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(The FASEB Journal. 2004;18:480-488.)
© 2004 FASEB

The effect of stress withdrawal on gene expression and certain biochemical and cell biological properties of peroxide-conditioned cell lines

WANCHAO MA1, DAYU LI1, FANG SUN, NORMAN J. KLEIMAN and ABRAHAM SPECTOR2

Department of Ophthalmology, College of Physicians & Surgeons, Columbia University, New York, NY 10032, USA

2 Correspondence: Department of Ophthalmology, Columbia University, 630 W. 168th St., New York, NY 10032, USA. E-mail as42{at}columbia.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maturity onset cataract is a disease that afflicts >25% of the U.S. population over 65. Oxidative stress is believed to be a major factor in the development of this disease and peroxides are suspected to be prominent stressing agents. To elucidate mechanisms involved in the protection of cells against oxidative stress, immortal murine lens epithelial cells ({alpha}TN4-1) have been conditioned to survive lethal concentrations of either tertiary butyl hydroperoxide, TBOOH (a lipid peroxide prototype) (T cells), or H2O2 (H cells). It was found that T cells survived exposure to H2O2 but H cells were killed by TBOOH. In this communication, biological characteristics of the T cells are reported. It is shown that the T cell’s ability to survive TBOOH is lost if the cells are grown in the absence of this peroxide (denoted as T- cells). By comparing the differential gene expression of 12,422 genes and ESTs from T and T- and the unconditioned control cells, 16 genes were found that may account for the loss of resistance to TBOOH. They include 5 glutathione-S-transferases, superoxide dismutase 1, zeta crystallin, a NADPH quinone reductase, as well as genes involved in detoxifying aldehydes, controlling iron metabolism, and degrading toxic lipoproteins.—Ma, W., Li, D., Sun, F., Kleiman, N. J., Spector, A. The effect of stress withdrawal on the gene expression and certain biochemical and cell biological properties of peroxide-conditioned cell lines


Key Words: microarrays • hydrogen peroxide • tertiary butyl hydroperoxide • GSH-S-transferase • catalase • cataract


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE LENS OF THE EYE is an avascular encapsulated tissue existing in an aqueous environment. It is held in position in the anterior chamber by zonular fibers, the only nonliquid contact with the rest of the eye. The tissue contains a single layer of epithelial cells on its anterior side that terminally differentiates in the equatorial zone into fiber cells that lose their nuclei and metabolic activity (1) . Thus, the lens is primarily dependent on the layer of epithelial cells to maintain homeostasis and transparency of the tissue. Injury to these cells by biochemical or physical insult leads to cataract and blindness. Cataract is the most common cause of blindness throughout the world (2 3 4) . Twenty-five percent of the U.S. population over 65 years of age is afflicted with this disease (5) .

It has been found that oxidative stress is a major contributor to cataract development and that peroxides are prominent oxidative stress agents (6 7 8 9 10 11 12 13 14 15 16) . That oxidative stress is associated with disease is not unique to the lens. Halliwell and Gutteridge (17) listed more than 340 references demonstrating the involvement of oxidative stress as a primary or secondary factor in causing over 100 diseases. If oxidative stress, and more specifically peroxide stress, is a primary factor in cataract development, they suggested that three criteria should be satisfied. 1) The agent should be found at the site of the disease (at least sometimes). 2) The agent, in the concentration range found in vivo, should be capable of reproducing the characteristics of the disease when applied to the tissue and 3) the degree of damage should be dependent on the concentration of the stressing agent, and removal of the agent should diminish the damage proportionally. Recent work has established that H2O2 is constantly generated in the aqueous humor and metabolized by surrounding tissue to give normal peroxide concentrations of ~1 µM (18) . The favored method for determining H2O2 uses dichlorophenol-indophenol, which Garcia-Castineiras et al. (19) have shown gives artificially high values in the presence of the high ascorbate levels found in the aqueous and lens. Based on H2O2 determinations that use methodology not affected by ascorbate, it appears that previous results may be ~20–25 µM too high (18) . However, even with such correction, remarkably high values have been observed in both the aqueous and lens of human and animal cataractous eyes (7 , 12 , 13 , 20) . Such levels of H2O2 or lipid peroxides will cause cataract and cause biochemical changes, based on in vitro studies, comparable to the changes found in cataract (8 , 11 , 14 , 21) . It has been found that lens epithelial cells are very sensitive to peroxide stress, and even short-term exposure to peroxide in the 25 to 50 µM range will cause disruption of normal metabolism particularly if a critical antioxidative defense gene has been compromised (22 23 24 25 26) . Furthermore, modulating the H2O2 concentration correspondingly affects the rate at which the opacity as well as biochemical changes develop (27) . Thus, the three criteria proposed by Halliwell and Gutteridge (17) are satisfied.

