Regulation of Thioltransferase Expression in Human Lens Epithelial Cells

Abstract

purpose. To study how the expression of thioltransferase (TTase), a critical thiol repair and dethiolating enzyme, is regulated in human lens epithelial cells under oxidative stress. Also to examine whether depleting the primary cellular antioxidant glutathione (GSH) in these cells has any influence on TTase expression under the same conditions.

methods. Human lens epithelial cells (B3) were grown to confluence (1.6 million) and gradually weaned from serum in the medium before exposing to 0.1 mM H₂O₂ for 2 hours. Cells were removed at the time intervals of 0, 5, 10, 15, 30, 60, and 120 minutes for protein measurements of GSH and TTase activity and for reverse transcription–polymerase chain reaction (RT-PCR) or Northern hybridization analysis to quantify TTase mRNA. The effect of GSH depletion on TTase mRNA expression was examined by treating the cells with buthionine S,R-sulfoximine (BSO); 1-chloro, 2,4-dinitrobenzene (CDNB); or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Lens epithelial cells, depleted of cellular GSH by treatment with BCNU, were subjected to oxidative stress to examine the effect on TTase activity and mRNA level.

results. A transient increase was detected in TTase mRNA after 5 minutes of H₂O₂ treatment. The upregulation reached a maximum of 80% above the normal level by 10 minutes and gradually decreased as the oxidant was detoxified by the cells. Manipulation of cellular GSH level by treatment with BSO, CDNB, and BCNU resulted in a minimum change in TTase expression. It is noteworthy that when cells depleted of GSH were subjected to oxidative stress, TTase expression was also found to be strongly upregulated.

conclusions. These observations suggest that the upregulation of TTase expression in the lens epithelial cells could be an adaptive response of the cells to combat oxidative stress to restore the vital functions of the lens proteins and enzymes. Such regulation is independent of cellular GSH concentration.

Generally, formation of reactive oxygen molecules is a natural metabolic consequence or, depending on cell type, is part of a system through which cells protect the organism against microbes. ¹ Left unchecked, these reactive oxygen radicals can be deleterious to the cells. In the case of the eye, both the lens epithelial cells and the surrounding aqueous humor can generate oxidants and free radicals. ² This creates an environment rich in oxidants, in which protein thiol groups can be thiolated readily. ³ This protein S-thiolation process (formation of protein-thiol mixed disulfide) triggers a cascade of events starting with protein or enzyme deactivation, alteration of protein conformation, protein–protein aggregation and the eventual opacification of the lens that is generally termed cataract. ⁴ Therefore, maintenance of the redox status of lens proteins is critical for the vital function of the lens. 

As a major defense, lens cells possess the ability to synthesize or acquire from the aqueous humor, quenchers (glutathione [GSH], ascorbic acid) that intervene either as sacrificial molecules in redox cycles or as producers of specific enzymes (i.e., catalase, superoxide dismutase, GSH peroxidase, glutathione S-transferase) that act directly or indirectly on the oxygen radical, thereby preventing oxidative damage. ⁵ Nevertheless, certain cellular processes and pathophysiological states can overwhelm these systems and produce oxidative stress, ⁶ during which protein S-thiolation takes place. 

Studies on cataractous lenses have shown that, although less than 10% of the protein thiol is present as disulfides in the normal lens, more than 70% is found to be in such linkage in human cataractous lenses. ^{7 8 9} In addition, levels of protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) were found to be elevated in H₂O₂-induced lens opacification in vitro ¹⁰ as well as in experimental cataracts such as those induced by naphthalene, ¹¹ UV light, ¹² and hyperbaric O₂. ¹³ Lund et al. ¹⁴ have also reported that the most distinctive difference between the soluble and insoluble proteins of the human cataractous lenses was the conversion of all the cysteine residues in the soluble proteins to their disulfide forms in the insoluble protein fractions. Liang and Pelletier ¹⁵ from their comparative studies on crystallin and crystallin-thiol mixed disulfides suggested that the conversion of native protein to the modified forms leads to conformational changes and proteolytic degradation. Recent mass spectrometry studies on the site of S-thiolation of γB-crystallin isolated from oxidatively stressed bovine lenses confirmed that conformational change takes place. ¹⁶  

