Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract

Free Radical Biology & Medicine, Vol. 20, No. 3, pp. 301-31 I, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00

SSDI 0891-5849(96)02050-0

ELSEVIER

Original Contribution LENS

EPITHELIAL

CELL

DEVELOPMENT

APOPTOSIS OF

IS AN

UVB-INDUCED

EARLY

EVENT

IN

THE

CATARACT

WAN-CHENG LI and ABRAHAM SPECTOR Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons of Columbia University, New York, NY, USA (Received 18 April 1995; Revised 6 July 1995; Accepted 14 August 1995) Abstraet--Epidemiological and experimental studies have revealed that exposure to UV can induce cataractogenesis. To investigate the mechanism of this induction, viability of the lens epithelial cells from UVB-treated rat lenses were examined. Irradiation of the cultured rat lenses with 8 J/s/m 2 UVB for 60 min triggers lens epithelial cell apoptosis as determined by terminal deoxyribonucleotide transferase (TdT) labeling and DNA fragmentation assays. The apoptotic lens epithelial cells were initially found in the equatorial region and then quickly appeared in both equatorial and central regions. The percentage of apoptotic cells continuously increased during the postirradiation incubation. After a 5-h post-UVB incubation, more than 50% of the lens epithelial cells were apoptotic. By 24 h, all of the lens epithelial cells in the irradiated lenses were dead through apoptosis. Associated with this apoptotic process is a large upregulation of the proto-oncogene, c-fos. Opacification appears to follow the death of lens epithelial cells occurring first in the equatorial region and then in the central area. This is also true of classical cataract parameters such as non-protein thiot and wet weight, which are significantly modified only after appreciable epithelial cell apoptosis. Together, these results suggest that the rapid apoptotic death of the lens epithelial cells induced by UVB initiates cataract development. Keywords--UVB irradiation, Generation of free radicals, Lens, Cataract, Epithelium, Cell apoptosis, Cell viability, c-fos, DNA fragmentation, Free radicals

wards the equatorial area (the germinative zone), and terminally differentiate into fiber cells in the equatorial region. 2°'2~ This single layer of lens epithelial cells is essential for maintaining the metabolic homeostasis and transparency of the entire lens. 22 Under normal physiological conditions, most of these cells have a relatively long life span. However, if normal conditions are altered or disturbed by factors such as UVB,6'z3 the viability of the lens epithelial cells may be effected, possibly resulting in opacification of the lens. Increasing evidence suggests that exposure of the eye to U V B irradiation m a y cause cortical and posterior subcapsular cataract in humans and animals. 24 28 U V B damage to the lens occurs in both lens fiber and epithelial cells. In the fiber cells, it has been observed that UVB irradiation leads to degradation and modification of lens crystallins, 4'29-34 and loss o f membrane voltage in the peripheral fiber cells. 35 In the lens epithelial cells, it has been shown that U V B induces unscheduled D N A synthesis, 36"37 D N A damage and repair, 23

INTRODUCTION It is generally accepted that UV irradiation generates active oxygen species including hydrogen peroxide and superoxide ion. 1-4 These reactive oxygen species induces various cellular responses in eukaryotes, which includes damage o f D N A and proteins, 5.6 triggering of signal transduction pathways, 7 9 and activation o f gene expression.8-~6 As a result of U V irradiation, carcinogenesis or other pathogenesis such as cataractogenesis m a y occur 6"17- Is The lens of the veterbrate eye is a unique organ in that it is nonvascular and contains only a single layer of epithelial cells on its anterior surface. ~9 The epithelial cells remain quiescent in the central section, divide to-

Address correspondence to: Abraham Spector, Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA. 301

