Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells
2005; Elsevier BV; Volume: 280; Issue: 30 Linguagem: Inglês
10.1074/jbc.m503698200
ISSN1083-351X
AutoresAglaia Pappa, Donald D. Brown, Yiannis Koutalos, James DeGregori, Carl W. White, Vasilis Vasiliou,
Tópico(s)Corneal surgery and disorders
ResumoAldehyde 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. 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. 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 (1Kruse F.E. Eye. 1994; 8: 170-183Crossref PubMed Scopus (144) Google Scholar). 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 (2Cotsarelis G. Cheng S.Z. Dong G. Sun T.T. Lavker R.M. Cell. 1989; 57: 201-209Abstract Full Text PDF PubMed Scopus (1188) Google Scholar, 3Kruse F.E. Tseng S.C. Investig. Ophthalmol. Vis. Sci. 1993; 34: 2976-2989PubMed Google Scholar). The intermediate cells of the corneal epithelium are post-mitotic and, together with the terminally differentiated cells, are incapable of cell division (4Lehrer M.S. Sun T.T. Lavker R.M. J. Cell Sci. 1998; 111: 2867-2875Crossref PubMed Google Scholar). The superficial epithelial cells are finally removed into the tear pool by a variety of mechanisms including apoptosis (5Ren H. Wilson G. Investig. Ophthalmol. Vis. Sci. 1996; 37: 1017-1025PubMed Google Scholar). 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 ALDH3A1 1The 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. (6Piatigorsky J. J. Ocul. Pharmacol. Ther. 2000; 16: 173-180Crossref PubMed Scopus (56) Google Scholar), an enzyme that is part of a well regulated defense system that protects the eye against oxidative damage (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar, 8Pappa A. Estey T. Manzer R. Brown D. Vasiliou V. Biochem. J. 2003; 376: 615-623Crossref PubMed Scopus (144) Google Scholar). 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 (6Piatigorsky J. J. Ocul. Pharmacol. Ther. 2000; 16: 173-180Crossref PubMed Scopus (56) Google Scholar, 9Piatigorsky J. Kozmik Z. Horwitz J. Ding L. Carosa E. Robison Jr., W.G. Steinbach P.J. Tamm E.R. J. Biol. Chem. 2000; 275: 41064-41073Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) 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 (10Manzer R. Pappa A. Estey T. Sladek N. Carpenter J.F. Vasiliou V. Chem. Biol. Interact. 2003; 143-144: 45-53Crossref PubMed Scopus (39) Google Scholar). 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) (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). In addition, we have reported that ALDH3A1 is expressed mainly in the suprabasal cells of the corneal epithelium (8Pappa A. Estey T. Manzer R. Brown D. Vasiliou V. Biochem. J. 2003; 376: 615-623Crossref PubMed Scopus (144) Google Scholar), which are known to be post-mitotic (4Lehrer M.S. Sun T.T. Lavker R.M. J. Cell Sci. 1998; 111: 2867-2875Crossref PubMed Google Scholar). 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. ALDH3A1 Stably Transfected Cell Lines—The SV-40-transformed corneal epithelial cell lines (HCE, ATCC CRL-11135) stably transfected with the mammalian expression vector ΔpCEP4 alone (Mock-HCE) or with the human ALDH3A1 cDNA (ALDH3A1-HCE) along with tissue culture conditions have been described elsewhere (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar, 11Araki-Sasaki K. Ohashi Y. Sasabe T. Hayashi K. Watanabe H. Tano Y. Handa H. Investig. Ophthalmol. Vis. Sci. 1995; 36: 614-621PubMed Google Scholar). 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 (GenBank™ accession number NM_000691) 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 (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). Primary antibodies used included monoclonal anti-ALDH3A1 (8Pappa A. Estey T. Manzer R. Brown D. Vasiliou V. Biochem. J. 2003; 376: 615-623Crossref PubMed Scopus (144) Google Scholar), 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-β-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 (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). 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 × 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 × 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 × 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 × 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 (12DeGregori J. Leone G. Ohtani K. Miron A. Nevins J.R. Genes Dev. 1995; 9: 2873-2887Crossref PubMed Scopus (202) Google Scholar). 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 40× 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 63× 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 × 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 (13Vasiliou V. Qamar L. Pappa A. Sophos N.A. Petersen D.R. Arch. Biochem. Biophys. 2003; 413: 164-171Crossref PubMed Scopus (17) Google Scholar). 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 × 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 × g for 10 min. DNA Fragmentation Assay—Cells (1 × 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). 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 (14Boesch J.S. Lee C. Lindahl R.G. J. Biol. Chem. 1996; 271: 5150-5157Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), 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. 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 (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). 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 (11Araki-Sasaki K. Ohashi Y. Sasabe T. Hayashi K. Watanabe H. Tano Y. Handa H. Investig. Ophthalmol. Vis. Sci. 1995; 36: 614-621PubMed Google Scholar), 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).Table IALDH3A1 retards cell proliferation in stably transfected HCE and NCTC-2544 cellsMock-HCEALDH3A1-HCEMock-NCTCALDH3A1-NCTCDoubling time (days)1.8 ± 0.114.1 ± 0.60ap < 0.001 (Student's t test; compared to mock-transfected cells).1.4 ± 0.093.8 ± 0.61ap < 0.001 (Student's t test; compared to mock-transfected cells).Length of cell cycle (days)2.7 ± 0.165.9 ± 0.87ap < 0.001 (Student's t test; compared to mock-transfected cells).2.0 ± 0.145.4 ± 0.89ap < 0.001 (Student's t test; compared to mock-transfected cells).a p < 0.001 (Student's t test; compared to mock-transfected cells). Open table in a new tab 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 (15Wunderlich M. Berberich S.J. Oncogene. 2002; 21: 4414-4421Crossref PubMed Scopus (20) Google Scholar) were also observed in ALDH3A1-expressing HCE and NCTC-2544 cells relative to their mock-transfected counterparts. 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 (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). Inhibition of apoptosis was attributable to the role of ALDH3A1 in metabolizing (and, thus, eliminating) toxic lipid peroxidation aldehyde byproducts (7Pappa A. Chen C. Koutalos Y. Townsend A.J. Vasiliou V. Free Radic. Biol. Med. 2003; 34: 1178-1189Crossref PubMed Scopus (105) Google Scholar). 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 β-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. 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
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