To define the defense mechanisms used by the cell in resisting peroxide stress, immortal lens epithelial cells have been conditioned to survive exposure to either TBOOH (T cells) or H2O2 (H cells) (28 29 30) . It is now shown that T cells lose their resistance to TBOOH when this peroxide is removed from the cell environment. Such cells, designated T- cells, retain a resistance to H2O2. Thus, we have a unique opportunity to define genes and biochemical systems that allow cells to survive TBOOH by comparing differential gene expression and biochemical parameters in T and T- cells and to elucidate systems protecting the cell from both H2O2 and TBOOH by comparing T, T-, H, and control (C) cell lines. Such experiments reveal a small number of genes that appear to be critical for defense against such oxidative agents. This information should be helpful in formulating antioxidative defense strategies for many tissues afflicted with oxidative stress associated disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell conditioning
Immortal murine lens epithelial cell {alpha}TN4-1 (generously provided by Paul Russell, National Eye Institute, Bethesda, MD, USA) was conditioned to resist TBOOH or H2O2 as described previously (28) . Briefly, cells at ~80% confluence were subcultured (~200,000 cells per 35 mm dish) in 2 mL of minimum essential medium, MEM (GIBCO BRL, Grand island, NY, USA; Cat#41500-034) supplemented with NaHCO3, 2.2 g/L (pH 7.2), 100 U penicillin and 100 µg streptomycin per milliliter (GIBCO BRL, Cat#15140-122), fungizone, 2.5 mg/L (GIBCO BRL, Cat#15295-017), and fetal bovine serum (Hyclone, Logan, UT, USA; Cat#5430070-02) to give a final concentration of 10% (the standard medium). After overnight incubation at 37°C in a 5% CO2 incubator, the medium was replaced with 4 mL of the standard medium, and various amounts of 10 mM H2O2 and/or TBOOH were added to give the desired concentration. Every 24 h, an additional aliquot of peroxide was placed in the medium. The culture medium was changed every 3 days. When cells were ~80% confluent, they were subcultured; after 16 h, exposure to peroxide was reinitiated. Over an ~6 to 8 month period, the peroxide concentration was gradually increased from 25 µM to 125–130 µM. The conditioned cells were maintained at this peroxide level as described above. Normal {alpha}TN4-1 cells were maintained in an identical fashion but without peroxide.

Enzyme assays
All enzyme assays were performed as described previously (28) . For GSH-S-transferase, two substrates were used: 1-chloro-2,4-dinitrobenzene and 4-hydroxynonenal. The method of Ålin, Danielson, and Mannervik (31) was followed to determine activity with 4-hydroxynonenal.

Other determinations
H2O2 was assayed essentially as described by Spector et al. (32) and protein as determined by Spector et al. (33) using a modification of the Bradford method (34) .

SDS-PAGE was performed as follows. 6–8 x 105 cells were homogenized in 70 µL of 0.15% Tritonix-100, 50 mM phosphate (pH 7.0) at 0°C, then centrifuged at 14,000 rpm at 4°C for 5 min. The supernatant was mixed with an equal volume of 2x sample buffer for SDS-PAGE as described by Smith (35) , then ~20 µL equivalent to 25 µg protein was used for electrophoresis with the Laemmli method (36) as modified by Wang and Spector (37) .

Molecular mass analysis of unknown bands found after SDS-PAGE was determined as described by Spector et al. (28) using methodology described previously (38 39 40) .

