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Originally published In Press as doi:10.1074/jbc.M503698200 on May 19, 2005

J. Biol. Chem., Vol. 280, Issue 30, 27998-28006, July 29, 2005
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Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells*

Aglaia Pappa{ddagger}§, Donald Brown¶, Yiannis Koutalos||, James DeGregori**, Carl White{ddagger}{ddagger}, and Vasilis Vasiliou{ddagger}§§

From the {ddagger}Molecular Toxicology and Environmental Health Sciences Program, University Colorado Health Sciences Center, Denver, Colorado 80262, Department of Ophthalmology, University of California, Irvine, California 92868, ||Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425, **Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, and {ddagger}{ddagger}Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, April 5, 2005 , and in revised form, May 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldehyde dehydrogenase 3A1 (ALDH3A1) is a NAD(P)+-dependent enzyme that is highly expressed in mammalian corneal epithelial cells and has been shown to protect against UV- and 4-hydroxynonenal-induced cellular damage, mainly by metabolizing toxic lipid peroxidation aldehydes. Here we report a novel function of ALDH3A1 as a negative cell cycle regulator. We noticed a reduction in ALDH3A1 gene expression in actively proliferating primary human corneal epithelium explant cultures, indicating that ALDH3A1 expression is inversely correlated with replication. To examine this further, a human corneal epithelial cell line (HCE) lacking endogenous ALDH3A1 was stably transfected to express ALDH3A1 at levels similar to those found in vivo. ALDH3A1-transfected cells exhibited an elongated cell cycle, decreased plating efficiency, and reduced DNA synthesis compared with the mock-transfected cells. These effects were associated with reduced cyclin A- and cyclin B-dependent kinase activities and reduced phosphorylation of the retinoblastoma protein (pRb) as well as decreased protein levels of cyclins A, B, and E, the transcription factor E2F1, and the cyclin-dependent kinase inhibitor p21. These observations were further expanded and confirmed on human keratinocyte cells (NCTC-2544) overexpressing ALDH3A1. Consistent with a protective role of an elongated cell cycle, ALDH3A1-transfected cells exhibited increased resistance to the cytotoxic effects of the DNA-damaging agents mitomycin C and Vp-16. Immunohistochemistry and biochemical fractionation revealed that ALDH3A1 is localized both in the nucleus and cytosol of ALDH3A1-transfected cells, implying a possible association between the nuclear localization of the enzyme and its proliferation-suppressive functions. In conclusion, these results suggest that ALDH3A1 may protect corneal epithelial cells against oxidative damage not only through its metabolic function but also by prolonging the cell cycle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corneal epithelium is a self-renewing stratified epithelial tissue that maintains transparency and protects the underlying structures of the eye. It is characterized by continuous cell turnover and contains proliferating basal cells and differentiating suprabasal and intermediate cells as well as terminally differentiated superficial squamous cells that eventually desquamate. The corneal epithelium is maintained by the centripetal migration of proliferating basal corneal epithelial cells derived from the stem cells located in the limbal epithelium (1). The proliferating basal corneal epithelial cells give rise to transient amplifying cells that can undergo a limited number of cell divisions before following a pathway of terminal differentiation (2, 3). The intermediate cells of the corneal epithelium are post-mitotic and, together with the terminally differentiated cells, are incapable of cell division (4). The superficial epithelial cells are finally removed into the tear pool by a variety of mechanisms including apoptosis (5).

The cornea serves as barrier between the external environment and the internal ocular tissues, protecting them against oxidative stimuli that include solar radiation and molecular oxygen. Mammalian corneal epithelial cells express high levels of ALDH3A11 (6), an enzyme that is part of a well regulated defense system that protects the eye against oxidative damage (7, 8). Because this enzyme represents nearly half of the total soluble protein in the corneal epithelium, levels that exceed those required for normal metabolism, it has been proposed to be a corneal crystallin, mimicking the situation in the lens (6, 9) where metabolic proteins are overexpressed for their intrinsic refractive properties. Additional functions have been hypothesized regarding the role of ALDH3A1 in corneal epithelium. These include direct absorption of UV radiation, scavenging of UV-generated reactive oxygen species (ROS), and a chaperone-like function preventing aggregation of partially denatured proteins (10). We have recently shown that ALDH3A1 protects corneal epithelial cells against oxidative damage induced by either UV radiation or the highly reactive product of lipid peroxidation 4-hydroxynonenal (4-HNE) (7). In addition, we have reported that ALDH3A1 is expressed mainly in the suprabasal cells of the corneal epithelium (8), which are known to be post-mitotic (4). The aim of the present study was to investigate a possible function of ALDH3A1 as a modulator of proliferation in the corneal epithelium. We report here a novel role of ALDH3A1 in retarding corneal epithelial cell cycle progression that might contribute to its ocular protective effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ALDH3A1 Stably Transfected Cell Lines—The SV-40-transformed corneal epithelial cell lines (HCE, ATCC CRL-11135) stably transfected with the mammalian expression vector {Delta}pCEP4 alone (Mock-HCE) or with the human ALDH3A1 cDNA (ALDH3A1-HCE) along with tissue culture conditions have been described elsewhere (7, 11). NCTC-2544 keratinocytes were grown in Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum (Sigma), 25 mM Hepes, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a humidified 5% CO2 incubator. NCTC-2544 cells were transfected with the same expression vectors used in the HCE cells by Lipofectamine 2000 (Invitrogen). Stable cell populations were selected by incubation in media containing hygromycin (0.4 mg/ml) (Invitrogen), and allowed to form colonies that were further expanded. ALDH3A1 expression was screened using Western blot analyses, enzyme activity assays, and immunofluorescence (as described below).

