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• W2025977442 abstract "The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad.proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells. The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad.proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells. Cathepsin D (CatD) is a ubiquitous enzyme that plays an important role in protein turnover, housekeeping and protein processing (1Yamamoto K. Cathepsin E and cathepsin D: Biosynthesis, processing and subcellular location.in: Takahashi K. Aspartic Proteinases: Structure, Function, Biology and Biomedical Implications. Plenum Press, New York1995: 223-229Crossref Scopus (29) Google Scholar). In the retinal pigment epithelial (RPE) cells of the eye, CatD participates in the digestion of the continuously growing and periodically shed photoreceptor outer segments (POS) (2Bok D. Young R.W. Phagocytic properties of the retinal pigment epithelium.in: Zinn K.M. Marmor M.F. The Retinal Pigment Epithelium. Harvard Univ. Press, London1979: 148-174Google Scholar). The digestion of the phagocytosed POS requires a high level of lysosomal enzyme activity. Of these lysosomal enzymes, CatD expression is the highest (3Yamada T. Hara S. Tamai M. Immunohistochemical localization of cathepsin D in ocular tissues.Invest. Ophthalmol. Vis. Sci. 1990; 31: 1217-1223PubMed Google Scholar, 4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar) and it plays the most important role in the digestive process, accounting for 80% of POS digestion-related enzymatic activity (5Regan C.M. de Grip W.J. Daemen F.J.M. Bonting S.L. Degradation of rhodopsin by a lysosomal fraction of retinal pigment epithelium: Biochemical aspects of the visual process. XLI.Exp. Eye Res. 1980; 30: 183-191Crossref PubMed Scopus (51) Google Scholar) in RPE cells. Due to the large phagocytic load, any imbalance in CatD activity in the RPE cells could result in the accumulation of undigested POS phagosomes and, ultimately, morphological changes in the retina (4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar, 6Rakoczy P.E. Lai C.M. Baines M. Di Grandi S. Fitton J.H. Constable I.J. Modulation of cathepsin D activity in retinal pigment epithelial cells.Biochem. J. 1997; 324: 935-940Crossref PubMed Scopus (56) Google Scholar). In contrast, the presence of enzymatically active CatD in the subretinal space has been implicated in the destruction of the retina (7El-Hifnawi E. Kuhnel W. El-Hifnawi A. Laqua H. Localization of lysosomal enzymes in the retina and retinal pigment epithelium of RCS rats.Ann. Anat. 1994; 176: 505-513Crossref PubMed Scopus (14) Google Scholar, 8Hayasaka S. Takahashi J. Mizuno K. Lysosomal behavior in the retina and choroid of spontaneously dystrophic rats.Exp. Eye Res. 1977; 24: 399-407Crossref PubMed Scopus (14) Google Scholar, 9Hayasaka S. Lysosomal enzymes in ocular tissues and diseases.Surv. Ophthalmol. 1983; 27: 245-258Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Considering its sensitivity to changes in CatD activity, the retina is an excellent system to study the biological and biochemical processes related to CatD production, regulation and maintenance. The optimal production of active CatD is maintained by transcription regulators (10Cavailles V. Augereau P. Rochefort H. Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 203-207Crossref PubMed Scopus (142) Google Scholar), by cell-specific promoters (10Cavailles V. Augereau P. Rochefort H. Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 203-207Crossref PubMed Scopus (142) Google Scholar, 11Cavaney-Brooker D.M. Rakoczy P.E. Cloning of a major human retinal pigment epithelial lysosomal aspartic protease and mapping its transcriptional start sites.Curr. Eye Res. 1999; 18: 310-318Crossref PubMed Scopus (7) Google Scholar), and by posttranslational modification of the protein precursor (12Richo G. Conner G.E. Proteolytic activation of human procathepsin D.in: Structure and Function of the Aspartic Proteinases. Plenum Press, New York1991: 289-296Crossref Scopus (37) Google Scholar). It is known that CatD is produced in the inactive form pre-proCatD, which is quickly converted into proCatD. The precursor, proCatD, is biologically inactive