In unstressed normal cells, the concentration of S-thiolated proteins is extremely low, suggesting that the dethiolation rate (breakdown of mixed disulfides by the reduction of disulfide bonds) may be sufficient to maintain the reduced status of proteins. This dethiolation process has been suggested to be mediated by low-molecular-weight thiols such as GSH. However, nonenzymatic reduction is usually slow and inefficient, suggesting the importance of catalytic dethiolation of mixed disulfides. Extensive studies on the dethiolation process led to the discovery of a new class of sulfhydryl enzymes called thiol disulfide oxidoreductases (TDORs) also known as protein sulfhydryl repair enzymes. Thioltransferase is a member of the TDOR enzymes and is an 11.8-kDa cytosolic protein that has been identified in liver, ¹⁷ red blood cells, ¹⁸ and ocular lens. ¹⁹ It contains an active site with redox-active dithiols. This functions in electron transport through a simple and elegant mechanism, the reversible oxidation of one protein SH group to a PSSG mixed disulfide. ²⁰ It is likely that this mechanism keeps the lens crystallins, enzymes, and membrane proteins in the reduced form by preventing their cross-linking or inactivation, an initial event in cataractogenesis. 

We hypothesized earlier ¹⁹ that, under conditions when the primary antioxidative defense system fails to protect the cells from oxidative damage, TTase acts as an antioxidant by its dethiolase activity to repair and regenerate the oxidatively damaged proteins. Studies conducted in our laboratory with purified recombinant human lens TTase on the dethiolation of radiolabeled crystallin-thiol mixed disulfides clearly support our hypothesis. ²¹ In addition to dethiolating the crystallin-thiol mixed disulfides, TTase has also been found to repair and regenerate important enzymes in the rabbit lens epithelial cells such as glyceraldehyde-3-phosphate dehydrogenase (G3PDH), glutathione reductase (GR), and glutathione peroxidase (GPx), which are experimentally inactivated by treatment with H₂O₂. ^{22 23} Studies on pig and rat lenses subjected to oxidative stress in organ culture have shown that the lens protein thiolation and dethiolation processes display a reciprocal relationship to that of TTase. ²⁴  

The present study was undertaken to learn how this critical repair enzyme responds to oxidative stress in the lens epithelial cells. Because TTase functions as the dethiolating and repair enzyme using GSH as reductant, the regulation of TTase in lens epithelial cells depleted of GSH was also studied. 

Materials and Methods

Materials

GSH; reduced nicotinamide adenine dinucleotide phosphate (NADPH); GR; 1-chloro, 2,4 dinitrobenzene (CDNB); buthionine S,R-sulfoximine (BSO); 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU); hydrogen peroxide (H₂O₂); minimum essential medium (MEM); fetal bovine serum (FBS); trypsin-EDTA (1×); and gentamicin solution were purchased from Sigma (St. Louis, MO). Hydroxyl ethyl disulfide (HEDS) was obtained from Aldrich-Chemie (Milwaukee, WI). Protein assay reagent was obtained from Pierce, (Rockford, IL). mRNA isolation and cDNA synthesis kits were purchased form Ambion (Austin, TX). Primers for polymerase chain reaction (PCR) amplification were custom made at IDT (Coralville, IA). A random primer labeling kit was obtained from Gibco (Gaithersburg, MD). β-Actin primer pair was obtained from Promega (Madison, WI). Taq DNA polymerase and PCR optimization buffers were obtained from Epicenter Technologies (Madison, WI). All other reagents and chemicals were of analytical grade. 

Human Lens Epithelial Cell Culture

Human lens epithelial cell line (HLE-B3) was immortalized by infecting with adenovirus 12-SV40 and was provided to us by Usha Andley (Washington University, St. Louis, MO). The cells were grown in 10 ml of MEM with 20% FBS and 50 μg/ml gentamicin in 100-mm tissue culture plates in a humidified atmosphere with 5% CO₂ at 37°C. Cells reached confluence with 6 × 10⁶ cells/plate in 4 days and were then used for the following studies. 

Harvest of Cells and Preparation of Cell Homogenate

The cells in each plate were harvested by scraping into 1 ml cold PBS buffer, and the suspension was centrifuged at 1700g for 15 minutes at 4°C. The pellet was resuspended in 5 ml EDTA (2 mM), sonicated to break the cells and centrifuged to collect the supernatant, which was used for protein analysis, GSH quantification, and TTase assay. Protein content was determined by the BCA protein assay protocol according to Smith et al. ²⁵  

Measurement of TTase Activity

TTase activity was determined with HEDS as substrate on a spectrophotometer (DU-640; Beckman, Carlsbad, CA) as described previously, based on the method of Raghavachari and Lou. ¹⁹  