Primary Explant Cultures of Human Corneal Epithelium—A normal corneal rim was obtained after the central cornea had been removed by a surgeon from patients presenting with penetrating keratoplasty (Institutional Review Board protocol 2004-3543). The remnant was trimmed in the laboratory after locating the scleral/corneal junction to 3 mm either side of this border. This was then cut into 16 equal-sized wedges and placed onto gelatin-coated plates. Media/keratinocyte-serum-free medium supplemented with epidermal growth factor (5 ng/ml) and bovine pituitary extract (50 µg/ml) containing penicillin (100 units/ml) and streptomycin (100 µg/ml) was placed into each dish and incubated at 37 °C and 5% CO2. Half of the dishes received the same media with the further addition of cholera toxin (0.1 µg/ml) and Me2SO (0.5%). The media were changed every other day, and the outgrowth of epithelial cells was monitored by microscopy. On day 5 the residual tissue was removed, and cultures continued until confluence was reached (~2 weeks). On days 5, 6, and 7 and at confluence cells were harvested and processed for RNA isolation, Western blot analysis, and immunofluorescence. Reverse transcription was performed using 100 ng of total RNA and random hexamers followed by PCR with primers designed from the ALDH3A1 sequence (GenBankTM accession number NM_000691 [GenBank] ) and spanning 3 exons. Primers were 5'-TGTTCTCCAGCAACGACAAG-3' and 5'-CTGACCTTCAGGCCTTCATC-3'. Primer specificities were confirmed by a BLAST Internet software-assisted search of the non-redundant nucleotide sequence data base. PCRs were carried out with 5-25 ng of reverse-transcribed RNA, Taq polymerase buffer (200 µM deoxyribonucleoside triphosphates, 1.25 units of Taq polymerase, and 200 nM forward and reverse primers), in a total volume of 50 µl. PCR controls without reverse transcriptase or with normal human genomic DNA (as a template) were routinely negative.

Western Immunoblotting—Whole cell lysates were subjected to electrophoresis and immunoblotted according to previously described methods (7). Primary antibodies used included monoclonal anti-ALDH3A1 (8), antibodies to cyclin A (sc-751), cyclin B (sc-245), cyclin E (sc-481), retinoblastoma (sc-102; recognizes both the unphosphorylated (pRb) and phosphorylated form (ppRb)), E2F1 (sc-193), and SV-40 large T antigen (obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-{beta}-tubulin (Sigma), anti-lamin B1 (Zymed Laboratories Inc., San Franscisco, CA). Anti-chicken, anti-rabbit, and anti-mouse immunoglobulin G-conjugated horseradish peroxidase antibodies were obtained from Jackson Laboratories (Jackson, West Grove, PA). When necessary, the antibodies were stripped from the membrane with stripping solution (62.5 mM Tris-HCl, pH 6.7, 2% SDS, 6 µl/ml 2-mercaptoethanol) and re-probed with an alternate antibody. Labeled proteins were detected by enhanced chemiluminescence.

ALDH3A1 Enzymatic Assay—ALDH3A1 activity was determined using 0.5 mM benzaldehyde (substrate) and 2.5 mM NADP+ (coenzyme) as described previously (7). ALDH3A1 activity was normalized to total cytosolic or nuclear protein concentration measured by the bicinchoninic acid method (Pierce).

Growth Curves and BrdUrd Incorporation Assay—Cells (1 x 104) of each clone were plated in a 12-well dish. The growth rate of cells was determined by counting the number of cells with a hemocytometer as a function of time. Cell growth curves were plotted, and cell population doubling times and length of cell cycles were calculated from the linear component of the exponential growth phase (GraphPad Prism 4.0, GraphPad Software Inc., San Diego, CA). For BrdUrd incorporation assays, cells (5 x 104) were plated in 96-well dishes and treated with 100 µM BrdUrd for 24 h. BrdUrd incorporation was determined using a non-isotopic enzyme immunoassay (Oncogene, Boston, MA) according to the manufacturer's procedure.

Colony Formation Assay—Cells (5 x 103) were plated in 6-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO2 in air to allow colony formation. The cultures were monitored on a daily basis, and when colonies were visible (~2 weeks later) cells were fixed and stained with 0.1% crystal violet. Colonies containing ≥50 cells were scored and counted.

Histone H1 Kinase Assay—Cells (2 x 106) grown in 100-mm plates were harvested and immunoprecipitated with antibodies specific for cyclin A, cyclin B, and cyclin E, and associated kinase activities were determined using histone H1 (Roche Diagnostics) as the substrate as described previously (12).