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W.-C. LI and A. SpECTO~

lowering of reduced glutathione, 38 enhancement of prostaglandin synthesis, 39 impairement of membrane pumps, 34.40 and inactivation of a number of metabolic enzymes.3S.41.42 Apoptosis is an active process of cellular self-destruction with distinctive morphological and biochemical features. 43"44 It is well established that cell apoptosis is involved in various human diseases. 45"46For example, the bacterial pathogen S. flexneri can induce apoptotic death of macrophages, potentially leading to human dysentery.a7 aIW- 1 can induce apoptosis of CD-4 ~ T lymphocytes, presumably contributing to development of AIDS.48 5~ This laboratory has recently demonstrated that lens epithelial cell apoptosis is closely associated with human cataract development. A significant percentage of the lens epithelial cells are found to be apoptotic in various kinds of human cataractous lenses but not in normal lenses. 52 Experiments with in vitro cultured rat lenses has revealed that the observed lens epithelial cell apoptosis can be induced by oxidative s t r e s s 52-54 and calcimycin. 55 Here, evidence is presented to show that after UVB irradiation, lens epithelial cells quickly lose their viability due to activation of apoptosis. Moreover, induced lens epithelial cell apoptosis precedes both temporally and spatially lens opacification, suggesting that activated lens epithelial cell apoptosis is an early and critical event during UVB induced cataract formation. MATERIALS AND METHODS

Chemicals In situ apoptosis detection kits were obtained from Oncor, Gaithersburg, MD; Radioactive compounds were purchased from Amersham Corporation, Arlington Heights, IL. Molecular biology reagents, various enzymes, and DNA size markers were obtained from Bethesda Research Laboratories, Gaithersburg, MD; Stratagene, La Jolla, CA; New England Biolabs, Beverly, MA, and Promega Biotech, Madison, WI. The culture medium, hydrogen peroxide, and most other chemicals and antibiotics were purchased from Sigma, St. Louis, MO.

Lens organ culture The rats used in this investigation were handled in compliance with the "Guiding Principles in the Care and Use of Animals" ( D H E W Publication, NIH 8623). Four-week-old S p r a g u e - D a w l e y rats, weighing about 100 g, were sacrified by CO2 inhalation. The eyes were removed and the lenses were carefully dissected by a posterior approach. Each of the dissected lenses was placed in a well of a 24-well culture plate

containing 1.5 ml medium 199 (M-3769, Sigma) for approximately 1 - 2 h. Transparent lenses (without surgical damage) were selected for experimentation. The medium 199 was supplemented with 26 mM NaHCO3 prepared with ion-exchange double-distilled water, sterilized by filtration through 0.22 # m filter (25942, Coming, NY). The pH was adjusted to 7.2 and had an osmolarity of 300 + 5 mosmol.

UVB exposure Transparent lenses were transferred into a 24-well culture plate containing 1.5 ml medium 199 and then irradiated for 1 h at room temperature with two 18," 15 W fluorescent bulbs (Spectronics Corp., Westbury, NY; Cat. No. BLE-1T158) mounted in an adjustable lamp housing (model 5002, Dazor Mfg. Co., St. Louis, MO) 6 inches above the culturing plate. The emitting energy levels were determined and found to be 0.154 J / s / m : at 254 nm, 10 J/s/m 2 at 300 nm and 1.8 J / s / m 2 at 365 nm. The absorbance of UV by the culture medium was 2 J / s / m 2 at 300 nm. Minimal values were found at the other wavelengths. Under this condition, the total exposed energy is 28.8 KJ/m 2. Experiments were also conducted with total emitting energy of 9 KJ/m 2 (2.5 J / s / m 2 for 1 h). Whenever this energy level was used, it was specified in the text. After UV exposure, all the ilradiated lenses were transferred into a 10 cm Petri dish containing 30 ml medium 199 and incubated at 37°C with a 5% CO2 gas phase for the required time (0, 5, 11, 17, 23, 47, 95, 119, and 143 h).

In situ apoptosis detection In situ detection of lens epithelial cell apoptosis was carried out by modification of the procedures of Schmitz et al. 56 and Gavrieli et al. 57 using an Oncor detection kit ($7100). Lens capsule epithelial cell samples were dissected from normal and UVB-treated rat lenses under a dissecting microscope and flat mounted on glass slides. Each sample was then fixed with 500 /.tl 10% neutral-buffered formalin followed by three changes of phosphate-bufferd saline (PBS) wash. Later, the fixed samples were preincubated with 1 × equilibration buffer ($7100-1, Oncor) at room temperature for 10 min and then incubated with terminal deoxynucleotidyl transferase in the presence of digoxigenin-1 1 dUTP and dATP at 37°C for 60 min. The enzymatic reaction was terminated by incubating the samples in stopping buffer ($7100-4, Oncor) for 30 min. Then the samples were washed with three changes of PBS followed by 30-min incubation with antidigoxigenin-peroxidase at room temperature. After this incu-