Measurement of GSH

An aliquot of the fresh lens cell extract was treated with an equal volume of 20% trichloroacetic acid (TCA) and centrifuged, and supernatant was used for the estimation of GSH immediately, based on the method of Lou et al. ¹⁰  

H₂O₂ Treatment of Lens Epithelial Cells

The cells were grown to confluence and trypsinized, and 1.6 million cells were subcultured in 60-mm plates with 5 ml of MEM and 2% FBS at 37°C overnight. The medium was removed, and 5 ml of fresh MEM without serum was added to the plates. After 30 minutes of incubation, the cells were treated with 0.1 mM H₂O₂ in 5 ml of MEM for 120 minutes. At different time intervals (0, 5, 10, 15, 30, 60, and 120 minutes) cells were harvested and used for the measurements of protein, GSH, and TTase activity and for reverse transcription–polymerase chain reaction (RT-PCR) ²⁶ and Northern blot ²⁷ analyses to quantify TTase mRNA. 

Treatment of Cells with BSO, BCNU, and CDNB to Deplete Cellular GSH

The lens epithelial cells, as before, were grown to confluence and 1.6 million cells were subcultured in 60-mm plates with 5 ml of MEM containing 2% FBS overnight at 37°C. The medium was removed, and 5 ml of fresh MEM without serum was added to the plates. After 30 minutes of incubation, the cells were treated with 0.02 mM CDNB in 5 ml of MEM and incubated for 10 minutes. In another experiment 1.6 million cells were incubated in 5 ml of MEM containing 2% FBS with 0.5 mM BSO for 20 hours in a humidified CO₂ incubator. Cells were also treated with 0.5 mM BCNU in 5 ml of MEM without serum for 30 minutes. The choices of the concentration and time of treatment were based on the maximum ability of these agents to deplete the free cellular GSH level. In all these experiments, cells without any treatment with BSO, BCNU, or CDNB remained as control samples. At the end of each experimental period, cells were harvested as described and used for the estimation of protein, GSH, and TTase activity and for RT-PCR, as before. 

Induction of Oxidative Stress in GSH-Depleted Cells

Lens epithelial cells that were depleted of cellular GSH by treatment with BCNU (0.5 mM for 30 minutes) were subjected to oxidative stress by incubating the BCNU-pretreated cells with 0.1 mM H₂O₂ in MEM for 60 minutes. At different time intervals (0, 5, 10, 15, 30, and 60 minutes), cells were harvested and analyzed. 

Quantification of TTase mRNA by RT-PCR and Northern Blot Analysis

mRNA from HLE B3 cells was isolated and reverse transcribed (Micro PolyA Pure kit and cDNA Cycle kit; Ambion), according to the manufacturer’s recommendations. Aliquots containing equal amounts of synthesized cDNAs (0.1 μg, confirmed by PCR with β-actin and cyclophilin primers) were used for quantification of TTase mRNA. The cyclophilin primer pair was 5′-CCATCGTGTCATCAAGGACTTCAT-3′ (forward) and 5′-TTGCCATCCAGCCAGGAGGTCT-3′ (reverse). The TTase primer pair was 5′-TTCATCAAGCCCACCTGCCC-3′ (forward, position 51 from ATG start codon) and 5′-TCCAATCTGCTTTAGCCGCG-3′ (reverse, position 309), and the size of the amplification product was 258 bp. To distinguish amplification of TTase cDNA from the TTase gene (Medline accession number AF115104 U40574) in case of genomic DNA contamination of RNA samples, the forward and reverse primers were located in exons 1 and 2 of the gene, respectively, divided by a 1-kbp intron. Primers were designed using the software (Oligo; MB Insights, Cascade, CO). 

Conditions for PCR reactions were 94°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute. Aliquots were taken and analyzed by agarose gel electrophoresis (1.5% gel) after 10, 15, 20, 25, and 30 cycles to establish the feasibility of using RT-PCR for quantification of mRNA. 

For Northern blot analysis, the total RNAs were isolated from control and H₂O₂-treated cells (Pure Script RNA isolation kit; Gentra, Minneapolis, MN). The aliquots, containing 20 μg of total RNA were separated on a denaturing gel by the method of Chomczynski and Sacchi, ²⁸ transferred to a nylon membrane (Amersham, Arlington Heights, IL), probed with ³²P-labeled TTase cDNA fragment in hybridization solution (Express Hyb; Clontech, Palo Alto, CA) and exposed with x-ray film (Optimum; Life Science Products, Denver, CO). Hybridization and subsequent washing were performed according to the manufacturer’s protocol. 