Preparation of cRNA
Approximately 30 µg total RNA (isolated from ~80% confluent culture with a RNA isolation kit, Cat# 74104, Qiagen, Valencia, CA, USA) was used to prepare double-stranded cDNA using a commercial system (SuperScript Choice; Gibco BRL) and a T7-(dT)24 primer: 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3' for first-strand cDNA synthesis. After synthesis of the second-strand cDNA, the double-stranded cDNA was purified with Phase Lock Gels (Brinkmann Instruments, Inc., Boulder, CO), phenol-chloroform extraction and ethanol precipitation. An RNA transcript labeling kit (Cat#42655; Enzo Diagnostic, Inc., Farmingdale, NY, USA) was then used to prepare biotin-labeled cRNA. The cRNA was purified using a spin column (RNeasy; Qiagen), followed by ethanol precipitation quantified and examined by gel electrophoresis. It was then fragmented at 94°C for 35 min.

Microarray analysis
Affymetrix murine chip MG-U74A, version 2, was used in this study. For each cell line, five independently isolated RNA samples were used for preparation of fragmented biotin-labeled cRNAs. Microarray hybridization was performed using protocols described by Affymetrix, Inc. (Santa Clara, CA, USA) at the Genome Center of the College of Physicians and Surgeons, Columbia University. The chips were stained with streptavidin phycoerythrin and detected by fluorescence laser scanning and confocal localization. Data were analyzed using Affymetrix Microarray Suite 5.0 and Microsoft Excel software. Standard linear one-way ANOVA was applied to the gene population with a stringent cutoff of one false positive per 1000 genes. To differentiate between cell lines, these genes were further subjected to the Tukey test for contrast using {alpha} = 0.01. Gene expression was considered to be significantly changed if 1) ANOVA P value <=0.001 and Tukeys for specified comparisons were significant, 2) expression increased or decreased by at least 1.5-fold with at least 40% "increased" or "decreased" change call in the same direction assigned by the Affymetrix software for the specified comparisons, 3) mRNAs were assigned at least one "present" detection call for the gene by the Affymetrix software in all experiments. Genes were annotated using NetAffx (see www.affymetrix.com).

Statistical analysis of the expression microarray data revealed a small population of antioxidative genes with a significant increase in expression in the conditioned cell lines. Confirmation of some of these data was undertaken with real-time PCR (41) . Total RNA was used to synthesize first-strand cDNA. One microgram of total RNA from a given sample was reverse transcribed using oligo-p(dT)15 and avian myeloblastosis virus reverse transcriptase (first-strand cDNA synthesis kit for RT-PCR; Roche Molecular Biochemicals, Indianapolis, IN, USA). Real-time quantitative PCR was then used to determine cDNA and the change in gene transcription. A fluorescein PCR detection system (LightCycler; Roche Molecular Biochemicals) was used for this purpose.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T- cells lose their resistance to TBOOH
{alpha}TN4-1 cells conditioned to survive H2O2 stress (H cells) are killed by the lipid peroxide prototype, tertiary butyl hydroperoxide (TBOOH); similar cells conditioned to survive TBOOH (T cells) can resist H2O2 (Fig. 1 ). These cells are maintained by daily exposure to peroxide except for the period when they are subcultured. Do these conditioned cells maintain their characteristics if the peroxide stress is removed? To answer this question, cells were grown in the absence of peroxide for up to 8 wk. Since the culture cycle procedure is to plate 200,000 cells and subculture when the population reaches 800,000 cells, this represents two cell doublings per culture cycle. It usually takes ~4 days for a cycle. Thus, the cells have gone through a minimum of 28 doublings in 8 wk. As shown in Fig. 1A , cells removed from stress for 8 wk (H- and T- cells) grow as well as the H and T cells when stressed with H2O2. However, the T- cells lose their resistance to TBOOH (Fig. 1B ) and die.



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  		Figure 1. Cell growth of conditioned cell lines subjected to peroxide stress. A) 125 µM H2O2, B) 130 µM TBOOH. The results represent the average of 3 or more experiments ± SD.