UVB-induced apoptosis and cataract bation, the samples were washed three times with PBS and allowed to react with 0.05% diaminobenzidine (DAB, D5637, Sigma) followed by three washes with distilled water and then counterstained with 0.5% methyl green (M-8884, Sigma). After washing with distilled water three times, the stained samples were dehydrated in butanol (Sigma), cleared in xylene (Sigma), and mounted with Permount (Fisher). After these procedures, the apoptotic cells in each sample were labeled brown or dark brown, while normal cells were stained light green.

DNA fragmentation assays DNA fragmentation was determined by a modification of the methods of Hogquist et al. 58 and Prigent et al. 59 Lens capsule epithelial cell samples from normal or UVB-treated rat lenses were combined in a microfuge tube (each combined sample contains eight whole rat lens epithelial cell capsule preparations). Five hundred microliter extraction buffer ( 10 mM Tris, pH 8.0; 10 m M EDTA, pH 8.0; 75 mM NaCI; 0.5% SDS and 150 # g / m l proteinase K) was added into each combined sample, which was incubated at 50°C for 3 h. After incubation, the sample was microfuged for 20 min at room temperature. The supernatant was recovered for DNA precipitation by 2 vol of 100% ethanol (200 proof, Pharmaco Products Inc.) with 0.1 M NaC1. The precipitated D N A was washed with 70% ethanol and then treated with DNAse-free RNAse (Gibco BRL 18030-015) for 60 min. Finally, the DNA sample was separated by 2.0% agarose gel electrophoresis and stained by ethidium bromide and photographed under UV illumination.

DNA probe preparation Rat c-fos 6° and mouse G A P D H cDNAs 61 were amplified in bacterial strain DH 5 a and purified by two continuous CsCI ultracentrifugations according to Ausubel et al. 62 The c D N A inserts were recovered by double gel purification 62 and labeled with a-32p-dATP (Amersham PB 10204) according to Feinberg and Vogelstein. ~3

RNA preparation and analyses Total RNAs were extracted from the treated or untreated rat lenses, lens epithelial cells, or lens fiber cells according to Chomczyaski and Sacchi 64 using a RNA buffer kit (CS-102, Biotex Laboratories, T X ) . For Northern blots, 25 #g of total RNA from the whole lens or lens fiber cells, or 7 / z g of total RNA from the lens epithelial cells was denatured, electrophoresed on

303

formaldehyde-agarose (1.2%) gel, and transferred to a supported nitrocellulose membrane (Gibco BRL #1465MC) according to Thomas. 65 The RNA blot was then UV crosslinked for 5 min and baked at 80°C under vacuum for 2 h. Prehybridization was conducted according to Li and Riddiford 66 at 42°C for 4 to 12 h in the following buffer: 50% formamide, 6× SSPE (0.9 M NaCI, 72 mM NaPO4, pH 7.4, 7.2 mM E D T A ) , 5× Denhardt's solution, 67 1% SDS, 200 #g/ml denatured and sheared herring sperm DNA. Hybridization was conducted at the same temperature and buffer for 36 to 42 h with a-32p-dATP-labeled specific probes at concentrations of 5 x 1 0 6 cpm/ml. After hybridization, the filter was washed once in 1 liter of 1 × SSPE, 0.2% SDS for 30 min at room temperature, then once in 1 liter of 0.1 x SSPE, 0.1% SDS for 15 min at room temperature, and finally once in 1 liter of 0.1 × SSPE, 0.1% SDS at 60°C for 15 min, and exposed to Kodak XAR-5 film for 12 to 48 h. For reprobing, bound radioactive probes were removed as previously described. 68

Analyses of nonprotein thiol Non-protein thiol was determined by modification of the Ellman method. 69'7° The lens was homogenized in 0.3 ml ice cold acetate buffer (100 mM HAc, pH 2.0, 2 mM E D T A ) . Homogenate (133 #1) was mixed with 7 #1 of ice-cold 100% trichloroacetic acid. The chilled preparation was centrifuged at 10,000 rpm for 10 min at 4°C. Supernatant (40 #1) was added to a tube containing 660 #1 Tris-EDTA buffer (100 mM Tris-C1, pH 8.2, 2 mM E D T A ) and 35 #1 10 mM Dithiol-bis (2-nitrobenzoic acid). Two minutes after mixing, the absorption at 412 nm was determined. The non-protein thiol values were normalized by utilizing glutathione ( G S H ) standards.