The intensities of bands observed in RT-PCR and Northern blot analysis experiments were determined and compared using image analysis software (Scion Image; Scion, Frederick, MD). 

Statistical Analysis

The statistical analysis was performed using a two-sample Student’s t-test, assuming equal variances. 

Results

Treatment of Lens Epithelial Cells with a Bolus of H₂O₂

We chose 0.1 mM H₂O₂ for inducing oxidative stress in human lens epithelial cells based on the observation that this concentration does not cause DNA damage, retardation of growth, or morphologic changes in the cells. ²⁹ Cellular GSH concentration was found to be lowered by approximately 20% at 60 minutes compared with control concentrations (untreated). The cells recovered this loss slowly, and at 120 minutes, when H₂O₂ was completely detoxified, the GSH concentration approached near-normal levels (Fig. 1A ). 

Treatment of Cells with BSO, BCNU, and CDNB

The cells were experimentally depleted of GSH by treatment with BSO (an inhibitor of GSH synthesis), BCNU (an inhibitor of GSSG reductase), and CDNB (a conjugator of GSH). After the experimental period, the cells were analyzed for GSH content, and the results are summarized in Table 1 . As shown in this table, the GSH content was found to be lowered by more than 90% in each experimental group. 

TTase Activity

Figure 1B and Table 1 summarize the specific activity of TTase in cells stressed with H₂O₂ and in cells depleted of GSH, respectively. As shown in Figure 1B , there was a sharp increase in TTase activity within 5 minutes in cells treated with H₂O₂. The activity was nearly doubled by 10 minutes before returning to the basal level by 30 minutes. However, cells depleted of GSH by three different methods showed little change in the activity of TTase (Table 1) . 

Quantification of TTase Transcript in Cells Treated with H₂O₂

To quantify the differences in the concentration of TTase mRNA precisely, the aliquots of PCR reactions were taken at different time points of PCR log phase after 10, 15, 20, 25, and 30 cycles and analyzed in 1.5% gels. After 10 cycles only traces of bands were visible. After 15 and 20 cycles, sharp bands were detected, corresponding to an amplified 258-bp TTase cDNA fragment with notable differences in their intensities in different time points after H₂O₂ treatment. After 25 cycles, the reactions had reached the saturation phase, and intensities of bands became virtually equal at all time points. Computer image analysis (Scion Image; Scion) revealed a strong similarity in band intensity ratios between samples after 15 and 20 cycles. The same procedure was used for amplification of internal controls. Through these experiments, 20 cycles were chosen for all the RT-PCR studies. These data are summarized in Figure 2 . Additional bands, corresponding to amplified fragment of TTase gene (containing an additional 1-kbp intron 1 sequence) never appeared in our experiments. Control amplifications without reverse transcription were also performed, and no bands were obtained (data not shown). 

Figures 3 represent the amplification of TTase mRNA by RT-PCR in HLE B3 cells treated with H₂O₂. As can be seen from Figures 3A and 3C , there was an increase in expression of TTase in cells within 5 minutes of H₂O₂ treatment. This increase was nearly two times above the basal level at 10 minutes and gradually returned to normal level at 60 minutes. Amplification of internal controls, β-actin (Fig. 3B) and cyclophilin (data not shown) cDNAs, confirmed that all the samples used in RT-PCR experiments contained equal amounts of cDNA. 

To corroborate the activation of TTase gene, the total RNAs from a separate experiment were analyzed by Northern blot analysis hybridization. The RNAs from different time points after H₂O₂ treatment were separated by denaturing gel electrophoresis and stained with ethidium bromide (Fig. 4A ). The RNAs were then transferred to a membrane and hybridized to ³²P-labeled probe. The result of this experiment is shown in Figure 4B , in which all the treatment time points were duplicated, and the sample applied in each lane contained equal amounts of total RNA. Figure 4C depicts the integrated volumes of the hybridization signals, compared with the control at different time points of hydrogen peroxide treatment. It is obvious from these data that the contents of TTase mRNA increased after H₂O₂ treatment, and the pattern of gene activation was the same as that in the RT-PCR experiment. 

Quantification of TTase Transcript in Cells Depleted of GSH

Figures 5A and 5B show the amplification of TTase transcript and the internal control (β-actin), respectively, in cells depleted of GSH by treatment with BCNU, BSO, or CDNB. The level of TTase expression remains almost constant under these conditions (also see Fig. 5C ). 