and it is subsequently converted into enzymatically active forms (12Richo G. Conner G.E. Proteolytic activation of human procathepsin D.in: Structure and Function of the Aspartic Proteinases. Plenum Press, New York1991: 289-296Crossref Scopus (37) Google Scholar). The presence of proCatD has been demonstrated in a variety of cells (4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar, 13Rochefort H. Biological and clinical significance of cathepsin D in breast cancer.Cancer Biol. 1990; 1: 153-160PubMed Google Scholar). One of the challenges of gene therapy is to control the amount of therapeutic transgene. The amount of therapeutic gene has been successfully controlled by a variety of cell-specific, inducible promoters (14Huang C.J. Lee M.M. Spinella F. Dopp J.M. Nazarian R. de Vellis J. Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system.Mol. Med. 1999; 5: 129-137PubMed Google Scholar, 15Houston P. Campbell C.J. White B.P. Braddock M. Delivery and expression of fluid shear stress-inducible promoters to the vessel wall: Applications for cardiovascular gene therapy.Hum. Gene Ther. 1999; 10: 3031-3044Crossref PubMed Scopus (34) Google Scholar). Here we propose that an intranuclear posttranslational control might also be suitable to limit the production of biologically active therapeutic genes. Naturally, this approach is only suitable for genes that are subjected to posttranslational modification in order to produce the biologically active form. In this work we investigated in vitro and in vivo whether the production of active CatD can be controlled posttranslationally following the upregulation of proCatD production in RPE cells by a recombinant adenovirus (16Rakoczy P.E. Shen W-Y. Lai M. Rolling F. Constable I.J. Development of gene therapy based strategies for the treatment of eye diseases.Drug Dev. Res. 1999; 46: 277-285Crossref Scopus (12) Google Scholar). Tissue culture. The 293-cell line (human embryo kidney cells transformed by sheared adenovirus type 5 DNA) was obtained from the American Tissue Type Culture Collection (ATCC, Rockville, MD). The human RPE cell lines, D407 (17Davis A.A. Bernstein P.S. Bok D. Turner J. Nachtigal M. Hunt R.C. A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.Invest. Ophthalmol. Vis. Sci. 1995; 36: 955-964PubMed Google Scholar) was a gift from Dr. Hunt and RPE51 cells were primary RPE cells isolated from a 51-year old donor eye as described previously (4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar). 293 cells and D407 cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and RPE51 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a humidified CO2 incubator. Cell culture media and sera were obtained from Gibco (Grand Island, NY). Cells were passaged every 4 days and were harvested by trypsin dispersion. Construction of recombinant adenovirus Ad.proCatD. A 2038-bp CatD cDNA fragment, isolated from human breast cancer MCF7 cells, was cloned into M13mp10 (18Augereau P. et al.Cloning and sequencing of the 52 kD cathepsin complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11.Mol. Endocrinol. 1988; 2: 186-192Crossref PubMed Scopus (87) Google Scholar). The 1616 bp HindIII fragment of this CatD cDNA was excised and subcloned into the HindIII-linearized pGem-11Zf(+) vector (Promega, Madison, WI) so as to create a XbaI site downstream of the CatD fragment. This then enabled the removal of the CatD fragment by XbaI restriction for subcloning into the XbaI site of the adenovirus vector pAd.MLP. The orientation of the CatD fragment was checked by restriction digest with KpnI. The recombinant adenovirus Ad.proCatD was constructed by homologous recombination. 