Immunofluorescence Microscopy—HCE and NCTC-2544 cells were grown in the appropriate media on glass coverslips, rinsed in phosphate-buffered saline (PBS), and then fixed with 4% buffered formalin in PBS. Cells were permeabilized in cold methanol, washed with PBS, and incubated with a monoclonal antibody against human ALDH3A1 (1:1) for 1 h. The cells were subsequently washed with PBS and stained with Texas Red-conjugated anti-mouse secondary antibody (1:20). Enzyme distribution was visualized using a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY), a xenon continuous arc light source (Sutter Instrument Co., Novato, CA), and a Zeiss 40x Plan Neofluar objective lens (numerical aperture = 1.3) and monitored through a CCD camera (Sensicam, Cooke Corp., Auburn Hills, MI). Texas Red fluorescence was excited at 555 nm, and emission was measured at 617 nm. 4',6-diamidino-2-phenylindole (DAPI) fluorescence was excited with 360 nm light, and emission was measured at 457 nm. Immunostaining of human corneal epithelial explants was conducted as follows. Corneal epithelial explant cultures (n = 2) were washed in media and then fixed in 1% paraformaldehyde in PBS for 5 min. Fixed cells were then permeabilized with acetone/methanol (-20 °C) and then incubated in PBS for 15 min. Primary antibody was applied to the cells and incubated in a moist chamber for 60 min. Cells were washed with PBS and incubated with rhodamine-conjugated secondary antibody (Chemicon International, Temecula, CA) for 60 min in a moist, dark chamber. After a washing step with PBS, the cells were examined and photographed using a Leica inverted fluorescent microscope equipped with a digital camera.

Confocal Microscopy—Confocal images were acquired using a Zeiss 63x oil immersion lens (numerical aperture = 1.4) on a Zeiss Axioskop 2FS MOT that is part of a Zeiss LSM 510 system (Carl Zeiss, Thornwood, NY). The pixel size for the laser scans was 0.29 x 0.29 µm. For nuclear staining with DAPI, fluorescence was measured using 458 nm of excitation (argon laser) and 480-520 nm of emission. Texas Red fluorescence was measured using 543 nm of excitation (He-Ne laser) and >560 nm of emission. The overall z axis resolution was 0.8 µm (confocality <0.8 µm for Texas Red and <0.6 nm for DAPI). Intensity profiles were generated from image analysis using ImageJ software (rsb.info.nih.gov/ij).

Preparation of Cellular and Nuclear Extracts—Total cell lysates were prepared as described previously (13). For the preparation of cytosolic and nuclear extracts, freshly collected cells were washed three times in ice-cold PBS and incubated in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% Nonidet P-40, 1.0 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml pepstatin A, 2.5 µg/ml leupeptin) for 15 min. The nuclei were pelleted by centrifugation at 600 x g for 10 min and washed 4 times in buffer A. The nuclear extracts were isolated by incubation of the nuclei in buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.1 mM EDTA, 1.0 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml pepstatin A, 2.5 µg/ml leupeptin) for 30 min at 4 °C followed by centrifugation at 10,000 x g for 10 min.

DNA Fragmentation Assay—Cells (1 x 106) were treated with either Vp-16 (50 µM, Sigma) or mitomycin C (5 µg/ml, Sigma) for 16 h. After treatment cells were collected and resuspended in lysis buffer (10 mM Tris-HCl, pH 7.4, containing 10 mM EDTA, 10 mM NaCl, 0.5% SDS, and 0.1 mg/ml proteinase K) and incubated overnight at 50 °C. DNA was extracted using phenol-chloroform and subsequently incubated with RNase A (0.1 mg/ml) for 1 h at 37 °C.DNA samples were analyzed using conventional electrophoresis in 1.5% (w/v) agarose gel and visualized under UV illumination.

Statistical Analysis—All values are expressed as the mean ± S.E. Comparison of results between different groups was performed by Student's t test using SigmaPlot (Version 7.0, 2001).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ALDH3A1 in Primary Explant Cultures of Corneal Epithelium—ALDH3A1 gene expression was studied in primary explant cultures of corneal epithelium. As shown in Fig. 1, ALDH3A1 expression appears to be down-regulated during corneal epithelial proliferation. Explant cultures at days 5 and 6 and confluence had progressively less product regardless of whether cholera toxin, known to help maintain expression of ALDH3A1 in primary rat corneal explants (14), was included in the medium (Fig. 1A). When examined by Western analysis, cultures at days 5 and 6 did have detectable ALDH3A1, although these levels appeared to be decreasing (Fig. 1A); at confluence, ALDH3A1 was not detectable (not shown). The reverse transcription-PCR analysis revealed the expected 230-bp product as detected in direct isolates of corneal epithelium (Fig. 1B, lanes a-d). When day 7 cultures were examined by immunofluorescence (Fig. 1C), the staining pattern was non-uniform in the cell and appeared to be associated with discreet vesicles and cellular remnants not containing nuclei (note arrowheads). These data demonstrate a progressive loss of the ALDH3A1 gene expression in the actively proliferating primary explant cultures of corneal epithelium.



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  		FIG. 1.
Expression of ALDH3A1 in primary explant cultures of corneal epithelium. A, Western blot analysis of paired explant cultures grown with (+ Toxin) or without (-Toxin) cholera toxin supplementation and harvested 5 or 6 days after initiation of the culture. Blots were probed with a monoclonal antibody to ALDH3A1 and developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. Each sample showed staining of a specific band of ~ 54 kDa, but in each case the staining was markedly reduced on day 6. B, reverse transcription-PCR analysis of ALDH3A1 in paired explant cultures grown with (+ Toxin) or without (-Toxin) cholera toxin supplementation and harvested 5 or 6 days after initiation of the culture. In both cultures and regardless of cholera toxin (+ Toxin) supplementation, no product was visualized from these samples. As a positive control, corneal epithelial RNA was harvested from four autopsy corneas (lanes a-d). As expected, reverse transcription-PCR of these samples resulted in a band of the expected size. The lane marked -RT indicates a control reaction of RNA (from dissected corneal epithelium) processed without reverse transcriptase. All samples showed a high level of product when primers specific for {beta}2-microglobulin were used (not shown). C, immunofluorescence staining of corneal explant cultures on day 7 grown without cholera toxin (panels a-c) or with cholera toxin (panels d-f). Panels a and d show the appearance of the cultures by light microscopy, whereas panels b and e show the immunofluoresence obtained using the monoclonal antibody to ALDH3A1 and a rhodamine-conjugated secondary antibody. Panels c and f represent the merged image showing that the fluorescent signal is associated with cell remnants (arrowheads) and is non-uniformly distributed within the cell. M, molecular weight standards.