Quantification of TTase Transcript in GSH-Depleted Cells Subjected to Oxidative Stress

To determine how the GSH-depleted cells would respond further to oxidative stress with respect to TTase, we treated the BCNU-pretreated cells with 0.1 mM H₂O₂ for 60 minutes. Figures 6A and 6C show the result of RT-PCR of mRNAs from these cells at different time intervals of H₂O₂ treatment. As can be seen in Figure 6C , at 5 minutes of treatment, there was an increase in expression of TTase that persisted for approximately 15 minutes and then gradually returned to a near-normal level. β-Actin (internal standard) mRNA showed no change under the same experimental conditions (Fig. 6B) . This result suggests that GSH depletion does not significantly affect the TTase gene activation under oxidative stress. 

Discussion

It is well established that oxidative stress is associated with the development of cataract, and it is generally believed that H₂O₂ is the major oxidant producing this stress in the ocular lens. ³⁰ Sulfhydryl proteins are the main targets for oxidative modifications, because they contain oxidizable thiol groups in a normally reducing environment. TTase, a sulfhydryl repair enzyme, the major function of which is to repair the oxidatively damaged proteins, may by itself be vulnerable to oxidative damage because it contains readily oxidizable cysteine residues at its active site. Damage to TTase would lead to a dysfunctional repair system in the lens resulting in irreversible damage to the ocular lens proteins. Generally, to counteract the oxidant effects and to restore a state of redox balance, cells have the ability to reset critical homeostatic parameters. The changes associated with oxidative damage and with restoration of cellular homeostasis often lead to activation or silencing of genes. ³¹  

In this study we report the ability of the human TTase gene to be overexpressed under oxidative stress conditions. When the lens epithelial cells were stressed by H₂O₂ treatment, we observed a significant transient increase in TTase mRNA concentration after 5 minutes that reached a maximal point almost two times higher than in untreated cells, and the upregulation subsided once the oxidant was totally detoxified. This suggests that the lens cells have a very sensitive mechanism that responds to the need for protection and repair of oxidizable sulfhydryl groups of proteins by a rapid up- and downregulation of TTase gene expression to dethiolate and restore the functions of the damaged enzymes and other proteins. Increased level of TTase mRNA after H₂O₂ treatment points to the activation of gene expression, but not to the activation of presynthesized protein. Although the increase of TTase mRNA half-life could also contribute to the increased mRNA level, our preliminary study suggests that AP 1 transcription factor binding to the 5′-end of the human TTase gene triggers its expression in the cells exposed to a low amount of H₂O₂. ³² Downregulation of the gene expression after its upregulation is probably caused by depletion of H₂O₂ from the media and, most probably, is controlled by the same mechanism. 

Similar fast activation of genes, particularly as a response to oxidative stress, has been demonstrated by others. For instance, Toone et al. ³³ have found that the gene coding for thioredoxin, a similar thiol-regulating enzyme in the same oxidoreductase family as TTase, was also upregulated two- to threefold after 20 minutes of treatment of the yeast cells with 0.2 mM H₂O₂. The upregulated thioredoxin expression was returned to normal level after 60 minutes, similar to our present study of TTase. Engelberg et al. ³⁴ described the activation of the xis4 gene in yeast caused by UV irradiation. It reached a maximum of 10-fold over control levels in 15 minutes and then gradually decreased. A 25-fold upregulation of c-jun and c-fos genes within 30 minutes, because a response to oxidative stress was demonstrated in rat lenses. ³⁵ Therefore, when the fast cellular response for stress is crucial for cell survival, the expression of the necessary genes can be activated in a very short time. 

It is well known that GSH is a key regulator of the redox state of protein cysteinyl thiols. ³¹ By far, GSH is the major form of cellular GSH and small increases in the oxidation of GSH to oxidized GSH (GSSG) resulting from reactive oxygen species and H₂O₂ metabolism have been shown to regulate many transcription factors such as AP1, MAF, and NRL. ³¹ In this study, we manipulated the GSH levels of cells by treating them with BSO, CDNB, and BCNU to study the response of these cells in terms of TTase expression. Under these conditions, the cellular GSH level did not have a significant influence on the regulation of TTase expression. Although TTase functions as a dethiolating enzyme with GSH as the major reductant, this function of TTase does not seem to be limited when only trace amounts of cellular GSH were present (see Table 1 ) as evidenced by the unchanged TTase activity. This suggests that TTase in the absence of GSH may use other reducing agents in the cell for its dethiolating function. Terada et al. ³⁶ have shown that TTase in the absence of GSH could use cellular cysteine or cysteamine and function normally as a dethiolating enzyme. Of note, when these GSH-depleted cells were stressed in the presence of H₂O₂, the cells responded by upregulating the TTase expression by approximately twofold above normal and then gradually returned to near normal levels at 60 minutes, a pattern that was similar to the TTase expression when cellular GSH was not deprived (Fig. 3C) . This suggests strongly that oxidative stress rather than GSH modulates the expression of TTase. 