293 cells grown to 80% confluence in 6-cm-diameter tissue culture dishes were cotransfected by using the calcium phosphate-precipitation method with 3 µg of PvuI-linearized pAd.proCatD and 3 µg ClaI-digested Ad.dl324 DNA. Twenty hours after transfection, the medium was replaced with MEM supplemented with 10% FBS. After a further 2-day incubation, the cells and medium were harvested and subjected to 3 cycles of freezing and thawing. The resultant cell lysate was then plated onto 293 cells grown in 96-well tissue culture plates. Cells in wells showing cytopathic effect (cpe) were harvested and used to infect 293 cells grown in 25-cm2 flasks. At the appearance of cpe, the cells were harvested and viral DNA was extracted. Restriction analysis using AatII, EcoRI, EcoRV, KpnI, and XbaI was performed on the viral DNA to select for Ad.proCatD. The orientation and the and size of the CatD fragment was confirmed by Southern blot hybridization with a CatD-specific [γ-32P]ATP-labeled oligonucleotide probe (5′-TACTTGTGGTGGATCCAGCA-3′) before large-scale preparation of virus stock. Propagation and purification. Ad.proCatD and Ad.LacZ (recombinant adenovirus carrying the LacZ reporter gene) were used to infect fifty 150-cm2 tissue culture flasks of 293 cells. After 36–40 h, the cells were harvested and resuspended in 25 mL of 10 mM Tris–HCl (pH 8). Ad.proCatD was purified by two-cesium chloride density gradient centrifugations (19Engelhardt J.F. et al.Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses.Nat. Genet. 1993; 4: 27-34Crossref PubMed Scopus (284) Google Scholar). The resultant virus band was removed and dialyzed against 4 changes of phosphate-buffered saline (PBS) at 4°C. Glycerol was added to the purified virus to a final concentration of 10% and the virus was stored at –70°C. The virus was tested for the presence of wild type adenovirus contamination using a method described previously (20Zhang W. Koch P.E. Roth J.A. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.Biotechniques. 1995; 18: 444-446PubMed Google Scholar) before use. Transduction of D407 cells. D407 cells were seeded into 75-cm2 tissue culture flasks. When the cells were 80% confluent, the medium was removed and the cell monolayers washed with PBS. Recombinant adenoviruses Ad.proCatD and Ad.LacZ was added separately or together to 1 mL of MEM and then plated onto the cell monolayers to result in a final multiplicity of infection (m.o.i.) required. After 1 h incubation at 37°C in a humidified CO2 incubator, 10 mL of media supplemented with 5% FCS was added and the cells were incubated for a further 48 h. The transduced D407 cells were harvested by trypsin dispersion and washed twice with PBS. Analysis of CatD expression by Western blot analysis. RPE51 cells, D407 cells transduced with Ad.LacZ, D407 cells transduced with Ad.proCatD and D407 cells transduced with both Ad.LacZ and Ad.proCatD were harvested and resuspended at a concentration of 105 cells per 100 µl sample reducing buffer. The cells were prepared for SDS/polyacrylamide gel electrophoresis by heating to 100°C for 5 min before being loaded on to a 12.0% polyacrylamide gel. Electrophoresis and blotting were performed as described previously (6Rakoczy P.E. Lai C.M. Baines M. Di Grandi S. Fitton J.H. Constable I.J. Modulation of cathepsin D activity in retinal pigment epithelial cells.Biochem. J. 1997; 324: 935-940Crossref PubMed Scopus (56) Google Scholar). After blotting, the membrane was incubated for 1 h in blocking solution containing 5% skim milk powder in Tris-buffered saline (pH 7.4) with 0.1% Tween 20 (TBS-T) and then subjected to one 15-min wash followed by two 5-min washes in TBS-T. A mouse anti-human CatD antibody (Calbiochem-Novabiochem Corp., San Diego, CA) was applied to the membrane at a concentration of 0.5 µg/mL for 1 h. Following washing, the membrane was incubated for 1 h with donkey anti-mouse IgG antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Uppsala, Sweden) at a dilution of 1/2000. Two extra 5-min washing steps were performed before incubation with ECL Western blotting detection solution (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 min. Excess solution was then drained from the membrane before exposing it to autoradiograph film (X-OMAT AR) (Eastman Kodak Company, Rochester, NY) for various time periods ranging from 15 s to 20 min. All incubations were performed at room temperature and all washing steps were carried out using TBS-T. Antibodies were diluted in the blocking solution. Analysis of bovine POS phagocytosis and digestion. The effects of CatD upregulation on the ability of D407 cells to phagocytose and digest bovine POS was measured using a fluorescence-activated cell sorter (Becton–Dickinson, San Jose, CA). D407 cells were seeded into 24-well tissue culture plates. When