Measurement of lens wet weight The wet weight of pairs of rat lenses (one for control, one for experimentation) were weighed at varying times. The lenses were carefully removed from the culture plate and placed on a small piece of preweighed parafilm. Excess water was carefully removed with Whatman paper and then the lens was quickly weighed in a analytical balance (Mettler Instrument Inc., Hightstown, NJ). RESULTS

UVB triggers epithelial cell apoptosis, which precedes development of lens opacification The change in lens epithelial cell viability during UVB irradiation was investigated with terminal deox-

304

W.-C. Ll and A. SPECTOR

yribonucleotidyl transferase (TdT) labeling, a previously established in situ hybridization procedure for identifying apoptotic c e l l s . 56'57 When transparent lenses were irradiated by UVB (8 J / s / m 2 at 300 nm) for 60 min, TdT labeling revealed that apoptosis was quickly activated in the irradiated lens (Fig. 1 ). Although after 1 h irradiation less than 5% of the lens epithelial cells underwent apoptosis, this number was significantly increased during post-UVB incubation (Fig. 1 ). By the end of 5 h post-UVB incubation, more than 50% of the lens epithelial cells were apoptotic (Fig. 1 ). After 24 h post-UVB irradiation, all the lens epithelial cells were dead through apoptosis (Fig. 1 ). Control preparations subjected to similar culture periods showed essentially no apoptosis. To determine the temporal and spatial relationships between lens epithelial cell apoptosis and development of opacification after UVB irradiation, we have analyzed the percentage of apoptotic cells in both equatorial region and central area, and monitored the gradual change of lens transparency. As shown in Fig. 2, by the end of a 60-min irradiation, the lens is almost as transparent as a control lens. At this time, apoptosis has already started. In the equatorial region, about 4% apoptotic lens epithelial cells were observed, while in the central area, 2% apoptotic cells were found (Table 1 and Fig. 2). In the next 5-h post-UVB incubation, 80% of the lens epithelial cells in the equatorial region and 30% of the epithelial cells in the central area have become apoptotic (Table 1 and Fig. 2, 6 Hr-E). At this time, cortical opacification has started to develop in the equatorial region, while the

central area still remained relatively transparent (Fig. 2, 6 Hr-E). By 17 h after irradiation, almost all of the cells in the equatorial region and 94% of cells in the central part were apoptotic (Table 1 and Fig. 2, 18 Hr-E). With the death of the majority of the lens epithelial cells in the central area, lens opacification quickly developed towards the center, but about one-third of the lens surface remains transparent (Fig. 2, 18 Hr-E). By 23 h after irradiation, all the epithelial cells were dead through apoptosis, and many apoptotic cells had detached from the capsule, leading to loss of the apoptotic cells in the TdT labeled samples (Fig. 2, 24 Hr-E). By this time, the cortical lens opacification had spread to the entire surface (Fig. 2, 24 Hr-E). These results demonstrate that activated lens epithelial cell apoptosis precedes both temporally and spatially UVB-induced lens opacification. Similar results have been obtained with oxidative stress and calcimycin-induced cataract) 2-55 To determine whether the observed relationship between epithelial cell apoptosis and lens opacification development is also true under treatment of lower level of UVB, we also irradiated rat lens with 2.5/s/m2 UVB for 1 h (total 9 KJ/m 2 exposure) and examined the change of epithelial cell apoptosis and lens opacification development. As shown in Table 2, under this condition, it takes much longer time for the lens epithelial cells to commit apoptosis and for the lens to develop complete opacification. However, the fact that lens epithelial cell apoptosis precedes development of opacification is even more distinct.