Microarray gene expression analysis reveals 16 antioxidative defense genes that may be necessary for TBOOH resistance
Since the T cells withdrawn from TBOOH stress (T- cells) are no longer able to survive exposure to TBOOH, this provided an opportunity to define the mechanisms used by the T cells to exist in a TBOOH environment. The investigation was initiated by examining the gene expression of the T- and T cells using control cells (C) cells as reference. Since H cells resistant to H2O2 are vulnerable to TBOOH, genes with increased expression in T and H cells may contribute to TBOOH resistance but are not sufficient. Therefore, to obtain a more complete picture of the changes in gene expression, the H and H- cells were also examined. Affymetrix murine expression microarrays (MG-U74A, version 2) were used for this analysis. Five arrays were used for each cell type. The results were analyzed using Microsoft and Affymetrix software programs and statistical examination with ANOVA (P<=0.001) and Tukey ({alpha}=0.01) analysis (see methods section for additional details). If the gene expression ratios of the cell lines being compared were between 1.50 and 0.67, they were not included. It is assumed that if the gene expression is not changed by >50% it is not significant.

Using the above criteria, gene expression in T/C and T/T- was determined. 244 genes and ESTs were found (see Supplemental Data, Table 1, http://www.fasebj.org/cgi/content/full/18/3/480/DC1). Examination of this group for antioxidative defense (AOD) genes revealed 16 genes (see Table 1 ) with significant change in gene expression. All of these genes may contribute to the T cells resistance to TBOOH. In comparison with the H cell lines, it was found there are nine genes that have increased expression only in T cells and have significantly decreased expression in T- cells. These genes may be of particular importance in protecting T cells. They are indicated by italics and underlines in Table 1 . The other genes are induced by both TBOOH and H2O2. They are not sufficient to protect against TBOOH since H cells are killed by TBOOH, but they may be necessary and together with other genes, contribute to their protection.


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  		Table 1. Antioxidative defense genes that contribute to TBOOH resistance

The change in expression of these 16 genes is shown in Fig. 2 , where the T/T- gene expression ratios are presented. Almost one-third of the genes in this group are GSTs, genes involved in the reduction of oxidized components or detoxification of xenobiotics. Some are located in the cytoplasm ({alpha}2 and {alpha}3), others in the mitochondria (Mu2 and Mu5) and one, microsomal (GST2). Two genes involved in detoxifying aldehydes and one associated with modulating cell metal ion concentrations (hephaestin) have decreased expression in the T- cells. Other genes include heat shock protein 2, which is similar to {alpha}B-crystallin and a putative chaperone; macrosialin, a transmembrane glycoprotein scavenger of low density lipoproteins; acyl protein thioesterase 1, which functions as a lysophospholipase controlling the level of lysophospholipids in the membrane (lysophospholipids in the high concentrations found in inflammation can disturb membrane integrity); another inhibitor of inflammatory reactions, secretory leukocyte inhibitor; superoxide dismutase 1, which detoxifies superoxide generating H2O2; and thioredoxin interacting protein, which contributes to the control of cellular redox levels. GARG16, a glucocorticoid attenuated response gene with potent anti-inflammatory characteristics, and zeta crystallin an NADPH-dependent quinone oxidoreductase, are also included among these AOD genes.



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  		Figure 2. Gene expression signal ratios (T/T-) of 16 AOD genes selected from T/C and T/T- gene expression microarray analyses. See Table 1 for additional information, including complete gene names.

The reliability of some of the AOD gene microarray data was further analyzed by real-time PCR. As shown in Table 2 , results confirm the microarray data in most cases. However, because of the semiquantitative nature of these results and since increased gene expression is not necessarily reflected in increased biological activity, it is important to assess changes occurring at the protein level.


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  		Table 2. Real-time PCR confirmation of microarray data

Enzyme activities of major AOD enzymes confirm gene expression analyses
A few of the major AOD enzymes were examined to determine whether the change in activity in the different cell lines reflected the conclusions obtained from the gene expression analyses. Since in most cases activity assays are not specifically for a particular gene product, it is generally not possible to match enzyme activity results as with the gene expression data. Based on activity measurements (Table 3 ), there are four enzymes or enzyme families with high enzyme activity T/C ratios (>=1.5).


