S Ribosomal RNA

18S Ribosomal RNA

28S Ribosomal RNA

M 44 4* «• «* •« 18S Ribosomal RNA

FIGURE 5. Composite of northern blot analysis showing time courseof mitochondrial gene expression. Cells were exposed to DMEM with-out ( - ) or with (+) 200 /xM H2O2 and then harvested 1, 3, 5, or 24hours later. Gene expression was also evaluated in cells grown inDMEM containing 20% fetal bovine serum and harvested at 0- and24-hour time points. Bottom panel: 28S and 18S rRNA bands in theethidium bromide-stained gel before blotting. Each lane contains 5 ^gtotal RNA. Relative changes in gene expression were quantified usingImageQuaNT Software after normalization to 18S rRNA using KodakBioMax ID Image Analysis Software. No degradation of RNA wasobserved.

H2O2/107 cells for 8 minutes to 10 hours) caused a decrease ina number of mitochondrial transcripts.'2 However, this latterstudy reported multiple bands and degraded mitochondrialgene products, which were suggested to be due to a mediatormitochondrial component, such as RNase or the direct degra-dation of RNA by H2O2. In addition, our data showed a broadercellular response to oxidative stress because we observeddownregulation of mitochondrial and cytoplasmic RNAs.

Using DD, we have shown that oxidative stress induceschanges in gene expression in a human lens epithelial cell line.Several pathways, including cellular respiration and mRNA andpeptide processing, are involved in this stress response.

Lens cells use a number of strategies to maintain ROS atlow levels, including activation of the ROS scavenger enzymescatalase and glutathione peroxidase. However, with age thereis a diminution of these protective systems, placing the lens atrisk for oxidative damage and cataract.37 H2O2 is believed toplay a role in oxidative damage of the lens and the develop-ment of maturity-onset cataract.1 Its involvement in cataractformation has been well documented in animal models.4"8 Inaddition, oxidative damage occurs in the lens epithelium, andthe effects of this damage precede lens opacification.9 Thepresent DD study was carried out to assess the oxidative stressresponse of the lens, at the molecular level, using a trans-formed cell line originating from human lens epithelium.19

These cells maintain certain lens characteristics and are usefulin that they are of human origin. The response of these cells toH2O2 may help reveal some of the same mechanisms and

pathways that are involved in the development of maturity-onset cataract. Using the genes in these pathways as targets,we can assess their expression in normal and cataractoushuman lenses and monitor their response in the presence oftherapeutic compounds.

Acknowledgments

The authors thank Julia Birch and Sharon Kiang for excellent technicalassistance.

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oxidative stress induces differential gene expression in a ... m urea gel using the genomy lxr dna...

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