UVB induces prolonged expression of the protooncogene, c-fos in cultured rat lenses tO0-

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P o s t - U V B Incubation T i m e (Hrs)

Fig. 1. Time course of UVB-induced lens epithelial cell apoptosis as determined by TdT labeling. Transparent rat lenses were irradiated with UVB for 60 min followed by incubation in medium 199 for 5 to 23 h. The time shown on the abscissa represents the time of the post-UVB incubation (0 to 23 h). After culture, capsule lens epithelial cell sections were processed for TdT labeling. The percentage of apoptotic cells were determined by counting 600 cells in three different fields. The presented data is the average of two set of experiments.

The fact that prolonged expression of c-fos may predict which cells are programmed to die was initially demonstrated by Smeyne et al. 7~ using transgenic mice containing a fos-lacZ transgene. In the lens system, it has recently been shown that expression of c-fos is closely associated with apoptosis activated by oxidative stress 52 54 and calcimycin, s5 To determine whether expression of c-fos is also upregulated during UVBinduced apoptosis, mRNA from control and UVB-irradiated rat lenses were analyzed by Northern blots. As shown in Figs. 3 to 5, the c-fos mRNA level in the 4week-old unirradiated rat lens is very low. After irradiation by UVB for 30 min, the c-fos expression did not show clear change (Fig. 3). UVB irradiation for 60 rain caused a twofold increase in the c-fos mRNA level (Figs. 3 and 4). This low level of upregulation remained unchanged in the first hour of post-UVB incubation (Fig. 3 ). During the longer post-UVB incubation (from 5 to 23 h), expression of c-fos was dramatically upregulated, more than 40-fold (column 5 to 7

UVB-induced apoptosis and cataract

A

B

305

C

1 Hr-E

6 Hr-E

18 Hr-E

24 Hr-E

24 Hr-C

Fig. 2. Temporal and spatial pattern of lens epithelial cell apoptosis and development of lens cortical opacification. The dissected rat lenses were either maintained under normal conditions in medium 199 (24 Hr-C) or irradiated for 60 min with UVB followed by culture in medium 199 for varying lengths of time (0 to 23 h). Column A depicts either control (24 Hr-C) or irradiated lenses (1 h irradiation plus 0 to 23 h post-UVB incubation, 1 Hr-E to 24 Hr-E). Magnification: 8.1x. Column B shows the TdT labeling results of the lens epithelial cells in the equatorial region from the lens shown in column A. Magnification: 426.6X. Column C shows the TdT labeling results of the lens epithelial cells in the central area from the same lenses shown in column A. Magnification: 426.6x. Because the control lens is transparent for the entire experimental period and contains very few apoptotic cells, only the result from a 24-h time point is shown. Hr, Hours; E, experiment; C, control.

W.-C. L] and A. SPECTOR

306

Table 1. Apoptosis Induced by UVB Irradiation Followed by Post-UVB Culture Post-UVB Period

0 h

5 h

11 h

17 h

23 h

Equatorial Region (%) Central Region (%) Whole sample (%)

4 _+ 1.0 2 _+ 0.5 3 ± 0.5

80 _+ 5.1 30 ± 6.5 55.5 _+ 5.5

90 ± 5.5 70 _+ 5.1 85.5 + 4.6

97 + 3.0 94 _+ 4.0 95 ± 4.5

100 _+ 0 100 ± 0 100 ± 0

All the lenses were UVB irradiated for 1 h and then were cultured for 0 to 23 h after UVB incubation. Percentage of apoptotic cells were determined by counting 400 cells in either equatorial region (for equatorial region percentage) or central region (for central region percentage) in two different fields and 600 cells in both equatorial and central region in three random fields (for the whole sample percentage). The presented result is an average of two separate experiments and the standard deviation is also included.

of Fig. 3; lanes 3 to 5 of Fig. 4A; column 3 to 5 of Fig. 4B ) against the control level of the lenses cultured for 1 h (the c-fos mRNA level in the controls of 6, 12, 18, and 24 h was undetectable with the present experimental conditions). When lens preparations were separated into epithelial cell and fiber cell fractions, UVB-induced upregulation was found not only in the epithelial cells but also in the fiber cells, although c-fos was undetectable in the control lens fibers (Fig. 5 ). The level of c-fos mRNA in the epithelial cells is approximately eightfold higher than that in the fibers after UVB irradiation based on calculation with equal amounts of total mRNA. Associated with this dramatic upregulation of c-fos in the lens epithelium, massive apoptosis was observed (Figs. 1 and 2).

