Regulation of Human Corneal Epithelial Cell Proliferation and Apoptosis by Dexamethasone

Abstract

purpose. To investigate whether human corneal epithelial cells express the glucocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells.

methods. Human corneal epithelial cells were cultured in medium supplemented with various concentrations of DEX (ranging from 10^{− 10} to 10^{− 4} M). Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 days of culture. Apoptosis was studied by nucleus labeling using a fluorescent dye and immunostaining by APO 2.7 at 6 days of culture. GR mRNA was detected in corneal epithelium and cultured corneal epithelial cells by means of reverse transcription–polymerase chain reaction (RT-PCR). Immunocytochemical staining of the epithelial cells was performed with a monoclonal anti-human GR.

results. RT-PCR and immunocytochemistry showed the expression of GR (mRNA and protein) in corneal epithelial cells. DEX significantly increased corneal epithelial cell proliferation with concentrations ranging from 10^{− 10} to 10^{− 6} M, with a maximum effect at 10^{− 7} M (P < 0.005). However, DEX also induced apoptosis of cultured corneal epithelial cells at any concentration used.

conclusions. These results indicate that human corneal epithelial cells express the GR and proliferate in response to DEX stimulation which also induces corneal epithelial cell apoptosis.

Understanding the factors that control corneal wound healing, corneal cell proliferation, apoptosis, differentiation, and the modulation of these effects by specific drugs is critical for clinically relevant problems and successful therapeutic interventions. 

Dexamethasone (DEX) is an anti-inflammatory glucocorticoid commonly used after cataract and penetrating keratoplasty surgeries. It is also used after refractive surgery (photorefractive keratectomy or laser in situ keratomileusis) in an attempt to reduce ocular surface inflammation and to delay corneal wound healing. ¹  

Despite the widespread use and demonstrated clinical effectiveness of steroids, little is known regarding the specific effects of glucocorticoids on the function of corneal cells. DEX has been shown to inhibit inflammation, through inhibition of phospholipase A2 activity and inhibition of transcription of metalloprotease genes. By preventing cellular division, glucocorticoids have been shown to decrease extracellular matrix and scar tissue formation. ² Conflicting data were reported on corneal wound healing, but investigations have shown that steroid eyedrops impair stromal and epithelial wound healing. ^{3 4 5 6 7 8} High DEX concentrations have been shown in vitro to inhibit keratocyte proliferation. ^{9 10 11 12}  

We recently demonstrated that cultured human keratocytes proliferate in response to low concentrations of DEX (10⁻⁹ to 10⁻⁵ M). ¹³ DEX also induces keratocyte apoptosis ¹³ which is probably an initiating factor in the wound-healing response after refractive surgical procedures. ^{14 15}  

Although the glucocorticoid receptor (GR) mRNA sequence has been detected in corneal epithelium by polymerase chain reaction and hot blot analyses, ¹⁶ little is known about the effect of glucocorticoids on corneal epithelial cells and the role of GR in the regulation of corneal wound healing. This study was initiated to determine whether human corneal epithelial cells express GR mRNA and the corresponding protein. DEX’s effects on corneal epithelial cell proliferation and apoptosis were also investigated. 

