Interferon-γ Induces Apoptosis of Lens αTN4–1 Cells and Proteasome Inhibition Has an Antiapoptotic Effect

Abstract

purpose. Targeted ectopic expression of interferon-γ (IFN-γ) in the eyes of transgenic mice disrupts lens differentiation, upregulates immunoproteasomes, and causes cataract. In this study, the hypothesis that IFN-γ induces proteasome-dependent apoptosis of lens epithelial cells was tested.

method. Murine lens epithelial αTN4-1 cells were treated with IFN-γ. Apoptosis was measured using annexin V-FITC and propidium iodide (PI) staining, and DNA fragmentation. IFN-γ–inducible mRNA and protein expressions were measured by RT-PCR and Western blot analysis. Caspase activities were measured using colorimetric substrates and poly (ADP ribose) polymerase (PARP) cleavage. The effect of proteasome inhibition was tested with MG132 and lactacystin.

results. IFN-γ treatment at a concentration that induces immunoproteasome expression causes an approximately 20% increase in early apoptotic cells as observed by annexin V-FITC/PI staining and the increase in DNA fragmentation. IFN-γ–induced apoptosis was accompanied by upregulation of apoptosis-related genes, including a dramatic increase in signal transducer and activator of transcription (STAT)-1 and interferon consensus sequence binding protein (ICSBP), a more than 2-fold increase in IRF-1, and a 1.7- to 2-fold increase in caspase-1 mRNA. Bcl-2 mRNA decreased 2.4- to 3.0-fold, whereas Bax mRNA was unchanged. The Bax-to-Bcl-2 protein ratio increased by 1.6-fold. Caspase-1 and -8 activities were higher, but there was no increase in caspase-3 activity. Proteasome inhibitors MG132 and lactacystin protected the cells against IFN-γ–induced apoptosis. A positive control treatment with staurosporine (STP) caused increased caspase-3 activity, which was inhibited by MG132.

conclusions. IFN-γ causes apoptosis of αTN4-1 cells, accompanied by upregulation of known effectors. IFN-γ–induced apoptosis involves Bcl-2 family proteins and caspases. Proteasome inhibition has antiapoptotic effects on IFN-γ–induced apoptosis. It also inhibits the STP-induced increase in caspase-3 activity. If IFN-γ–induced apoptosis of lens epithelial cells contributes to cataractogenesis, the proteasome may be a therapeutic target.

Apoptosis, or programmed cell death, is a mechanism by which unwanted or damaged cells are removed from the tissues of multicellular organisms. It is an essential process for normal development, homeostasis, and maintenance of multicellular organisms ^{1 2 3} and has also been implicated in certain pathologic conditions. ⁴ Apoptosis can be induced by various stimuli, including IFN-γ, an inflammatory cytokine produced by activated T lymphocytes and natural killer (NK) cells that plays an important role in immune surveillance and host defense against infection. ⁵ IFN-γ can induce apoptosis as demonstrated in several in vitro models. ^{6 7 8 9} In most studies so far, IFN-γ has been shown principally to activate the JAK/STAT pathway. The IFN-γ–activated JAK phosphorylates STAT-1, which is then translocated to the nucleus, resulting in transcriptional activation or repression of specific target genes, mediated by a family of transcription factors known as interferon regulatory factors (IRFs). Among the nine IRFs characterized, IRF-1, a transcriptional activator, has been reported to be involved in IFN-γ–induced cell cycle arrest or apoptosis through activation of caspase-1 gene expression, ^{8 9 10} and interferon consensus sequence binding protein (ICSBP), a transcriptional repressor, has been shown to regulate apoptosis. ¹¹  