 
Expression of ALDH3A1 in Human Corneal Epithelial Cells Inhibits Cell Growth—We have generated a stable-transfected human corneal cell line (ALDH3A1-HCE) expressing a metabolically active human ALDH3A1 at levels comparable with those found in vivo (7). Fluorescence microscopy (Fig. 2A) and Western immunoblotting (Fig. 2B) confirmed expression of the enzyme in ALDH3A1-HCE cells and the absence of the protein from parental (naïve) HCE cells or from HCE cells transfected with empty vector alone. One of the distinctive phenotypic changes that we have observed previously in ALDH3A1-HCE cells is a slower proliferation rate than the mock-transfected HCE cells or parental HCE cells (data not shown). For this reason the characteristics of growth curve, cell population doubling time, DNA synthesis, and plating efficiency were examined. ALDH3A1-HCE cells demonstrated a significantly slower growth rate compared with the mock-HCE cells (Fig. 2C). The doubling time and the length of cell cycle in mock-HCE cells were found to be 1.8 and 2.7 days, respectively (Table I). Similar values were determined for the parental HCE cells (data not shown). Expression of ALDH3A1 in HCE cells increased the doubling time to 4.1 days and the length of cell cycle to 5.9 days (Table I). In addition, expression of the enzyme was associated with a significantly reduced DNA synthesis as indicated by BrdUrd incorporation assays (Fig. 2D). Consistent with these findings, ALDH3A1-HCE cells showed a 70% decrease in colony formation efficiency relative to mock-HCE cells (Fig. 2E). Because the HCE cell line is SV-40-transformed (11), we examined if the decreased cell proliferation resulted from disruption of the SV40 large tumor T antigen after ALDH3A1 transfection. There were no apparent differences between any of the cells lines in expression of SV40 large tumor T antigen (Fig. 2F).


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  		TABLE I
ALDH3A1 retards cell proliferation in stably transfected HCE and NCTC-2544 cells

Cell population doubling time and length of the cell cycle for each cell line were calculated from the cell growth curves (Figs. 2B and 3C) using Graph Pad Prism 4.0 according to formulas provided by the manufacturer. Data are the mean ± S.E.; n = 3.

 



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  		FIG. 2.
Expression of ALDH3A1 negatively regulates cell proliferation in HCE cells. A, immunohistochemical detection of ALDH3A1 in HCE cells transfected with empty vector (Mock-HCE) and HCE cells stably transfected with human ALDH3A1 (ALDH3A1-HCE). After fixation cells were permeabilized and stained for microscopic analysis using a polyclonal antibody against the human ALDH3A1 (red). Nuclei were also stained with DAPI (blue). Bars, 10 µm. B, detection of ALDH3A1 in Mock-HCE and ALDH3A1-HCE cells by Western blot analysis. Whole cell lysates (30 µg) of parental HCE, mock-HCE, and ALDH3A1-HCE cells were analyzed for the expression of ALDH3A1 and {beta}-actin by Western blotting. C, cell growth curves of Mock-HCE (closed squares) and ALDH3A1-HCE (closed triangles). Cells (1 x 104) were seeded in 12-well dishes, and cell populations were counted at the indicated time points. Data are presented as the mean ± S.E. from three independent determinations. *, p < 0.001, compared with Mock-HCE at the same time point. D, DNA synthesis in the Mock-HCE and ALDH3A1-HCE cells. Cells (5 x 104) were seeded in 96-well plates and monitored for DNA synthesis by the BrdUrd incorporation assay, as described under "Material and Methods." Incorporated cellular BrdUrd (BrDU) was detected by measuring the fluorescence product created by peroxidase-conjugated IgG bound to anti-BrdUrd antibody. Data are expressed as relative fluorescence signal units (RFU) and presented as the mean ± S.E. from three independent determinations. (*, p < 0.001). E, expression of ALDH3A1 reduces colony formation efficiency in HCE cells. Mock- and ALDH3A1-HCE cells (5 x 103) were plated in 6-well culture dishes, and colonies were stained with crystal violet after ~2 weeks. Representative plates are shown in the left panel. Colony numbers obtained for Mock-HCE and ALDH3A1-HCE cells are shown in the right panel, with data presented as mean ± S.E. from three independent experiments (*, p < 0.001). F, SV40 large T-antigen is present in ALDH3A1-transfected HCE cells. Whole cell lysates (30 µg) of parental HCE, Mock-HCE, and ALDH3A1-HCE cells were analyzed for the expression of SV40 large T-antigen and {beta}-actin by Western blotting.