Materials and Methods

Human Corneal Epithelial Cell Culture

This study was performed according to the tenets of the Declaration of Helsinki. Human corneal epithelial cell primary cultures were obtained using human donor corneas that were discarded before transplantation because of low endothelial cell counts. Primary cultures of human corneal epithelium were started using explants, as previously described. ^{17 18} Under a tissue culture hood, Descemet’s membrane, endothelium, and posterior stroma were removed with forceps using a dissecting microscope. The anterior cornea with intact epithelium was covered with 1.2 U/ml Dispase II (Boehringer Mannheim, Mannheim, Germany) in calcium and magnesium-free phosphate-buffered saline (PBS) and incubated at 37°C in a humidified 5% CO₂ incubator. One hour later, corneas were placed in culture medium and 1 to 2 mm² full-thickness epithelial explants were gently peeled off with forceps from the peripheral areas (1–2 mm inside the limbus). Four to six explants were removed from the peripheral cornea. Explants were placed epithelial side up on 12-well tissue culture plates (Costar, Cambridge, MA). A 12-mm diameter sterile glass coverslip was placed on the explant in the well before the addition of culture medium. One milliliter of medium was then added to the wells. The culture medium was supplemented hormonal epithelial medium (SHEM) ¹⁹ and consisted of a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 (Gibco–Life Technology, Cergy-Pontoise, France) with 10% fetal calf serum (FCS; Gibco), 5μ g/ml insulin (Sigma–Aldrich, Saint Quentin Fallavier, France), 0.5 mg/ml cholera toxin (Sigma), 10 ng/ml human recombinant epidermal growth factor (Sigma), 0.5% dimethylsulfoxide, 2 mM l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. Steroids were removed from the FCS by means of a dextran charcoal treatment to eliminate serum steroids. ²⁰ The cultures were incubated at 37°C in 5% CO₂, and the medium changed twice a week. Corneal epithelial cells were allowed to migrate from the explants onto the surface of the wells. The cells reached confluence within 21 to 28 days. They were then enzymatically detached using 0.05% trypsin (Gibco) at 37°C for 2 minutes, after which TC199 medium with 20% FCS was added to stop the trypsinization. The suspended epithelial cells were then centrifuged at 400g for 10 minutes. The supernatant was removed, and fresh medium was added again. The single-cell suspension was counted in a hemocytometer and 2 × 10⁵ cells/well were plated using 24-well tissue culture plates (Costar). Second-passage human corneal epithelial cells were used in all the experiments. They were incubated in 1 ml of SHEM at 37°C (5% CO₂) and were allowed to attach to the bottom of the well for 24 hours before DEX was added. Epithelial cells were then cultured in SHEM supplemented with various concentrations of DEX (10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, or 10⁻⁴ M) for 6 days. 

Drug Preparation and Addition

DEX was purchased from Sigma–Aldrich. It was dissolved and serially diluted in absolute ethanol before addition to the culture medium. On the second day, SHEM was replaced by 1 ml SHEM containing various concentrations of DEX (10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰ M). DEX was added to the culture medium every day at the same concentration. In all experiments, the ethanol concentration in the culture media was maintained at 0.1%. All the solutions were filter sterilized and stored at 4°C in light-protected containers. 

The control group consisted of epithelial cells cultured in SHEM with 0.1% absolute ethanol and no DEX. The culture media were renewed every day. All the experiments were repeated six times. For each experiment—3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assays, apoptosis assays, and immunocytochemistry—all cultures were obtained from the same donor cornea. Cultured epithelial cells were studied daily by means of phase-contrast microscopy. 

RNA Preparation

Total RNA extraction was performed on primary and secondary passages of human corneal epithelial cultures. RNA samples were also prepared from ex vivo corneal epithelium that was mechanically removed from donor corneas stored in preservative medium (Inosol; Chauvin–Opsia, Toulouse, France) for less than 1 month. 

RNA extraction was performed by the acidic phenol-chloroform guanidine thiocyanate method described by Chirgwin. ²¹ Total RNAs were suspended in sterile deionized diethylpyrocarbonate (DEPC)-treated water, and aliquots were prepared and stored at− 80°C. Total RNA recovery was measured by spectrophotometric absorbance at 260 nm. 

RT-PCR for GR

For reverse transcription–polymerase chain reaction (RT-PCR), total RNA (0.25 μg) was reverse transcribed in a 30-μl reaction mixture containing 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl₂, 10 mM dithiothreitol, a 1 mM concentration of each deoxyribonucleoside triphosphate (dNTP), 1 μg oligo (dN), 1μ g oligo (dT), 24 U RNAsin (Promega, Madison, WI), and 200 U Moloney murine leukemia virus reverse transcriptase (Gibco) at 37°C for 1 hour. The reaction was terminated by heating at 90°C for 5 minutes, and samples were quick chilled on ice. 

One fifth of the RT reaction was used for the PCR amplification of the human GR. The reaction mixture consisted of 16 mM Tris-HCl (pH 8.3), 40 mM KCl, 1 mM MgCl₂, a 0.2 mM concentration of each dNTP, 50 picomoles of sense primer (ATGAGACCAGATGTAAGCTC), 50 picomoles of antisense primer (AATGCCATAAGAAACATCCA), and 1 U Taq polymerase (Perkin Elmer–Cetus, Norwalk, CT). The primers were synthesized by Gibco–Life Technology from previously published sequences. ²² As previously described, a 30-cycle amplification was completed followed by a final extension step at 72°C for 10 minutes. ¹³ The amplification was performed in a DNA thermal cycler (model 480; Perkin Elmer-Cetus). The PCR product (588 bp) was sequenced and exhibited 100% homology with the sequence from 1262 to 1949 of the α and β glucocorticoid receptor, named HSGCRAR and HSGCRBR respectively in GenBank. 