Under normal conditions the production of IFN-γ in the eye remains suppressed, ^{12 13} but IFN-γ has been demonstrated in inflammatory intraocular disorders from multiple stimuli, including viral infection, ^{14 15} retinopathy, ^{16 17} and uveitis. ¹⁸ Orderly development of the lens is necessary for transparency. Targeted ectopic expression of IFN-γ in the lens of transgenic mice completely disrupts lens development, and the normal spherical shape of the lens is lost. Normal lens fiber cells, the anterior subcapsular monolayer epithelia, and the equatorial nuclear bow region become unidentifiable. The malformed cells replace normal fibers, and the lens becomes cataractous. ^{19 20} Recently, it has been shown that IFN-γ inhibits lens epithelial cell proliferation. ^{21 22}  

We have shown the upregulation of the immunoproteasome in the lens of IFN-γ–expressing transgenic mice, demonstrating a mechanism by which IFN-γ could affect the lens. ²³ Multiple studies have shown that the proteasome takes part in the apoptotic processes, sometimes acting as an antiapoptotic agent and sometimes as a proapoptotic agent, depending on cell type, proliferating activity of cells, type of apoptotic stimulus or availability of growth factors. ^{24 25} Proteasome involvement in apoptosis is based on its capacity to degrade or process certain apoptosis-related regulatory proteins. ²⁶  

The lens epithelium is a single layer of cuboidal cells at the anterior surface of the lens. The epithelial cells remain quiescent in the central section, divide toward the equatorial area, and terminally differentiate into fiber cells in the equatorial region. ²⁷ This single layer of lens epithelial cells is important for maintaining metabolic homeostasis and transparency of the lens. ²⁸ Apoptosis-like events play an important role in lens development. ^{29 30} Under normal conditions lens epithelial cells have a long life span. When apoptosis of lens epithelial cells is observed, it has been associated with cataract formation. ^{31 32 33} In the present study, IFN-γ caused apoptosis of lens epithelial cells, and proteasome inhibition protected the cells from IFN-γ–induced apoptosis. 

Materials and Methods

Cell Culture and Treatment

The murine lens epithelial cell line, αTN4-1, ³⁴ (kindly provided by Paul Russell, National Eye Institute, Bethesda, MD), was grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mM glutamine, and gentamicin (100 μg/mL) in a 37°C humidified incubator in an atmosphere of 5% CO₂. The cells were grown in 25-cm² flasks, up to 80% to 85% confluence, and treated with 100 U/mL murine recombinant IFN-γ (Invitrogen-Life Technologies, Gaithersburg, MD), 0.125 μM staurosporine (STP), 10 μM MG132, and 10 μM or 1 μM clasto-lactacystin β-lactone (Sigma-Aldrich, St. Louis, MO) for 12 hours, alone or in combination. The αTN4-1 cell line was selected, because it responds to IFN-γ treatment by upregulating immunoproteasome subunits similar to IFN-γ–expressing transgenic mice. ²³  

Phase-Contrast Microscopy

The morphology of αTN4-1 cells was analyzed by phase-contrast microscopy. Briefly, cells were treated for 12 hours, with 100 U/mL IFN-γ alone, 10 μM MG132 (proteasome inhibitor) alone, or 100 U/mL IFN-γ+10 μM MG132 together, and observed by phase-contrast microscopy. 

Apoptosis Assay by Annexin V-FITC and PI Staining

Annexin V-FITC and PI staining were used to detect apoptosis at an early stage in IFN-γ–treated αTN4-1 cells. The annexin V–positive and PI-negative cells were considered to be early apoptotic cells. The assay procedure was as follows: IFN-γ–treated and untreated cells were washed with phosphate-buffered saline (PBS) and harvested using a commercial formulation (Accutase; Innovative Cell Technologies Inc., San Diego, CA). Cell pellets were washed and resuspended in 1 mL of binding buffer (Caltag Laboratories, Burlingame, CA). The 100-μL cell suspension was stained with 10 μL annexin V-FITC (Caltag Laboratories) and 5 μL PI (Sigma-Aldrich). After incubation for 10 minutes at room temperature in the dark, the samples were immediately analyzed by flow cytometry (FACSCalibur system; BD Biosciences, Franklin Lakes, NJ). 