the cells were about 80% confluent, they were transduced with Ad.proCatD or Ad.LacZ at an m.o.i. of 100. One day after adding the recombinant adenoviruses, 1 mL of MEM supplemented with 10% FBS and containing 107 FITC-labeled POS (FITC-POS) for the measurement of phagocytosis (6Rakoczy P.E. Lai C.M. Baines M. Di Grandi S. Fitton J.H. Constable I.J. Modulation of cathepsin D activity in retinal pigment epithelial cells.Biochem. J. 1997; 324: 935-940Crossref PubMed Scopus (56) Google Scholar) and containing 107 POS for the measurement of digestion was added to each well. Controls were not challenged. The cells were harvested by trypsin dispersion at 6 h postchallenge for the phagocytosis assay and at 3 days postchallenge for the digestion assay. Cells were washed 3 times with PBS and resuspended in 300 µL of PBS containing 0.4% paraformaldehyde. Analysis was performed using the fluorescence-activated cell sorter and the means (n = 3) and standard deviations were calculated. Aspartic protease activity measurement. RPE51 cells, D407 cells and D407 cells transduced with Ad.proCatD and Ad.LacZ were harvested and aspartic protease activity in these cells was measured using acid denatured hemoglobin as substrate according to the method described previously (6Rakoczy P.E. Lai C.M. Baines M. Di Grandi S. Fitton J.H. Constable I.J. Modulation of cathepsin D activity in retinal pigment epithelial cells.Biochem. J. 1997; 324: 935-940Crossref PubMed Scopus (56) Google Scholar). Eyes injected with Ad.proCatD or Ad.LacZ were enucleated at 3 days postinjection. After removal of the cornea, the sclera-choroid-RPE layers were gently separated from the neuroretina. The sclera-choroid-RPE layers and the neuroretina were placed into 1 mL of 0.1 M sodium acetate (pH 3.4)/0.1% Triton X-100 for 30 min at room temperature to lyse the cells. The lysates were passed through a pipette several times. After a brief centrifugation, the supernatant was removed and aspartic protease activity was performed as described previously (6Rakoczy P.E. Lai C.M. Baines M. Di Grandi S. Fitton J.H. Constable I.J. Modulation of cathepsin D activity in retinal pigment epithelial cells.Biochem. J. 1997; 324: 935-940Crossref PubMed Scopus (56) Google Scholar). Subretinal injection. All animal work was performed following the Statement for the Use of Animals in Ophthalmic and Vision Research. Subretinal injections were performed as described earlier (16Rakoczy P.E. Shen W-Y. Lai M. Rolling F. Constable I.J. Development of gene therapy based strategies for the treatment of eye diseases.Drug Dev. Res. 1999; 46: 277-285Crossref Scopus (12) Google Scholar, 21Rolling F. et al.Evaluation of AAV mediated gene transfer into the rat retina by clinical fluorescent photography.Hum. Gene Ther. 1999; 10: 641-648Crossref PubMed Scopus (56) Google Scholar, 22Lai C.-M. Shen W.-Y. Constable I.J. Rakoczy P.E. Preferential adenovirus-mediated transduction of cells at the sites of laser photocoagulation in the rat eye.Curr. Eye Res. 1999; 19: 411-417Crossref PubMed Scopus (10) Google Scholar). Briefly, 6- to 8-week-old nonpigmented Royal College of Surgeons (RCSrdy+) congenic control rats were subretinally injected with 2 × 108 pfu/mL Ad.proCatD or Ad.LacZ in 2 µL vehicle (PBS containing 10% glycerol). Controls were injected with vehicle only. For experiments to investigate the effect of active CatD on the eye, 2 µg (2 × 10–2 unit) active bovine CatD (Sigma Chemical Co., St. Louis, MO) or 2 µg heat-inactivated bovine CatD were injected into the subretinal space. At 4 days postinjection, the animals were euthanized and the eyes were enucleated, fixed in 2% paraformaldehyde and paraffin embedded. Five-micrometer-thick paraffin sections were prepared for immunodetection of CatD with a mouse anti-human CatD antibody (Calbiochem). CatD immunohistochemistry. The sections were deparaffinized with xylene, then rehydrated through graded alcohol to distilled water. All subsequent incubations were carried out at room temperature in a humidified atmosphere, using Tris-buffered saline (TBS) (pH 7.2) for all washing steps and as the diluent. Sections were blocked with 10% normal goat serum for 30 min then subjected to three 5-min washes. Either a polyclonal rabbit