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  		Table 3. Enzyme activity of some antioxidative defense genes that contribute to TBOOH resistancea

The highest value of 70 was obtained with catalase. However, since the T/T- ratio as well as H/C indicated that catalase activity is high in cell lines vulnerable to TBOOH, it is obvious that catalase is not sufficient to protect cells from TBOOH. This is not surprising since TBOOH is not a substrate for catalase. However, as shown recently by this laboratory (42) , inhibition of catalase by the specific inhibitor 3-amino-triazole causes T cells to lose their resistance to TBOOH. Thus, catalase is probably necessary but not sufficient for protection against TBOOH. The increase in catalase activity is ~10-fold greater than the increase at the RNA level (see Supplemental Data, Table 2, http://www.fasebj.org/cgi/content/full/18/3/480/DCI). This indicates that the process by which the mRNA is converted to active enzyme contains additional controlling factors.

Glucose-6-phosphate dehydrogenase (G-6-P dehyd) has a T/C activity value of 1.6. In contrast, the average gene expression signal ratio was 5. The overall G-6-P dehyd activity and gene expression data support the viewpoint that the enzyme is necessary but not sufficient. Unlike catalase, the cell contains more than one G-6-P dehyd. The gene shown in Table 1 is the major one but the activity assay does not differentiate between the various members of this family of enzymes and thus represents an average activity measurement.

A third type of interpretive problem is illustrated by the GST family. There are no specific assays for a given GST, but rather substrates that are preferred by certain members. The most general substrate is 1-chloro-2,4-dinitrobenzene, and a substrate preferred by GSTs that detoxifies lipid peroxides is 4-hydroxy nonenal. The T/C activity ratio with the latter substrate is 8.7 and with CDNB, 2.0. Based on enzyme activity in both cases, the GSTs may be necessary but not sufficient. The microarray results obtained with GST, {alpha}4, which detoxifies lipid peroxide and its products, parallel the enzyme data. GSH-Px also has increased activity. Some GSH-Pxs are not differentiated by the assay. Based on activity, the GSH-Pxs may be necessary but not sufficient. In contrast, the gene expression data suggest no protective role for most of the GSH-Pxs (electronic file 2). This is the only case where gene expression and activity results differ.

SDS-PAGE analysis of conditioned cell line proteins confirms importance of GSTs
Since there is no direct biological activity assay available for many of the gene products activated in these conditioned cell lines, they were examined by SDS-PAGE. There are some regions where significant differences are found between T and T- cells. Of particular interest is the 25 kDa region, where GSTs might be expected to be located (Fig. 3 ). Here a prominent doublet is found in the T preparation that is markedly reduced in T- samples. The 25 kDa bands were cut out and the extracted polypeptides subjected to mass spectroscopy and sequencing. The major components were shown to be GST{alpha}1 and {alpha}2. However, other GSTs may be present in low concentrations and contribute to the difference between these results and the observed enzyme activities. The results again emphasize the prominence of GSTs in these preparations. In the H lines, these GSTs also appear to be prominent and are comparable to T cell results. Thus, all analyses support the conclusion that GSTs are major AOD enzymes involved in protecting the conditioned cell lines but, although necessary, do not appear to be sufficient to assure survival.



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  		Figure 3. 25 kDa region of the SDS-PAGE profiles of protein from various cell lines. 25 µg of 0.15% Triton X-100-soluble protein was used for each cell preparation. Typical results from two or more experiments are shown. C-{alpha}TN4-1 control cells, H–H2O2-conditioned {alpha}TN4-1 cells, H-–H cells withdrawn from H2O2 stress, T–TBOOH-conditioned {alpha}TN4-1 cells, T-–T cells withdrawn from TBOOH stress.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The 16 genes found in Table 1 all appear to contribute to the T cells’ resistance to TBOOH. They can be placed in a few groups based on general function. Five of the 16 are GSTs, enzymes that use GSH to detoxify oxidized components. Such enzymes as well as a few others (including thioredoxin-interacting protein, aldehyde dehydrogenases, and zeta crystallin) can be placed in the general category of reductants. The other genes are involved in controlling cellular metal ion concentration and degrading toxic lipoproteins and phospholipids, suppressing inflammation, and include a chaperone and an SOD. Genes with increased expression in only the T line include many that are involved with lipid metabolism.