PCR Product Analysis

Five microliters of PCR samples were electrophoresed on 1% agarose gels in 90 mM buffer containing Tris borate and 2 mM EDTA. A 100-bp DNA ladder was routinely introduced (Gibco–Life Technology) as a size marker. Gels were stained with ethidium bromide and photographed under UV illumination (665 film; Polaroid, Cambridge, MA). ²³  

RT-PCR Control Samples

A negative control was routinely introduced in all assays to confirm the absence of contamination. For these controls, RNA was omitted from the RT reaction mixture and the RT was performed as described. PCR amplification was performed in the same conditions as used for the samples. Human corneal keratocytes known to express the GR were used as a positive control. ¹³  

Immunocytochemistry

Primary cultures and second-passage corneal epithelial cells were studied by indirect immunoperoxidase staining. A monoclonal mouse anti-human GR (dilution 1:100; MCA 1390; Serotec, Oxford, UK) was used. A monoclonal anti-human cytokeratin (dilution 1:400, M821; Dako, Glostrup, Denmark) was used to confirm that cultured cells were epithelial cells (positive staining for cytokeratin) and not keratocytes (negative staining for cytokeratin). Monoclonal mouse anti-human APO 2.7 (dilution 1:50; 2087; Immunotech, Marseille, France), a mitochondrial protein reliably expressed by cells involved in the apoptotic pathway, was used. ²⁴ Epithelial cells were grown onto glass slides, washed two times in PBS, and fixed with methanol. Incubation with the primary anti-GR (or anti-APO 2.7) monoclonal antibody dilution was followed with the peroxidase-labeled anti-mouse antibody (dilution 1:100; PO447; Dako). After three washings, the color reaction was developed (VIP; Vector, Burlingame, CA). Seven-micrometer cryostat sections of donor corneas were also processed with anti-GR monoclonal antibody. Negative controls consisted of cryostat section incubated with no primary antibody and section incubated with anti-vimentin monoclonal antibody (dilution 1:20; M725; Dako). 

MTS Assay

MTS and phenazine methosulfate (PMS) were obtained from Promega and Sigma, respectively. MTS is a tetrazolium salt that undergoes a color change caused by its bioreduction of MTS into a water-soluble formazan. The conversion of MTS into the aqueous-soluble formazan is accomplished by dehydrogenase enzymes found in active mitochondria and is such that the reaction occurs only in living cells. ²⁵ The quantity of formazan product measured by the amount of 490-nm light absorbance is directly proportional to the number of living cells in culture. MTS (2 mg/ml; pH 6.5) was dissolved in PBS and filter sterilized. A 3-mM PMS solution was also prepared (in PBS) and filter sterilized. These solutions were stored at −20°C in light-protected containers. To enhance the cellular reduction of MTS, PMS was added to MTS immediately before use (MTS-to-PMS ratio, 1:20). The mixture (150μ l) was added to each well. After incubation at 37°C in a humidified atmosphere with 5% CO₂ for 2 hours, 100 μl supernatant was diluted in 1 ml deionized water. The optical density was measured at 490 nm by means of spectrophotometry. Epithelial cell growth was analyzed by means of MTS assay after 2, 4, and 6 days of culture. Epithelial cell proliferation was analyzed with a hemocytometer and a cell counter (Coulter, Hialeah, FL). 

Apoptosis Assay

We also looked for DEX-induced cell apoptosis by nucleus labeling using a fluorescent dye for nuclei after 6 days of culture. Corneal epithelial cells were fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature. After a wash in PBS, the cells were incubated in a solution of 10 μg/ml Hoechst 33258 (Sigma) in PBS for 15 minutes. After another wash in PBS, the specimens were mounted in glycerol and were examined using an epifluorescence microscope (Diaphot TDM; Nikon, Tokyo, Japan) with UV filters. Six to 12 photographs of each specimen were taken using the same instrument. Photographs were analyzed by two observers in a blind fashion. The number of apoptotic cells and the total number of cells were counted. Condensed and/or fragmented nuclei appeared highly fluorescent in apoptotic cells. Cells with ruptured cytoplasmic membranes were considered to be necrotic cells. ²⁶  

Primary cultures and second-passage epithelial cells were treated each day for 6 days with three concentrations of DEX (10⁻¹⁰, 10⁻⁷, and 10⁻⁴ M) before the apoptosis assay. Control samples consisted of corneal epithelial cells cultured in SHEM medium with no ethanol and corneal epithelial cells cultured in SHEM with 0.1% absolute ethanol. All experiments were reproduced three times. 