To test for the involvement of the proteasome in IFN-γ–induced apoptosis, cells were treated with 10 μM of MG132 alone or cotreated with 100 U/mL IFN-γ and 10 μM MG132. The early apoptotic cells were measured by annexin V-FITC and PI staining. 

DNA Fragmentation Assay

We also measured DNA fragmentation after IFN-γ treatment, which is another hallmark of apoptosis. For DNA fragmentation detection, DNA was isolated from IFN-γ–treated and untreated cells using a minikit (QIAamp; Qiagen Inc., Valencia, CA), according to the manufacturer’s protocol. The DNA samples were electrophoresed by 2% agarose gel containing ethidium bromide and photographed under ultraviolet (UV) illumination. Relative amounts of different sized DNA bands were determined by scanning the gel using a UV illuminator (Imager 2000; Alpha Innotech Corp., San Leandro, CA). 

RT-PCR Analysis

αTN4-1 cells were treated with 100 U/mL of IFN-γ and incubated for 12 hours. Total RNA was isolated from IFN-γ–treated and untreated cells with RNAzol (Tel-Test, Friendswood, TX). To quantitate mRNA, an RNA PCR core kit (GeneAmp; Applied Biosystems, Foster City, CA) was used for RT-PCR. Total RNA from each sample was reverse transcribed at 42°C for 60 minutes followed by heat denaturation at 95°C for 5 minutes and cooling at 4°C for 5 minutes. The reaction mixture (40 μL) included 1 μg of total RNA, 2.5 μM oligo (dT)₁₆, 40 U RNAase inhibitor, and 100 U MuLv reverse transcriptase. The PCR mixture (50 μL) included 5 μL from reverse transcription reaction, 0.3 μM each sense and antisense primer and 1.25 U DNA polymerase (AmpliTaq; Applied Biosystems). The PCR was performed with an initial denaturation for 2 minutes at 95°C, followed by 25 amplification cycles, each comprising 1 minute at 94°C, 30 seconds at 55°C (63°C for ICSBP), and 1 minute at 72°C, followed by cooling to 4°C. The PCR products were analyzed by separating them by 2% agarose gel electrophoresis and visualized using ethidium bromide. The bands were quantitated by densitometry. The sequences of mouse-specific primers, corresponding references or GenBank accession numbers, and expected product sizes are shown in Table 1 (GenBank is available at http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). 

Western Blot Analysis

The cells were washed in PBS and lysed in lysis buffer containing 25 mM HEPES (pH 7.5), 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.05% TritonX-100, 20 mM β-glycerophosphate, 1 mM orthovanadate, 0.5 mM dithiothreitol [DTT] and one protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) per 10 mL of lysis buffer. After centrifugation for 15 minutes at 13,000 rpm, supernatant protein concentrations were measured by bicinchoninic acid (BCA) assay. ³⁷ Proteins were separated on 15% Tris-Cl SDS-polyacrylamide gels (50 μg lysate protein for Bcl-2, 25 μg for Bax and poly(ADP-ribose) polymerase [PARP]) and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C in TBS-T (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat milk powder and incubated with mouse anti-Bcl-2 antibody at a 1:500 dilution and rabbit anti-Bax and -PARP antibody at a 1:1000 dilution, for 1 hour at room temperature. These blots were then incubated with horseradish peroxidase (HRP)–conjugated secondary antibody for 1 hour at room temperature, and specific bands were detected using enhanced chemiluminescence (ECL) reagent (Perkin Elmer Life Sciences, Boston, MA) on autoradiographic film. All the antibodies were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. 

Measurement of Caspase Activities

Caspase-1–, -3–, and -8–like activities were measured by a colorimetric method, using the substrates Ac-YVAD-pNA, Ac-DEVD-pNA, and Ac-IETD-pNA, respectively (Biomol Research Laboratories, Plymouth Meeting, PA). Briefly, cells were washed three times with PBS and dissolved in lysis buffer containing 50 mM HEPES (pH 7.4), 0.1% CHAPS (3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate), 5 mM DTT, and 0.1 mM EDTA. The samples were centrifuged at 13,000 rpm for 20 minutes at 4°C, and supernatant protein concentrations were measured by BCA assay. ³⁷ Then, protein lysate (100 μg) was mixed with the corresponding substrate (400 μM) and diluted to 100 μL with assay buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, and 10% glycerol and incubated at 37°C. The optical density (OD) at 405 nm was measured by microplate reader (model 450; Bio-Rad, Richmond, CA). 