In previous studies of conditioned cell lines in which T- and H- cells were not available, many AOD genes were found to have increased expression in response to H2O2, TBOOH or a combination of both peroxides (29 , 30) . All AOD genes revealed in this investigation are in that group. The remainder, of course, do not show a significant change in their T/T- gene expression ratio. However, some (such as catalase) show very significant increases in T/C gene expression ratios. This group of genes may be necessary for defense against TBOOH but are not sufficient. They are listed in Table 4 . Two other observations support this conclusion with respect to catalase. When catalase is inhibited by the specific inhibitor 3-amino-triazole, T cells are killed by TBOOH. Second, preliminary experiments with {alpha}TN4-1 cells enriched in the catalase gene survive H2O2 stress but not TBOOH.


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  		Table 4. Antioxidative defense genes that may be necessary but not sufficient for TBOOH resistance

To get some sense of the function of all 244 genes that appear in the gene group showing significant change of expression in T/T- and T/C comparisons, the gene ontology database has been used. This provides an ordered semantics for defining function (43) The genes have been categorized on the basis of biological process or molecular function, as shown in Fig. 4 . Genes may be involved and thus assigned to more than one biological process or molecular function. In the biological processes delineation, 152 genes are annotated and assigned to one or more categories, resulting in 217 assignments. The AOD category represents 5.2% of the assignments. Most aspects of biological activity are reflected in these categories. One hundred and sixty-seven genes are assigned to one or more molecular functions, resulting in 271 assignments encompassing a wide variety of functions. There are 61 genes that have not been assigned to either a biological process or molecular function.



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  		Figure 4. Distribution of gene functions of 244 genes with significant change in expression in T/C and T/T- comparisons. Gene functions were assigned according to Gene Ontology Consortium definitions (see http://www.geneontology.org).

Many classical AOD genes do not appear in this or previous analyses and, therefore, do not seem to contribute to the cells’ defense against these particular stresses (see electronic file 2 and ref 29 ). There are a few other considerations that deserve mention. It is possible that some genes performing an AOD function have not been recognized. In some cases, changes in cell biology may impose new requirements with respect to constituent levels, reflected in changes in gene expression that do not contribute to the cell’s AOD. There may be modification in chromatin structure in response to the conditioning that results in changes in the accessibility of particular gene sequences. Changes in levels of regulatory factors that control gene expression have probably also occurred.

Though it is clear that after 8 wk without TBOOH stress, the T cells lose their TBOOH resistance, it is not apparent from the present work how quickly this resistance is lost. Unpublished work has shown that the resistance is almost completely lost within 5 days. However, the cells retain a capacity to quickly regain resistance. Furthermore, the loss of resistance is not complete in that at low TBOOH concentrations (60 µM) that would kill unconditioned cells in ~24 h, the T- cells survive. The mechanism involved in regaining resistance is not apparent at this time.

Although a number of studies have examined the response of the lens to oxidative stress, we are not aware of any investigations from other laboratories in which the gene expression of conditioned stable lens epithelial cells has been studied. Using a human lens epithelial cell system in which gene expression changes with time from the onset of the stress, Goswami et al. (25) found from cDNA microarrays 1171 genes that were significantly up- and down-regulated. Using differential display, Kantorow et al. (44) examined epithelia from human cataract and normal lenses and found metallothionein II overexpressed and a protein phosphatase 2A regulatory subunit underexpressed, respectively. Other genes have been defined as responding to oxidative stress, including thiol transferase (45) , GSTs (46 , 47) Na, K ATPase (48) , APT (45 , 49) , and GSH-Px (50) . Whereas some of these genes are found in the present work, further investigation in which the genes are shown to specifically protect the lens from oxidative stress is needed in order to be certain of their protective contribution. Such experiments have begun with catalase (unpublished experiments) and some of the GSTs (46) . However, the present work has considerably reduced the number of genes to be considered as likely protectors against TBOOH stress.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Eye Institute, Research to Prevent Blindness, Research to Cure Cataract Foundation, and the Department of Ophthalmology, College of Physicians and Surgeons of Columbia University. The assistance of Elaine Bluberg in the preparation of this manuscript is gratefully acknowledged.


   FOOTNOTES
 
1 Contributed equally to this investigation.

Received for publication August 8, 2003. Accepted for publication November 21, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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