Statistical Analysis

Data were analyzed by analysis of variance and Wilcoxon rank sum test. Commercial software (SPSS ver. 6.1.3; SPSS, Chicago, IL) was used for statistical analysis. 

Results

Expression of GR mRNA was detected in human corneal epithelium and cultured epithelial cells. A unique PCR product of 640 nucleotides was detected with ethidium bromide after gel electrophoresis (Fig. 1) . The size of this band was consistent with the expected fragment size, determined from the human GR cDNA. ²⁷ Analysis of this qualitative profile showed that GR mRNA was present in corneal epithelium and cultured epithelial cells. 

Specific nuclear intracellular staining of GR was observed in cultured epithelial cells (Fig. 2A 2B ) and, ex vivo, in the whole cornea (Fig. 3) . Both basal epithelial cells and keratocytes showed positive staining for GR. Negative controls showed no staining (data not shown). 

After 6 days of culture, DEX induced a biphasic dose-dependent effect on epithelial cell proliferation. It increased cell proliferation at concentrations ranging from 10⁻¹⁰ to 10⁻⁶ M (Fig. 4) . The maximum proliferative effect was observed at 10⁻⁷ M (P < 0.005). In contrast, 10⁻⁴ M DEX induced an inhibitory effect on cell growth (P < 0.005). There were no significant effects at days 2 and 4. All the results of the MTS assay were confirmed by the cell counter and hemocytometer proliferation assays (data not shown). 

In the nucleus labeling assay, the percentage of viable cells decreased when DEX was added to the culture medium (Figs. 5 6A 6B ). There was no statistical difference in epithelial cell viability between the SHEM group and the ethanol group (control group). Epithelial cell apoptosis and necrosis were significantly enhanced by addition of DEX, whatever the concentration used (10⁻¹⁰ M, 10⁻⁷ M, and 10⁻⁴ M; P < 0.05). APO 2.7 immunostaining confirmed the morphologic nucleus labeling assay data. There was an increased expression of the apoptotic marker APO 2.7 in the DEX groups compared with that in the control group (Fig. 6C 6D) . 

There were differences of cell morphology among the different groups. Cell density was higher in the 10⁻⁷ M DEX group (Fig. 7A ) compared with density in the control group (Fig. 7B) . Most of the epithelial cells cultured in the presence of 10⁻⁷ M DEX were small polygonal cells in the central area of outgrowth, and elongated cells with filopodia in the peripheral area. On the contrary, most of the epithelial cells cultured in 10⁻⁴ M DEX had a elongated morphologic appearance with interconnected filopodial processes (Fig. 7C) . 

Discussion

Human corneal epithelium is a stratified squamous epithelium that forms a barrier between the external environment, tears, and the intraocular environment. In addition, it contributes to the maintenance of normal stroma transparency by transporting fluid out of the stroma. It is known to have a rapid self-renewing capacity. The epithelium is frequently injured through physical or chemical insult. Wound closure after corneal abrasions involves a complex series of cellular changes in both the epithelium and the stroma. Pharmacologic control of epithelium proliferation appears to offer the potential to regulate corneal wound healing. It has been hypothesized that stimulation of epithelial cell proliferation and inhibition of cell differentiation could promote epithelial hyperplasia associated with regression after photorefractive keratectomy (PRK). ¹⁵ However, there is little specific information regarding the effects of corticosteroids on corneal epithelial cells. 

We observed a dose-dependent effect of DEX on epithelial cell proliferation with growth stimulation at low concentrations (10⁻¹⁰ M to 10⁻⁶ M) and inhibition at high concentrations (10⁻⁴ M). The similar dose–response curves for the hemocytometer cell count and the MTS assay provided evidence that they measured the same proliferative effect under our experimental conditions. The proliferative effect of DEX was observed in the presence of FCS from which steroids had been removed, indicating that it depended only on exogenous steroids. 