Statistical Analysis

Statistical significance was analyzed by two-tailed Student’s t-test. P < 0.05 was considered to be statistically significant. 

Results

IFN-γ-Induced Apoptosis of αTN4-1 Cells

We evaluated apoptosis induced by IFN-γ using two different methods. After IFN-γ incubation, the percentage of apoptotic cells was determined, first by flow cytometry using double-staining with FITC-conjugated annexin V and PI. IFN-γ effectively induces apoptosis after 12 hours’ incubation. There were 22.8% ± 3.0% early apoptotic events in IFN-γ–treated samples compared with 5.0% ± 0.9% in untreated samples (Fig. 1) . Fragmentation of genomic DNA was also observed after IFN-γ treatment. An increase in ∼1900-, ∼1600-, and ∼700-bp DNA fragments (1.5- to 3-fold) was observed in IFN-γ–treated cells (Fig. 2) . The classic DNA laddering pattern of 180- to 200-bp multiples was not observed in our study. 

Effect of IFN-γ on Morphology of αTN4-1 Cells

IFN-γ–treated and untreated cells were analyzed by phase-contrast microscopy. Compared with the untreated control culture, IFN-γ–treated samples had a markedly higher number of small, adherent, rounded, or bubbling and shrunken cells, a typical apoptotic morphology (Fig. 3) . 

Effect of IFN-γ on the Expression of Apoptosis-Related Genes

To investigate the altered regulation of apoptotic-related gene transcription in IFN-γ–induced apoptosis, RT-PCR assays were performed to record the expression of STAT-1, ICSBP, IRF-1, and caspase-1. In our studies, a dramatic increase in STAT-1 and ICSBP, a more than 2-fold increase in IRF-1, and a 1.7- to 2.0-fold increase in caspase-1 mRNA expression were observed (Fig. 4) . To identify the involvement of Bcl-2 family proteins in IFN-γ–induced apoptosis, we examined expression of Bcl-2 (antiapoptotic) and Bax (proapoptotic) mRNA expression by RT-PCR assay. A 2.4- to 3.0-fold decrease in the expression of Bcl-2 mRNA was observed, whereas Bax mRNA levels remained the same in IFN-γ–treated cells, compared with untreated cells (Fig. 4) . At the protein level, a decreased expression of Bcl-2 protein, but no significant change in the expression of Bax protein occurred in IFN-γ–treated cells compared with untreated cells (Fig. 5) . This led to a 1.6-fold increase in the Bax-to-Bcl-2 ratio in IFN-γ–treated cells. 

Upregulation of Caspase-1 and -8 Activity

Caspases are widely involved in the execution of apoptosis. ³⁸ In our studies, we observed increased activities of caspase-1 and -8 by IFN-γ treatment. Using cleavage of colorimetric substrate as the assay procedure, 2.9- and 2.7-fold increases in caspase-1 and -8 activity, respectively, were observed in IFN-γ–treated cells (Fig. 6) . 