 



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  		FIG. 3.
Overexpression of ALDH3A1 negatively regulates cell proliferation in NCTC-2544 cells. A, Western blot analysis of ALDH3A1 in parental (lane 1) and mock-NCTC-2544 (empty vector) (lane 2) and ALDH3A1-NCTC (lane 3) cells. Purified human ALDH3A1 (1 µg) was also included (lane 4). Equal protein loading was confirmed by {beta}-actin. B, cell growth curves for mock- (closed squares) and ALDH3A1-NCTC-2544 (closed triangles). Cells (1 x 104) were seeded in 12-well dishes, and cell populations were counted at the indicated time points. Data are presented as the means ± S.E. from three independent determinations. *, p < 0.001, compared with mock-NCTC cells at the same time point. C, inhibition of DNA synthesis in ALDH3A1-NCTC cells. Mock- and ALDH3A1-NCTC-2544 cells (5 x 104) were seeded in 96-well plates and monitored for DNA synthesis using the BrdUrd incorporation assay (as described under "Materials and Methods"). Incorporated cellular BrdUrd (BrdU) was detected by measuring the fluorescence product created by peroxidase-conjugated IgG bound to anti-BrdUrd antibody. Data are expressed as relative fluorescence signal units (RFU) and presented as means ± S.E. from three independent determinations (*, p < 0.001). D, expression of ALDH3A1 reduces colony formation efficiency in NCTC-2544 cells. Mock- and ALDH3A1-NCTC-2544 cells (5 x 103) were plated in 6-well culture dishes, and colonies were stained with crystal violet after ~2 weeks. Representative plates are shown in the left panel. Mean colony numbers obtained with mock- and ALDH3A1-NCTC-2544 cells are shown in the right panel with data presented as the means ± S.E. from three independent experiments (*, p < 0.001).

 



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  		FIG. 4.
Asynchronous cultures of ALDH3A1-transfected HCE and ALDH3A1-transfected NCTC-2544 cells exhibit reduced cyclin-dependent kinase activities. A, cyclin A, cyclin B, and cyclin E were immunoprecipitated from the lysates of asynchronous growing cultures of mock-transfected (lane 1) and ALDH3A1-transfected (lane 2) HCE cells. B, cyclin A, cyclin B, and cyclin E were immunoprecipitated from the lysates of asynchronous-growing cultures of mock-transfected (lane 1) and ALDH3A1-transfected (lane 2) NCTC-2544 cells. The cyclin A-, cyclin B-, and cyclin E-associated kinase activities were determined using histone H1 as the substrate by mixing 5 µg of histone H1 and 5 µCi of [{gamma}-32P]ATP with the immunoprecipitates and incubating at 37 °C for 30 min. Phosphorylation of histone H1 (indicated by an arrow) by cyclin-dependent kinases was analyzed by SDS-PAGE.

 
Overexpression of ALDH3A1 in Human Keratinocytes Inhibits Cell Growth—To test whether the effect of ALDH3A1 on cell growth was specific to HCE cells, we overexpressed ALDH3A1 in a human keratinocyte cell line NCTC-2544, which is a spontaneously transformed human epithelial cell line that expresses ALDH3A1. Transfection of ALDH3A1 into NCTC-2544 cells resulted in a 4-fold overexpression of ALDH3A1 (Fig. 3A, lane 3) relative to parental (Fig. 3A, lane 1) or mock (empty vector)-transfected (Fig. 3A, lane 2) cells. The enzymatic activity of ALDH3A1 in cellular extracts was also significantly elevated in ALDH3A1-NCTC-2544 cells (123 ± 18 nmol of NADPH/min/mg of protein) when compared with parental cells (26 ± 4 NADPH/min/mg of protein) or mock-transfected cells (28 ± 5 nmol of NADPH/min/mg of protein). Overexpression of ALDH3A1 increased the doubling time and the length of the cell cycle in ALDH3A1-NCTC-2544 cells to 3.8 and 5.4 days, respectively, whereas mock-transfected cells were estimated to be 1.4 and 2.0 days, respectively (Table I). BrdUrd incorporation revealed that expression of ALDH3A1 is associated with reduced DNA synthesis (Fig. 3C). Furthermore, ALDH3A1-NCTC-2544 cells exhibited a significantly lower plating efficiency relative to mock-transfected cells (Fig. 3D).

Expression of ALDH3A1 Inhibits Cyclin-dependent Kinase Activities—To gain further insight into the mechanism by which ALDH3A1 affects the cell cycle progression, we examined the activities of the cyclins A, B, and E in extracts from mock- and ALDH3A1-transfected HCE and NCTC-2544 cells. A significant reduction in all cyclin-associated activities was observed in HCE and NCTC-2544 cells transfected to express ALDL3A1 when compared with their respective mock-transfected cells (Fig. 4).

Altered Expression Profiles of Cell Cycle Regulatory Proteins in ALDH3A1-transfected Cells—Because the cell cycle is controlled by expression and activation of several cyclins and cyclin-dependent kinases, we addressed whether their expression levels were altered by the presence of ALDH3A1. Protein expression of cell cycle regulatory proteins in mock-HCE, ALDH3A1-HCE, and ALDH3A1-NCTC-2544 cells were examined (Fig. 5). When compared with mock-transfected cells, expression of cyclin A was decreased, whereas expression of cyclins B and E were abolished in ALDH3A1-expressing cells compared with mock-transfected cells. Expression of ALDH3A1 was also associated with reduction of the phosphorylated form of pRb. Decreased levels of E2F1 and its target p21 (15) were also observed in ALDH3A1-expressing HCE and NCTC-2544 cells relative to their mock-transfected counterparts.