The apparent paradoxical biphasic effect of DEX on human corneal epithelial cell (i.e., proliferation after exposure to low doses of DEX and inhibition of growth after exposure to high doses) has been already observed in rabbit conjunctival cells, dermal fibroblasts, ¹⁰ retinal pigment epithelial cells, ²⁸ and human corneal keratocytes. ¹³ The originality of the present work is in the possible effect of low concentrations of DEX on cultured human corneal epithelial cells. Thus, low doses of DEX could also have a mitogenic effect on cultured human epithelial cells. 

It has been shown recently that AP1 components (c-Fos, c-Jun, and Fra-2) are expressed in normal ocular surface epithelia and dysplastic epithelium. ²⁹ These proto-oncogenes may play an important role in modulating epithelial cell functions (e.g., proliferation, migration, and differentiation) during epithelial wound healing. It has been suggested that immediate expression of nucleoprotein encoding proto-oncogenes could represent the molecular response that initiates the healing process. ³⁰ Moreover, proto-oncogenes of the Fos/Jun family have been shown to be massively upregulated in many basal cell layers of the corneal epithelium after UV exposure. ³¹ DEX and other steroids could interact through the AP1 proto-oncogenes. GR and AP1 interactions have recently been described in the glucocorticoid response elements (GREs), where these two transcriptional factors are adjacent. ³² Products of these proto-oncogenes may bind to DNA and act to regulate the expression of other genes that encode structural proteins and enzymes. 

Apoptosis in the corneal epithelium has been detected after mechanical injuries (e.g., corneal trauma, PRK), infection, and UV exposure. ^{33 34} This event could be considered an initiating factor in the wound-healing response. ³⁵ Epithelial cell apoptosis has also been shown to reflect physiologic epithelial turnover. A strict equilibrium can be observed, because epithelial cells that die are thought to be replaced by proliferation of activated epithelial cells. 

Glucocorticoid-induced cell death or apoptosis has been described in many cell systems including lymphocytes. ³⁶ However, glucocorticoids have been shown to protect other cell types, such as epithelial cells of the mammary gland, ³⁷ hepatocytes, ³⁸ and thymocytes. ³⁹ Our results showed that epithelial cell apoptosis and necrosis were significantly enhanced by addition of DEX. Compared with the results of MTS proliferation assays, there were no dose-dependent effects of DEX. By increasing epithelial cell apoptosis, DEX could paradoxically increase cell activation and the wound-healing response. Moreover, apoptosis which is considered as a controlled form of cell death, induces little or no inflammation compared with necrosis. The surrounding tissue could thus be protected from the release of degradative cytokines, and apoptosis could constitute a mechanism for epithelial cells to regulate cell proliferation and loss. The mechanism of apoptosis induced by glucocorticoids can fall roughly in two categories: induction of“ death genes” by the activated GR or repression of survival factors. In any case, glucocorticoid-induced apoptosis provides further evidence for the existence of GR in corneal epithelial cells. 

Glucocorticoids mediate their effects after binding to a specific intracellular receptor belonging to the steroid receptor superfamily: the GR. Once activated, the GR can mediate its effects through direct binding on the DNA or through protein–protein interactions with transcription factors. ⁴⁰ Wilson identified GR mRNA sequences in each of the three major cell types of the cornea (i.e., corneal epithelial cells, keratocytes, and endothelial cells), ¹⁶ as well as in human lacrimal gland. ⁴¹ Previous studies have identified GR binding sites in rabbit corneal epithelium ^{42 43} and cytosol prepared from whole bovine cornea. ⁴⁴ In the rabbit, it was demonstrated that cytoplasmic GR translocates to the cell nucleus after topical administration of DEX. ⁴³ We have used the RT-PCR technique to show that in vitro and ex vivo human corneal epithelial cells produced mRNA coding for the GR. Immunocytochemistry results showed the presence of receptor protein and confirmed that GR mRNA is physiologically relevant in human corneal epithelium. Moreover, identification of GR in each of the three major cell types of the cornea suggests that steroids may have autocrine and/or paracrine roles in the cornea and that endogenous steroids can be found in the cornea. Midelfart et al. ⁴⁵ recently used nuclear magnetic resonance (NMR) spectroscopy to study the penetration and metabolism of DEX phosphate in the rabbit cornea after topical administration. Many endogenous metabolites were detected among DEX in the extracts of rabbit cornea. These endogenous metabolites could be synthesized from cholesterol, which is present in the cornea. ⁴⁶ Further investigations are needed to determine whether these steroids are produced by corneal epithelial cells, keratocytes, endothelial cells, or all three cell types. 