Effect on Caspase-3 Activity

The activity of caspase-3, a general downstream effector caspase, was measured by the cleavage of the colorimetric substrate Ac-DEVD-pNA. No significant change in activity was observed in IFN-γ–treated compared with untreated cells, in cells treated with the proteasome inhibitor MG132 (10 μM) alone, or in cotreatment with IFN-γ and MG132. There was a significant increase in caspase-3 activity in cells treated with 0.125 μM STP (Fig. 7A) . Caspase-3 activity was also assayed by detection of PARP cleavage. PARP is a DNA repair enzyme, which is often degraded by caspase-3 during apoptosis. Western blot analysis was performed to detect the 85-kDa cleaved PARP fragment. As shown in Figure 7B , no cleavage product was detected in IFN-γ–treated and untreated samples. The cleavage product was observed in cells treated with 0.125 μM STP, which causes a comparable number of apoptotic cells in HLE B-3 cell cultures, as observed by flow cytometry. Apoptosis induced by STP was used as a positive control to demonstrate caspase-3 activity by cleavage of Ac-DEVD-pNA and PARP. Inhibition of the proteasome reduced these apoptosis-associated activities (Fig. 7) . 

Effect of Inhibition of Proteasome on IFN-γ–Induced Apoptosis

Incubation of cells with the proteasome inhibitor 10 μM MG132, protected the cells from IFN-γ–induced apoptosis (Figs. 3 8) . Phase-contrast microscopic analysis showed that a significant number of small, adherent, rounded, or bubbling and shrunken cells, indicative of dying apoptotic cells, were present in the IFN-γ–treated sample compared with the untreated sample. However, there was no significant difference in cell morphology among untreated, MG132-treated, and IFN-γ+MG132 cotreated cells (Fig. 3) , indicating that MG132 alone does not cause apoptosis but rather protects cells from IFN-γ–induced apoptosis. The antiapoptotic function of proteasome inhibition was verified using clasto-lactacystin β-lactone, a more specific and irreversible proteasome inhibitor. ³⁹ Phase-contrast microscopic analysis indicated that 10 μM clasto-lactacystin β-lactone prevented IFN-γ–induced apoptosis but that 1 μM concentration of the inhibitor was insufficient to provide protection (data not shown). 

The effect of MG132 on IFN-γ–induced apoptosis was confirmed by annexin V-FITC and PI staining. A significant increase in apoptotic cells was observed in IFN-γ–treated samples: there were 20.1% ± 3.87% apoptotic cells in IFN-γ–treated samples compared with 4.2% ± 2.3% in untreated sample. The samples treated with MG132 alone and cotreated with IFN-γ and MG132 had 1.5% ± 0.44% and 3.2% ± 1.9% apoptotic cells, respectively, which was not significantly different from the levels in untreated samples (Fig. 8) . 

Discussion

Apoptosis has been implicated in several ocular diseases, including glaucoma, retinitis pigmentosa, retinoblastoma, retinal ischemia, diabetic retinopathy, and cataract. ⁴⁰ Several animal lens studies have shown lens epithelial cell death under various stress conditions. There are also several reports showing a connection between apoptosis of lens epithelial cells and cataractogenesis, ^{31 32 33} although this has been disputed. ⁴¹ Because IFN-γ has been implicated in various inflammatory intraocular disorders ^{14 15 16 17 18} and ectopic expression of IFN-γ in the eyes of transgenic mice leads to various pathologic conditions including cataractogenesis, in the present study we tested the hypothesis that IFN-γ causes apoptosis of cultured lens epithelial cells, which is modulated by the proteasome. 

Our study demonstrated for the first time that IFN-γ causes apoptosis of lens epithelial cells. IFN-γ–induced apoptosis of lens epithelial cells was observed by phase-contrast microscopy, annexin V/PI staining and DNA fragmentation analysis. The annexin V/PI staining experiment was stringently selective considering only early apoptotic cells and excluding late apoptotic or necrotic cells. Another hallmark of apoptosis is the fragmentation of genomic DNA, in which DNA is cleaved at internucleosomal sites to produce the well-known ladder pattern. Other reports in the literature suggest that DNA is cleaved to release the chromatin loop domain, producing high molecular weight fragments before cleavage at internucleosomal sites, and the internucleosomal cleavage is not an essential step in the apoptotic process, in which case the typical ladder is not seen. ^{42 43 44} In our experiment, the classic DNA fragmentation pattern was not observed but an increase in high molecular weight DNA fragments was observed in IFN-γ–treated cells. This observation was consistent with a previous report of human lens epithelial cell cultures in which the apoptotic process was not accompanied with the classic DNA fragmentation pattern. ⁴⁵  