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  		FIG. 5.
Stable transfection of ALDH3A1 alters the expression profile of cell regulatory proteins in HCE and NCTC-2544 cells. Whole lysates (30 µg) of asynchronous cultures of Mock-HCE (lane 1) and ALDH3A1-HCE (lane 2) and Mock-NCTC-2544 (lane 3) and ALDH3A1-NCTC-2544 (lane 4) cells were subjected to Western blot analysis. Protein levels were identified using anti-cyclin A, anti-cyclin B, anti-cyclin E, anti-pRB (recognized both the non-phosphorylated and the phosphorylated form of pRb), anti-E2F1, and anti-p21 antibodies (as described under "Materials and Methods"). Detection of {beta}-actin was performed as an internal control for protein loading.

 



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  		FIG. 6.
Expression of ALDH3A1 protects HCE cells from apoptosis induced by Vp-16 or mitomycin C. Mock-HCE or ALDH3A1-HCE cells were incubated in the absence (-) or presence (+) of the DNA-damaging agents mitomycin C (5 µg/ml) or Vp-16 (50 µM) for 16 h. DNA fragmentation was evaluated as described under "Materials and Methods."

 
Expression of ALDH3A1 Protects HCE Cells against Apoptosis Induced by DNA-damaging Agents—We have shown that ALDH3A1 protects human corneal epithelial cells from either UV- or 4-HNE-induced apoptosis (7). Inhibition of apoptosis was attributable to the role of ALDH3A1 in metabolizing (and, thus, eliminating) toxic lipid peroxidation aldehyde byproducts (7). The association of ALDH3A1 with inhibition of cell growth led us to hypothesize that ALDH3A1 retards the cell cycle, thus promoting cell survival. To examine this possibility, ALDH3A1- and mock-HCE cells were treated with DNA-damaging agents (viz. mitomycin C or Vp-16), and apoptosis was evaluated by the DNA fragmentation assay. In the absence of drug treatment, no DNA laddering was apparent in either ALDH3A1-HCE or mock-HCE cells. Upon treatment with either mitomycin C or Vp-16, DNA fragmentation occurred to a significant extent in the mock-HCE cells but only to a minor extent in ALDH3A1-HCE cells (Fig. 6).

Cellular Localization of ALDH3A1 in ALDH3A1-HCE Cells—The involvement of ALDH3A1 in the regulation of cell cycle is intriguing, and as yet we do not know the exact mechanism by which this protein may exert its inhibitory effect on the cell cycle. The distribution pattern of a protein within the cell may provide important information regarding this function. We have, therefore, used a combination of confocal immunofluorescence microscopy and biochemical fractionation to visualize and determine the distribution of ALDH3A1 in ALDH3A1-HCE cells. Expression of ALDH3A1 was detected in both the cytoplasmic and the nuclear compartments in ALDH3A1-HCE cells (Fig. 7A, top panel). Although ALDH3A1 was mainly expressed in the cytoplasm, a significant amount of the protein appeared to be localized in the nucleus. Fig. 7A, bottom panel, also shows the intensity profile of nuclear and ALDH3A1 staining (expressed as arbitrary fluorescence units) generated by image analysis. By examining the fluorescence intensity at different focal planes and at the same confocality, we ensured that the fluorescence could not have originated from the cytoplasm present above or below the nucleus. To confirm the nuclear localization of ALDH3A1, we employed a biochemical fractionation procedure followed by Western blot analysis and enzymatic assays. Immunoblot analysis of cytosolic and nuclear fractions in ALDH3A1-HCE cells indicated the presence of ALDH3A1 in the cytosol and the nucleus in these cells (Fig. 7B). Tubulin was used as a cytosolic marker, whereas lamin B1 was used as a nuclear marker. The absence of lamin B1 and {beta}-tubulin from the cytoplasmic and nuclear fractions, respectively, verifies the effectiveness of the isolation processes and argues against the notion that ALDH3A1 in the nuclear compartment may be a cytoplasmic fraction contaminant. Enzyme activity analyses (Fig. 7C) revealed that ALDH3A1 activity was greater in the cytoplasmic compartment than in the nuclear compartment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we report a novel function of ALDH3A1 associated with inhibition of cellular growth. We used human HCE and skin keratinocytes (NCTC-2544) to investigate the role of ALDH3A1 in modulating cell proliferation. We provide evidence that ALDH3A1 serves as an inhibitor of cell growth. First, proliferating primary corneal epithelial cells exhibited a gradually decreasing ALDH3A1 expression. Second, induced expression of ALDH3A1 in HCE cells, which do not constitutively express the enzyme, resulted in growth inhibition. Overexpression of ALDH3A1 in NCTC-2544 cells, which constitutively express some ALHD3A1, was associated with significantly reduced cell proliferation. Third, the growth inhibition caused by ALDH3A1 was associated with decreased activities of cyclin-dependent kinases as well as reduced levels of critical cell cycle regulators. Cytosolic localization of ALDH3A1 is well established (16). The nuclear localization of this enzyme, reported here for the first time, provides additional support for its involvement in nuclear functions associated with replication. We have recently found that ALDH3A1 prevents UV- and reactive aldehyde-induced apoptosis in HCE cells mainly by metabolizing the highly reactive 4-HNE (7). We now present evidence that ALDH3A1 enhances resistance of HCE cells against apoptosis induced by DNA-damaging agents. Taken together, our data suggest that ALDH3A1 may play an important role in suppressing corneal epithelial cell proliferation and thereby promote increased cell survival after oxidative damage.