DNA fragmentation assays confirm that TdT-labeled cells were apoptotic To further confirm that UVB induces lens epithelial cell apoptosis, nuclear chromatin structure was analyzed. Nuclear chromatin fragmentation has been widely used as an index of cell apoptosis. 44'72 During apoptosis, dying cells often have their DNA cleaved by cellular endonucleases in the internucleosomal regions,

Table 2. Apoptosis and Lens Opacification Induced by 9 KJim ~- UVB Irradiation Post-UVB Period Apoptotic Cells (%)* Opacification Change*

47 h

95 h

119 h

143 h

30 ± 4.4

75 ± 5.6

100 _+ 0

100 ± 0

T

+

++

+++

All the lenses were UVB irradiated for 1 h (total exposure: 9KJ/ m 2) and then were cultured for 47 to 143 h after UVB treatment. * Percentage of apoptotic cells were determined by counting 300 cells in three random fields. T, completely transparent lens. For reference, the different degrees of opacification are equivalent to that observed in Fig. 2 where + = 6Hr-E, + + = 18 H r - E a n d + + + = 24Hr-E.

resulting in DNA fragments of both multimers and monomers of 180 base pairs. 72 When rat lenses were irradiated with UVB and then either examined immediately or cultured for additional periods, analyses of the genomic DNA from these irradiated or control lenses showed that DNA fragmentation was activated sometime after irradiation (Fig. 6). In a 5-h culture after UVB irradiation, fragmented chromatin was observed (lane 5 of Fig. 6). This fragmentation pattern became more definitive in the 17- to 23-h post-UVB culture samples. In comparison with the 100 base pair ladder marker, the observed DNA banding pattern had a 180 base pair increment typical of apoptotic chromatin fragmentation (Fig. 5). It is interesting to note that DNA fragmentation did not occur at the end of irradiation (lane 3 of Fig. 5), indicating that the observed DNA fragmentation pattern occurred at later times and is not directly due to UVB damage but results from activation of endonuclease.

Significant changes in classical biochemical parameters associated with cataract occur after death of most lens epithelial cells Our previous studies have shown that the classical parameters associated with cataract do not change extensively until substantial lens epithelial cell apoptosis is observed.52 54 To determine whether a similar relationship also occurs after UVB irradiation, non-protein thiol and wet weight were determined. As shown in Fig. 7, a significant change of lens wet weight or non-protein thiol did not occur until more than 85% of the lens epithelial cells were dead via apoptosis. Therefore, induced lens epithelial cell apoptosis precedes the change of the classical parameters associated with cataract. DISCUSSION

UVB irradiation triggers lens epithelial cell apoptosis Using TdT labeling and DNA fragmentation assays, it is demonstrated here that UVB irradiation of the

UVB-induced apoptosis and cataract

cultured rat lenses triggers epithelial cell apoptotic death. Our observation with rat lens organ culture is consistent with earlier studies where various cell lines were examined. 73 7,5 In the human promyelocytic leukaemia cell line, HL-60, 73 rat chloroleukaemia cells, 69 and human histiocytic lymphoma U937 cell line, 75'76 irradiation of these cells for 10 to 30 min with UVB (approximately 8 J / s / m 2) followed by postirradiation incubation all activated apoptosis. Furthermore, as shown in this report, UVB triggers the apoptotic program during irradiation although the actual apoptotic process occurs at a later time. This is illustrated by the finding that TdT labeling is hardly observed and DNA fragmentation is not detected at the end of the irradiation period but appears at a later time (Fig. 6). However, these earlier studies with various cell lines differ from our results in that UVB irradiation of the cultured cell lines led to massive apoptosis within a much short time. For example, UVB irradiation of HL-60 cells for 5 to 30 min followed by 4 h culture in normal control conditions causes 100% apoptosis of the treated cells, 73 while in our organ culture, UVB irradiation of rat lenses with 8 J / s / m 2 for 60 min leads to a relatively slower apoptotic death of lens epithelial cells (Figs. 1 and 2). Decreasing the exposed energy (with 2.5 J / s / m 2 for 1 h) leads to a much longer commitment period of apoptotic death of the lens epithelial cells (Table 2). These results suggest that the lens may have a better defense system or that cell culture systems are