IFN-γ has been shown to exert pleiotropic biological effects through the activation of STATs, and increase of several apoptosis-related molecules, including CD95 receptors (Fas/APO1), different members of the Bcl-2 family, and caspases such as caspase-1. ^{6 7 8 9 10 46} IFN-γ has also been shown to promote apoptosis through the upregulation of caspase-8 in a STAT-1/IRF-1–dependent manner. ⁴⁶ In our study also, during IFN-γ–induced apoptosis of lens epithelial cells, upregulation of STAT-1, IRF-1, and caspase-1 mRNA was observed, suggesting that IFN-γ causes apoptosis of lens cells through the STAT-1/IRF-1/caspase-1–dependent pathway. The IFN-γ–inducible transcription repressor ICSBP has been shown to regulate apoptosis. ¹¹ In our study, a dramatic increase in ICSBP mRNA was observed with IFN-γ treatment. An induced expression of STAT-1, IRF-1, and ICSBP has been demonstrated in transgenic mice, with constitutive expression of IFN-γ in the lens. ⁴⁷ There are several reports in the literature showing an altered ratio of pro- and antiapoptotic members of Bcl-2 family proteins during the apoptotic process. ^{33 46 48} In our study, the increased ratio of Bax (a proapoptotic member of Bcl-2 family) to Bcl-2 (an antiapoptotic member of Bcl-2 family), indicates IFN-γ–induced apoptosis of lens epithelial cells also involved Bcl-2 family members. It is interesting to note that the expression of Bcl-2 mRNA and its protein is markedly decreased in lens epithelial cells from patients with anterior polar cataract, whereas Bax levels remain the same. ³³ In our study, caspase-1 and -8 activity was also increased in IFN-γ–treated cells compared with untreated cells. Although several reports have shown caspase-3 as a major downstream effector caspase in the apoptotic process, including apoptosis of lens epithelial cells, ^{49 50 51} there are reports of studies in which apoptosis did not involve caspase-3 activation. ^{48 52} In our study, no detectable increase in caspase-3 activity was observed during IFN-γ–induced apoptosis, suggesting this apoptotic pathway to be caspase-3 independent. Our finding is in good agreement with other reports in the literature in which IFN-γ–induced apoptosis has been shown to be accompanied by upregulation of initiator caspase-1 and -8, but with no change in expression of effector caspase-3, indicating that the IFN-γ–induced apoptotic pathway is caspase-3 independent. ^{9 46}  

The 20S proteasome is a 700-kDa protease, responsible for the degradation of most cytosolic proteins and in particular, short-lived proteins such as p53, c-myc, c-jun, various cyclins, NF-κB, and I-κB. These proteins are critical in cell proliferation, cell cycle regulation, transcriptional regulation and, under certain conditions, are involved in regulation of apoptosis. ²⁶ There are several reports in the literature about proteasome involvement in the apoptotic process as both a pro- and an antiapoptotic agent, depending mainly on cell type or proliferating activity of the cells. Meriin et al. ⁵³ reported both pro- and antiapoptotic effects of proteasome inhibitor with the same cell line, depending on the conditions of treatment. We have observed upregulation of immunoproteasome in the lenses of IFN-γ–expressing transgenic mice. ²³ In the current study, we sought to verify the role of the proteasome in IFN-γ–induced changes in lens epithelial cells. We observed that MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome, at 10 μM concentration and 12 hours of incubation, did not cause lens epithelial cell apoptosis. When the cells were cotreated with IFN-γ and MG132, MG132 completely blocked IFN-γ–induced apoptosis, as observed by phase-contrast microscopy and flow cytometry. MG132 is highly potent, but also inhibits calpain and cathepsin B. ⁵⁴ Therefore, we also tested lactacystin, considered to be a more proteasome-specific inhibitor, ³⁹ although it has been shown to inhibit the human platelet cathepsin A-like protease. ⁵⁵ Lactacystin also prevents IFN-γ–induced apoptosis. The results from the two inhibitors together indicate that proteasome inhibitors can provide protection against IFN-γ–induced apoptosis of lens epithelial cells, or at least delay the onset of the apoptosis cascade. Lin et al. ⁵⁶ have reported that the concentration of a proteasome inhibitor is critical for its action as a pro- or antiapoptotic agent. The proapoptotic effect of lactacystin on bovine lens epithelial cells observed by Andersson et al. ⁴⁹ may have been due to the longer incubation time of 25 hours. We expect that at higher concentrations or longer incubation periods, proteasome inhibitors would be proapoptotic in most cells. We are currently investigating the mechanism by which proteasome inhibition provides protection against IFN-γ–induced apoptosis. 