Mammalian corneal epithelial cells express ALDH3A1 at very high concentrations (6). To explain the functional significance of such high levels of ALDH3A1 expression, several hypotheses have been postulated, and these include both structural and enzymatic properties (17). It was initially suggested that ALDH3A1 is involved in the transparency and the optical properties of the corneal cells (18), although ALDH3A1 null mice have no apparent corneal abnormalities (19, 20). We have previously shown that SWR mice, a strain prone to corneal clouding after exposure to UV radiation (21), lack ALDH3A1 due to mutations in the structural gene (19). In human corneal epithelial cells we have demonstrated that ALDH3A1 confers resistance to apoptosis induced by exposure to UV radiation and 4-HNE primarily through an action to metabolize aldehydes generated by lipid peroxidation (7). In the present study we showed that ALDH3A1 prevents apoptosis induced by Vp-16 and/or mitomycin C, two agents that damage DNA by direct binding but very different mechanisms. Vp-16 forms a complex with the free ends of DNA and with the nuclear enzyme topoisomerase II, thus allowing DNA double strand break formation (22). Mitomycin C, on the other hand, causes cross-linking of DNA and inhibits DNA synthesis (23). The protective effect of ALDH3A1 may derive from the observed growth inhibitory properties of ALDH3A1. It is well known that dividing cells are vulnerable to genotoxic insults and random events that impede the proper replication and segregation of their genomes to daughter cells (24). A DNA replication stress response pathway, known as the DNA replication check point, has evolved that enables cell survival. This pathway responds to replication interference by retarding DNA replication, thus allowing DNA repair before polymerases encounter more damage (25). Our findings of reduced levels of cyclin-dependent kinase activities and cyclin protein expression profiles in ALDH3A1-expressing cells may provide a molecular explanation for the observed growth inhibition and protection against DNA damage. Synthesis of cyclin A is initiated in late G1, and the formation of its active complex with cyclin-dependent kinase 2 is responsible for the progression through the S-phase of the cell cycle. Once there it appears to phosphorylate transcription factors and other proteins necessary for DNA synthesis (26). Active cyclin B is required for the entry into M phase, whereas cyclin E peaks in late G1 phase, and its association with cyclin-dependent kinase 2 is essential for entry into the S phase (26). Phosphorylation of retinoblastoma protein (pRb) has a critical role in the progression to S phase by inducing the expression of a number of genes required for S phase transition (27). We found that phosphorylation of the pRb is down-regulated by elevated ALDH3A1 expression in HCE cells, a result that is consistent with the reduced DNA synthesis we observed in these cells (Fig. 2D). Taking these results together, we propose that ALDH3A1-induced inhibition of cell growth may promote resistance of corneal epithelial cells to apoptosis induced by a variety of noxious stimuli, such as UV radiation, 4-HNE, and DNA-damaging agents. Our recent observation that resistance of ALDH3A1-HCE cells to apoptosis was associated with a lack of caspase-3 activation and poly(ADP-ribose) polymerase cleavage (7) is consistent with this proposal. Lack of caspase-3 expression has been implicated in the resistance of senescent fibroblasts to UV- or staurosporine-induced apoptosis compared with actively dividing cells (28).



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  		FIG. 7.
Expression of ALDH3A1 in the cytosol and the nucleus of ALDH3A1-HCE cells. A, localization of ALDH3A1 in ALDH3A1-HCE cells by confocal microscopy. Cells were fixed on glass coverslips and labeled with DAPI to stain nuclei (blue) and Texas Red-conjugated secondary antibody to anti-ALDH3A1 (red). The overlay (top) panel shows the localization of ALDH3A1 in the nucleus. The relative levels of ALDH3A1 and nuclear staining are presented as intensity profiles from computer-aided image analysis and given in relative fluorescence units (bottom panel). Profiles were generated from left to right through the center of the ALDH3A1-HCE cell (arrow). x axis is distance (µm), and y axis is relative fluorescent units (linear scale). a.u., arbitrary units. B, Western blot analysis of protein (15 µg) from cytosolic (C) and nuclear (N) extracts (prepared as described under "Materials and Methods") from mock- and ALDH3A1-HCE cells. Cytosolic and nuclear purity were confirmed by detection of the presence of marker proteins, tubulin and lamin B1, for cytosol and nucleus, respectively. Actin was used as an internal control for protein loading. B, enzymatic activity of ALDH3A1. The ability of ALDH3A1, contained in the cytosolic and nuclear extracts from mock- and ALDH3A1-HCE cells to oxidize benzaldehyde in the presence of NADP+, was monitored as described under "Materials and Methods." ALDH3A1 activity is expressed as nmol of NADPH produced/min/mg of total cytosolic or nuclear protein, respectively.