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Fig. 4. UVB-activated upregulation of c-fos. Twenty-five milligrams of total RNA extracted from rat lenses exposed to medium 199 or UVB for 1 h followed by incubation in medium 199 for varying lengths of time (5, 11, 17, and 23 h) were denatured, separated on 1.2% formaldehyde-agarose gel, transferred to supported nitrocellulose membranes ( B R L ) , hybridized to c-fos or glyceraldehyde-3phosphate dehydrogenase ( G A P D H ) cDNA probes, and washed under high stringency conditions. ~8 The c-fos m R N A in control lenses cultured for 6, 12, 18, and 24 h were undetectable and, therefore, were not shown. (A) Fluorogram of Northern blot. (B) Scanning results of (A).

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Hours Fig. 3. Scanning results of UVB-induced upregulation of c-fos. Twenty-five micrograms of total RNA extracted from rat lenses exposed to medium 199 (C) or UVB for 0.5 or 1 h only, or UVB for 1 h followed by incubation in medium 199 for varying lengths of time ( 1,5, and 17 h) were denatured, separated on 1.2% formaldehyde-agarose gel, transferred to supported nitrocellulose membranes ( B R L ) , hybridized to c-fos or glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) cDNA probes, and washed under high stringency conditions. 68 The Fluorograms of Northern blot were then scanned and analyzed by a computer-linked densitometer (model 300, ImageQuant).

more susceptible to the insult. It should be pointed out that prolonged UVB irradiation of cell lines (2-h irradiation) 73 or rat lenses (3 or more hours irradiation, data not shown) all lead to necrosis. Necrotic death often occurs when severe stress is present. In the case of photochemical stress, necrotic death has been observed as a major mode of cell death during induction of in vitro lens opacification, s3'54 This is also true of the treatment of rat lenses with high concentrations

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GAPDH mRNA Fig. 5. UVB-activated upregulation of c-fos in lens epithelial and fiber cells. Seven micrograms of total RNA extracted from the epithelial cells and 25 ,ug of total RNA from the fiber cells of the rat lenses exposed to medium 199 or UVB for 60 min followed by incubation in medium 199 for 11 and 17 h were denatured, separated on 1.2% formaldehyde-agarose gel, transferred to supported nitrocellulose membranes (BRL), hybridized to c-fos or glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA probes, and washed under high stringency conditions. 68 The mRNA blot for GAPDH in the lens epithelial cells was exposed 48 h while that in the lens fibers 15 h.

of calcimycin) 5 With normal physiological conditions, the ocular lens may be subject to long-term irradiation o f U V B . 27 However, the energy level from the solar source is much lower (approximately 3 × 10 2 J / s / m 2 calculated from data in ref. 27) than 8 J/s/m 2 or 2.5 J/s/m 2 used in this and other previous studies. 7~ 76 Therefore, it would be expected that apoptosis rather than necrosis would be the major mode of cell death associated with putative UVB-induced human cataractogenesis. Such an assumption is consistent with our previous observations with human cataract where a high level of apoptosis was found in all cataracts examined and no indication of necrosis was observed, s2 The quick upregulation of c-fos can be achieved through induction by a number of agents. 77 In the case of UV induction, it has been shown that UVC quickly activates c-fos expression in both Hela $3 and NIH 3T3 Cells. "~'~2 Such induction presumably occurs through activation of Src tyrosine kinase signal transduction pathway 8 with involvement of growth factor receptors.9 UVB irradiation of mouse JB6 epidermal cells ( 17.6 J/ s/m 2, total 6 KJ/m 2) induces a biphasic induction of the c-fos gene: the initial peak appears after 1 h, followed by attenuation to control level, then reaches another higher peak 8 h after irradiation.'5 UVB irradiation of transfected mouse JB6 epidermal cells ( 16.5 J / s / m 2 , total 12 KJ/m 2) also causes an enhanced expression of