In summary, the data in the current study demonstrate for the first time that IFN-γ, at a dose that caused the upregulation of immunoproteasome subunits, induced apoptosis of lens epithelial cells. This finding suggests a mechanism by which IFN-γ could affect the lens and lead to cataractogenesis. The finding that a proteasome inhibitor provides protection against IFN-γ induced apoptosis suggests that inhibition of the proteasome may be a useful therapeutic strategy. 

 Supported in part by National Eye Institute Grant EY02299.

 Submitted for publication June 6, 2003; revised August 11, 2003; accepted September 5, 2003.

 Disclosure: N. Awasthi, None; B.J. Wagner, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primers Used for RT-PCR Analyses

Gene Name	Primer Sequence (5′→3′)	Reference or GenBank Accession Number	Expected Product Sizes (bp)
STAT-1	Forward-TTGGGT ACGCACACAAAAG	NM_009283	227
	Reverse-ATCACCACGACAGGAAGAG
ICSBP	Forward-GCTGCGGCAGTGGCTGATCGAACAGATCG	35	627
	Reverse-AGTGGCAGGCCTGCACTGGGCTGCTG
IRF-1	Forward-CTTTCTCTGATGGACTCAGC	NM_008390	452
	Reverse-CGTGAAGACATGTTGTATGC
Caspase-1	Forward-CTCAGAGAAATGAAGTTGC	NM_009807	299
	Reverse-ATGCCTGAATAATGATCACC
Bax	Forward-GTTTCATCCAGGATCGAGCAG	BC053380	487
	Reverse-CATCTTCTTCCAGATGGTGA
Bcl-2	Forward-GACTTCTCTCGTCGCTACC	XM_123726	332
	Reverse-GAGAAATCAAACAGAGGTCG
β-Actin	Forward-TTGGCCTTAGGGTTCAGGGGGG	36	243
	Reverse-CGTGGGGCGCCCCAGGCACCA

Figure 1.

 

Treatment of αTN4-1 cells with IFN-γ for 12 hours increased the number of apoptotic cells measured by flow cytometry using annexin V-FITC and PI staining. Data presented are the mean ± SD of results in one of five independent experiments with similar results. n = 3; *significant difference from untreated control (P < 0.001).

Figure 2.

 

IFN-γ treatment of αTN4-1 cells for 12 hours caused an increase in large DNA fragments. Total genomic DNA was isolated from IFN-γ–treated and untreated cells and electrophoresed on 2% agarose gels containing ethidium bromide. Bands were visualized under UV illumination and quantitated by densitometry. (A) Agarose gel showing IFN-γ–induced DNA fragmentation. Lane M: standard size markers; lanes 1 and 2: control (untreated); lanes 3 and 4: treated with 100 U/mL IFN-γ. (B) Quantitation of DNA fragments expressed in arbitrary units. (□) Control; (▪) treated with 100 U/mL IFN-γ. Data are representative of results in one of two independent experiments (each performed in duplicate) with similar results.

Figure 3.