In conclusion, we have shown the expression of GR in human corneal epithelial cells. Such a receptor is functional because DEX significantly increases epithelial cell proliferation, apoptosis, and necrosis. 

 Supported by a grant from the Association Claude Bernard and Institut National de la Santé et de la Recherche Médicale, Equipe de Recherche Clinique Associée.

 Submitted for publication March 15, 2000; revised July 5, 2000; accepted July 19, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Electrophoretic profile of the human GR product obtained from RT-PCR. Total RNA (0.25 μg) from cultured human epithelial cells and 30 μg total RNA from cultured human keratocytes were reverse transcribed using oligo(dN) and oligo(dT) primers. The obtained cDNA were electrophoresed on a 1% agarose gel, and the bands were visualized by ethidium bromide staining. Lane 1: Human corneal epithelial cells; lane 2: human keratocytes, known to express GR (positive control); lane M: 100-bp DNA ladder.

Figure 2.

 

Immunostaining of cultured human corneal epithelial cells with monoclonal anti-human GR antibody. Cultured epithelial cells showed positive staining for GR (A) compared with control (B). Inverted microscopy. Magnification, ×200.

Figure 3.

 

Immunostaining of cryostat section of human cornea with monoclonal anti-human GR antibody. The immunoreactivity in the cornea was confined to basal epithelial cells and keratocytes. No significant immunoreactivity was observed in the superficial cells of the epithelium. Inverted microscopy. Magnification, ×200.

Figure 4.

 

Corneal epithelial cell proliferation studied by MTS assay. Human corneal epithelial cells were cultured with various concentrations of DEX diluted in 0.1% final absolute ethanol (10^{− 4}, 10^{− 5}, 10^{− 6}, 10^{− 7}, 10^{− 8}, 10^{− 9}, and 10^{− 10} M) for 6 days. Results are expressed in optical density, which was measured at 490 nm by means of spectrophotometry. Bars, SD (n = 6 for each group). The control group consisted of epithelial cells cultured in charcoal-treated SHEM with 0.1% absolute ethanol. After 6 days of culture, DEX induced a dose-dependent increase in epithelial cell proliferation at concentrations ranging from 10^{− 10} to 10^{− 6} M. The maximum proliferative effect was observed at 10^{− 7} M (P < 0.005). However, 10^{− 4} M DEX had an inhibitory effect on cell growth (P < 0.05). Significantly different from the control group by the Wilcoxon rank sum test:* P < 0.05; **P < 0.005.

Figure 5.

 

Apoptosis assay. Results are expressed for viable (A), apoptotic (B), and necrotic cells (C) in percentage of cells (mean ± SD). Apoptosis was significantly enhanced by addition of 10⁻⁴ M, 10⁻⁷ M, and 10⁻¹⁰ M DEX to the culture medium. Epithelial cell necrosis was also significantly enhanced by addition of DEX to the culture medium. There was no statistical difference in epithelial cell viability between the SHEM group and the ethanol group (0.1%). Significantly different from the control group by the Wilcoxon rank sum test: *P < 0.05.

Figure 6.

 

Apoptosis assay. The late phase of apoptosis was studied by nucleus labeling using a fluorescent dye for the nuclei after 6 days of culture. Condensed and/or fragmented nuclear appeared highly fluorescent in apoptotic cells (arrows). The percentages of apoptotic and necrotic epithelial cells were significantly higher in the 10^{− 7} M DEX group (A) than in the control group (B; P < 0.05). The early phase of apoptosis was studied by immunolabeling with APO 2.7 mAb. There was a much stronger expression of the apoptotic marker APO 2.7 in the 10⁻⁷ M DEX group (C) than in the control group (D). Magnification: (A, B) ×100; (C, D)× 200.

Figure 7.

 

Inverted phase-contrast micrographs of human epithelial cells after 6 days of culture and DEX treatment. There were differences in cell morphology among the groups. Cell density was higher in the SHEM + 10^{− 7} M DEX group (A) compared with density in the control group (SHEM + 0.1% ethanol; B). The epithelial cells cultured in the presence of SHEM culture medium + 10⁻⁷M DEX diluted in 0.1% final absolute ethanol were small polygonal cells in the central area of outgrowth, and elongated cells with filopodia in the peripheral area. On the contrary, most of the epithelial cells cultured in SHEM + 10⁻⁴ M DEX diluted in 0.1% final absolute ethanol had an elongated morphologic appearance with interconnected filopodial processes (C). Magnification,× 200.