²  Present affiliation: Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison.

³  NR and KK contributed equally to the work.

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1999.

 Supported by National Institutes of Health Grants EY10595 RO1-10595 (MFL).

 Submitted for publication September 27, 2000; revised December 12, 2000; accepted January 8, 2001.

 Commercial relationships policy: N.

 

Figure 1.

 

Effect of 0.1 mM H₂O₂ treatment on GSH level and TTase activity in human lens epithelial cells (HLE B3) as a function of time. (A) Concentration of GSH and H₂O₂. The data are expressed as mean ± SD, n = 3. (□) GSH level as a percentage of the control. (▪) H₂O₂ level in the medium as percentage of the initial concentration. (B) TTase activity. The data represent a typical pattern of over three separate experiments. *P < 0.01; **P < 0.001.

Table 1.

 

GSH Levels and Thioltransferase Activity in HLE B3 Cells Treated with BSO, CDNB, and BCNU

Treatment	GSH;0>(mmoles/1.6 × 106 cells)	Thioltransferase (mU/mg protein)
None (control)	11.5 ± 0.35	7.25 ± 0.09
BSO	0.60 ± 0.05*	7.04 ± 0.13*
CDNB	0.80 ± 0.09*	6.58 ± 0.12*
BCNU	0.95 ± 0.12*	6.52 ± 0.07*

 Data are expressed as mean ± SD; n = 3.

*  P < 0.001, significantly different from control.

Figure 2.

 

Calibration of RT-PCR for mRNA quantification. (A) The amount of PCR product in the reaction after 10 (▪), 20 (▴), and 30 (○) cycles. (B) Corresponding fragments of ethidium bromide–stained gels from which integrated volumes were quantified. A 258-bp amplified fragment of TTase cDNA is visible. The data are expressed as mean ± SD, n = 3.

Figure 3.

 

Quantification of TTase mRNA in H₂O₂-treated HLE B3 cells by RT-PCR as a function of time. The data represent a typical pattern of three separate experiments. (A) TTase mRNA expressed at the indicated intervals in 0.1 mM H₂O₂-exposed cells. M, DNA size marker. (B) β-Actin (internal control). (C) Integrated volumes of TTase expression.* P < 0.01, **P < 0.001, both are significantly different from the control (0 time).

Figure 4.

 

Quantification of TTase mRNA in H₂O₂-treated HLE B3 cells by Northern hybridization as a function of time. Data are expressed as a typical pattern of two separate experiments. (A) Total RNA from HLE B3 cells treated with 0.1 mM H₂O₂ after the intervals shown. Bands of 28S (top) and 18S (bottom) are visible. (B) Northern hybridization with TTase cDNA-containing probe. (C) Comparison of integrated volumes of hybridization signals. *P < 0.05;** P < 0.001, both are significantly different from the control (0 time).

Figure 5.

 

Quantification of TTase mRNA by RT-PCR in GSH-depleted HLE B3 cells. Data represent a typical pattern of three separate experiments. (A) TTase expression in normal cells (lane 1) and GSH-depleted cells by treating with CDNB (lane 2), BSO (lane 3), and BCNU (lane 4). M, DNA size marker. (B) β-Actin (internal control). (C) Comparison of integrated volumes of signals. *P < 0.01,** P < 0.001, both are significantly different from the control (lane 1).

Figure 6.

 

Quantification of TTase mRNA by RT-PCR in GSH-depleted HLE B3 cells at different time points of H₂O₂ treatment. Data represent a typical pattern of three separate experiments. (A) TTase mRNA expressed at the intervals shown in cells exposed to 0.1 mM H₂O₂. M, DNA size marker. (B) β-Actin (internal control). (C) Comparison of integrated volumes of signals.* P < 0.01, **P < 0.001, both are significantly different from the control (0 time).

The authors thank Usha Andley for providing the human lens epithelial cell line (B3).