 

Phase-contrast microscopic analysis of αTN4-1 cells. The cells were treated with 100 U/mL IFN-γ, 10 μM MG132, or 100 U/mL IFN-γ+10 μM MG132, incubated for 12 hours and photographed by phase-contrast microscope. A significant number of small, adherent, rounded, and shrunken cells were present in IFN-γ–treated samples. There was no effect on cells treated with MG132 alone, and the effect of IFN-γ was reversed when MG132 was present. Data shown are from one of three independent experiments with similar results.

Figure 4.

 

Agarose gel showing RT-PCR products of STAT-1, ICSBP, IRF-1, caspase-1, Bax, Bcl-2, and β-actin in IFN-γ–treated and untreated αTN4-1 cells after 12 hours’ incubation. Lanes 1 and 2: control (untreated); lanes 3 and 4: treated with 100 U/mL IFN-γ; lane M: standard size markers of 200, 300, 400, 500, and 650 bp. Data are representative of at least three separate experiments.

Figure 5.

 

Western blot analysis for the expression of Bcl-2 and Bax protein in IFN-γ–treated and untreated αTN4-1 cells after 12 hours’ incubation. (A) Total cell lysate was prepared from lens epithelial cells, proteins were separated by SDS-PAGE, and then the whole gel was immunoprobed. Lane M: standard size markers; lanes 1 and 2: control (untreated); lanes 3 and 4: treated with 100 U/mL IFN-γ. (B) The intensity of the bands was quantitated by densitometry. Data are expressed as the mean ± SD of triplicate determinations in a representative experiment from two independent experiments with similar results. n = 3; *significant difference from untreated control (P < 0.005).

Figure 6.

 

Upregulation of caspase-1 and -8 activities after IFN-γ–induced apoptosis in αTN4-1 cells. IFN-γ–treated cells were harvested after 12 hours’ incubation, and activity was assayed in all extracts. The experiment was performed in triplicate and the data are presented as specific activities taking the specific activity in untreated samples as 100%. Data are expressed as the mean ± SD of results in one of two independent experiments with similar results. n = 3; *significant difference from untreated control (P < 0.005).

Figure 7.

 

Measurement of caspase-3 activity by cleavage of colorimetric substrate (Ac-DEVD-pNA) and PARP cleavage. The αTN4-1 cells were treated with IFN-γ (100 U/mL), MG132 (10 μM), IFN-γ (100 U/mL)+MG132 (10 μM), STP (0.125 μM), and STP (0.125 μM)+MG132 (10 μM), incubated for 12 hours and harvested. (A) Cleavage of colorimetric substrate Ac-DEVD-pNA by caspase-3 enzyme. Equal amounts of protein were incubated with the substrate in a microtiter plate. The plate was read in a microplate reader at an absorbance of 405 nm after 1 (□), 2 (▪), 3 ( ), 4 ( ), 5 ( ), and 6 ( ) hours. (B) Western blot analysis for PARP cleavage. The total cell lysate was subjected to SDS-PAGE for protein separation and then immunoprobed using the PARP antibody, which recognizes uncleaved (116 kDa) and cleaved (85 kDa) PARP. The data are averages of duplicate determinations in one of two independent experiments with similar results.

Figure 8.

 

Prevention of IFN-γ–induced apoptosis by MG132. The cells were treated with 100 U/mL IFN-γ alone, 10 μM MG132 alone, and 100 U/mL IFN-γ+10 μM MG132, incubated for 12 hours. (A) The apoptotic cells were measured by flow cytometry after staining with FITC-conjugated annexin V and PI. Cells in the bottom left quadrant represent viable cells (low annexin V and PI staining), cells in the bottom right quadrant represent early apoptotic cells (high annexin V staining but low PI staining), and cells in the top right quadrant represent late apoptotic or necrotic cells (high annexin V and PI staining). The percentage of cells in each quadrant is indicated within the quadrant. Shown are representative data from one of two independent experiments with samples in triplicate. (B). Data are expressed as the mean ± SD of results in one of two independent experiments with similar results. n = 3 *significant difference from untreated control (P < 0.005).

The authors thank Harold I. Calvin and Raymond B. Birge for critical reading of the manuscript.