 
There are two possible mechanisms by which ALDH3A1 may inhibit cell growth: (a) through its well known metabolic functions, for example by metabolizing low levels of 4-HNE that stimulate growth and (b) by functioning as a signaling protein involved in mitosis. The first hypothesis, viz. metabolism of mitogenic levels of endogenous aldehydes, has some experimental support. Aside from its cytotoxic effects, 4-HNE has been shown to function as a physiological modulator of several cell signaling processes. At low micromolar concentrations, 4-HNE appears to modulate cell proliferation and differentiation by interfering with the activity of cyclins, protein kinases, and transcription factor E2F as well as with the cell apoptotic machinery (29, 30). As such, the ability of ALDH3A1 to metabolize the physiological levels of endogenously produced lipid-derived aldehydes would be anticipated to have an anti-mitogenic effect. Our data from NCTC-2544 cells, where overexpression of ALDH3A1 resulted in a dramatic inhibitory effect on cell proliferation, support this notion. However, the growth-suppressing effects of ALDH3A1 observed here may be independent of its role in metabolism and be consistent with the second hypothesis, as evidenced by its protective effects against mitomycin C- and Vp-16-induced DNA damage. Several members of the ALDH superfamily have been reported to possess noncatalytic properties as well (31). For example, ALDH1A1 has been identified as an androgen-binding protein in human genital fibroblasts (32), a cholesterol-binding protein in bovine lens epithelia (33), and a cytosolic thyroid hormone-binding protein (34). In addition, ALDH1A1 has been identified as a flavopiridol-binding protein in non-small cell lung carcinomas (35) and as a daunorubicin-binding protein in rat liver (36). Furthermore, the {omega}-crystallins in the lens of cephalopods (ALDH1C1 and ALDH1C2) do not have any catalytic activity due to the lack of the cysteine in their active site (37).

The similarity between the functions of ALDH3A1 in the cornea and those of crystallins in the lens extends beyond the ALDH protein family. The {alpha}-crystallins of the lens also exhibit anti-apoptotic (38-40) and growth control roles (41, 42). Although ALDH3A1 fulfills the corneal-crystallin profile (6), it appears to lack any structural-physical functions that would contribute to transparency, historically associated with crystallins (Ref. 43 and references therein). As mentioned above, ALDH3A1 null mice have transparent cornea (20). However, the lack of any obvious physical-structural functions does not eliminate ALDH3A1 as a corneal crystallin. Similarly, the {alpha}B-crystallin-deficient mice have transparent lenses (44). The parallel between the lens crystallins and the abundant corneal ALDH3A1 is in agreement with the notion that ALDH proteins may have multiple roles not necessarily restricted to their metabolic function.

Our observation that ALDH3A1, normally considered to be a cytosolic enzyme, was present in the nucleus is novel and lends support to a role for this protein in the nucleus. Analysis of the primary sequence for ALDH3A1 revealed that it contains a nuclear localization signal (NLS) that conforms to the "bipartite" type of NLS consisting of two clusters of basic amino acid groups separated by a spacer region of 10 residues (265KKSLKEFYGEDAKKSRD281). The three-dimensional structure of human ALDH3A1 is currently not yet available. Consequently, we utilized the crystal structure of rat ALDH3A1 and the Cn3D 3.0 program to explore the possibility that the nuclear localization signal motifs could be functional. These studies indicated that the nuclear localization signal-like sequences in ALDH3A1 are not distributed randomly along the helices but are located preferentially at the surface-exposed loops of the protein where they may have a functional role. The presence of cytosolic metabolic enzymes in the nucleus, although surprising, is not without precedent. The glycolytic metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase was recently identified in the nucleus as an essential component of a transcriptional co-activator complex involved in histone gene expression, with its function being to sense the redox state of NAD+ co-factor (45). C-terminal binding protein (CtBP), a transcriptional co-repressor, was shown to have NAD+-regulated dehydrogenase activity that may link enzymatic activity to protein-protein interactions associated with transcriptional repression (46). Currently, we have no evidence for involvement of ALDH3A1 in gene expression. Nevertheless, it is tempting to speculate that nuclear ALDH3A1 may be associated directly or indirectly with transcriptional repression affecting the gene expression of proteins involved in the cell cycle machinery. The location of ALDH3A1 in the nuclear compartment may have further implications in the protection of DNA damage demonstrated in this study. Interestingly, ferritin, another corneal protein with protective effects against UV-induced DNA damage, is almost exclusively expressed in the nucleus in avian corneal epithelial cells (47), whereas in most other cell types it is found in the cytoplasm. The actual mechanism of nuclear translocation as well as the precise role of ALDH3A1 in the nucleus remains to be elucidated.

In summary, our findings demonstrate a novel function of ALDH3A1 and provide compelling evidence that expression of ALDH3A1 at levels similar to those found in vivo in corneal epithelial cells inhibits cell growth and promotes survival against DNA damage.


   FOOTNOTES
 
* This work was supported by National Institute Grants EY11490 (to V. V.) and T32 AA07464 (to A. P.), by Discovery Fund for Eye Research (to D. B.), and by an unrestricted grant from Research to Prevent Blindness (to D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Molecular Biology and Genetics, Democritus University of Thrace, Dimitras 19, 68100 Alexandroupolis, Greece. Back

§§ To whom correspondence should be addressed: Molecular Toxicology and Environmental Health Sciences Program, Dept. of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262. Tel.: 303-315-6253; Fax: 303-315-6281; E-mail: vasilis.vasiliou{at}uchsc.edu.

1 The abbreviations used are: ALDH3A1, aldehyde dehydrogenase 3A1; ROS, reactive oxygen species; HCE, human corneal epithelial cell line; NCTC-2544, human skin keratinocyte cell line; 4-HNE, 4-hydroxynonenal; BrdUrd, bromodeoxyuridine; pRb, retinoblastoma protein; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alan Townsend for providing the ALDH3A1 cDNA. We also thank Dr. David Thompson for valuable discussions and critical reading of this manuscript.



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 DISCUSSION
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