Fig. 6. DNA fragmentation assays of the lens epithelial cells from control and UVB-irradiated rat lenses. Lens capsule epithelial cell samples from either control or UVB-irradiated rat lenses were combined for extraction of the genomic DNA as described in Materials and Methods. 5~'~4The extracted DNAs were separated in 2.0% agarose gels, stained with ethidium bromide, and photographed under UV illumination. DNA ladder ( 100 bp) (Gibco BRL) is shown on the far left and right lanes.

the report gene. j~' In our rat lens system, the biphasic induction has not been observed in repeated experimentation. UVB irradiation of rat lenses only upregulates expression of c-fos slightly within the first few hours (Fig. 3 ). However, during the longer post-UVB culture, expression of c-fos was dramatically increased by 5 h (Figs. 3 and 4). This level is maintained during the

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UVB Irradiation and Post-UVB Culture Time (Hrs) Fig. 7. Variation of non-protein thiol, wet weight, and viable cells with UVB treatment. Non-protein thiol and wet weight were analyzed as described in the Materials and Methods. Each point represents an average of two separate experiments after normalization against control values.

UVB-induced apoptosis and cataract

next 12 h. Thus, the UVB induction pattern of c-fos expression differs in different systems. A prolonged expression pattern of c-fos was also found during apoptosis of rat lenses induced by hydrogen peroxide, 52 photochemical stress, s4 and calcimycin. 55 Kinalc acidinduced apoptosis of neurons in the brain of the c-foslacZ transgenic mice was also associated with prolonged c-fos expression. 7~ Because c-fos encodes a transcriptional factor, 78 it is possible that the c-Fos protein may be required for mediating changes in gene expression that lead to the observed lens epithelial cell apoptosis, as suggested by Preston et al.79 in the preneoplastic Syrian hamster embryo cell lines. The upregulation of c-fos mRNA in the lens fiber cells suggests that the differentiating fiber cells may stop differentiation to undergo apoptosis.

Activated lens epithelial cell apoptosis is an early and critical cellular event during UVB-induced cataract formation We have recently proposed that induced lens epithelial cell apoptosis may be a common cellular basis for noncongenital cataract formation in both humans and animals. The conclusion is based on the observations that human cataract patients all have a significant percentage of apoptotic epithelial cells while normal human lenses of similar age do not, 52 and that lens epithelial cell apoptosis can be activated by oxidative s t r e s s 52-54 and calcimycin, 55 agents that cause cataract. The demonstration in this communication that UVB-induced lens epithelial cell apoptosis precedes both temporally and spatially development of cortical lens opacification provides further evidence for this hypothesis. Although UVB irradiation causes various kinds of modifications in the lens epithelial cells, the very early events probably are the damage to DNA 23 and membrane pumps. 4'~'8° It is conceivable that UVB-damaged membranes would allow influx of calcium into lens epithelial cells, which is consistent with the observation that UVBirradiated lenses contain high level of intracellular calcium. ~ Elevated levels of calcium are known to activate apoptosis. 82 The quick death of lens epithelial cells through apoptosis following UVB irradiation would eliminate homeostatic epithelial cell control of the underlying fiber cells, leading to impairment of the integrity of these underlying fiber cells. 22'83 It has been demonstrated that UVB causes generation of hydrogen peroxide and superoxide ion in biological systems. ~ 4 The degradation of tryptophan to N-formyl-kynurenine is believed to preceed the generation of these o x i d a n t s 4'29 Such reactions plus direct damage at the DNA and membrane levels would lead to the observed loss of epithelial cell viability. With death of the epithelial cells plus the UVB-induced dam-

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age to the fiber cell proteins, lens opacification rapidly develops. In the case of oxidative stress-induced cataract, recent work suggests a possible mechanism by which oxidative stress-induced sequential damage leads to cataractogenesis. 53'54 The first step is a definitive change in the redox setpoint of the epithelial cells quickly followed by damage at the DNA level and the plasma membrane. It appears that this damage initiates the loss of epithelial cell viability that precedes the development of cataract in the postinsult period by many days. The overall pattern of cataract development and cell death 52-54 is similar to that reported in this communication. Acknowledgements - - This work is supported by a NE1 fellowship

to W-C. Li and NEI grants and grants from Alcon Laboratory and Research to Prevent Blindness to A.S.

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