anti-bovine CatD antibody (4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar) or normal rabbit serum was applied to the sections for 1 h at a dilution of 1/200. Following washing, sections were incubated for a further hour with goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (Vector Laboratories Incorporated, Burlingame, CA) at a dilution of 1/1500. Sections were then washed and immunodetection was carried out by incubating the sections with SIGMA FAST Fast Red TR/Naphthol AS-MX (Sigma Chemical Co.) chromagen for 20 min resulting in the formation of a red/pink deposit. Sections were lightly counterstained with Meyer's hematoxylin then mounted for bright-field microscopy using a glycerol based mounting medium. Preparation of cells for resin section immunochemistry. D407 cells, Ad.proCatD-transduced D407 cells and RPE51 cells, received as a monolayer in a tissue culture flask, were washed with fresh media and fixed for 1 h at room temperature in warm 4% paraformaldehyde in 0.5 M cacodylate buffer (pH 7.4). The cells were then rinsed in three washes of cacodylate buffer, scraped off the plasticware and pelleted at 1000g in a centrifuge. The cell pellet was then transferred to a microfuge tube, pelleted again and the supernatant removed. The pellet was resuspended in a solution of 10% bovine serum albumin (BSA), repelleted and most of the BSA aspirated off. A solution of 2.5% glutaraldehyde/cacodylate buffer was gently layered over the remaining BSA, and left until the pellet firmly gelled. The pellet was sliced into 1-mm rings and processed for electron microscopy, embedded in araldite resin, and cured at 60°C for 48 h. Semithin sections were prepared mounted onto silanated slides and immunochemically reacted for CatD. Thin sections (60–80 nm) were taken on nickel grids for immunogold cytochemistry. Resin section immunocytochemistry for light microscopy. Sections were deplasticized in saturated sodium ethoxide/xylene mixture for 8 min, rehydrated to water and treated for 2 min in aqueous 0.1% hydrogen peroxide to remove endogenous peroxidase. Following copious rinsing in TBS, the sections were treated with blocking horse serum for 20 min and then reacted with a mouse antihuman CatD antibody (Calbiochem) for 1 h at 37°C then rinsed thoroughly in TBS. The sections were then incubated in rabbit anti-mouse biotinylated IgG (DAKO LSAB kit) for 20 min at room temperature (Dako Corp., Carpenteria, CA), washed well in TBS and treated with two drops of streptavidin/horseradish peroxidase (HRP) (DAKO LSAB kit) solution for 20 min. The reaction product was visualized using a Pierce metal enhanced diaminobenzidine (DAB) kit. Following a thorough wash in distilled water, the sections were stained with a Diff Quik staining solution (Lab Aids Prop. Ltd., Adelaide, Australia), dried on a hot plate and mounted in DePeX. Resin section immunocytochemistry for electron microscopy. Thin sections were rapidly etched for 5 s in Maxwell's solution (20% potassium hydroxide in ethanol containing 50% propylene oxide) and immediately rinsed in a beaker of double distilled water. The grids were placed section down for 10 min on a droplet of double distilled water and deosmicated using 1% aqueous hydrogen peroxide solution for 30 s. The grids were washed in several changes of distilled water and placed section down onto a droplet of 3% aqueous sodium meta-periodate for 30 min. Following substantial rinsing in at least six changes of distilled water, the sections were blocked for nonspecific staining and primary antibody incubation as mentioned in semi-thin protocol. The grids were then floated section down on droplets of biotinylated antibody (DAKO-LSAB kit) for 20 min washed on several droplets of TBS buffer. They were then incubated on droplets of strepavidin/gold (Zymed, 10 nm gold) (Zymed, San Francisco, CA) for an hour at room temperature, then washed in several changes of double distilled water. The tissue was stained in freshly Millipore-filtered 1% aqueous osmium tetroxide for 5–7 min to visualize membranes, rinsed clean in distilled water, blotted dry, and stained for 5 min each in both uranyl acetate and lead citrate, carbon coated and viewed on a Philip's 410LS transmission electron microscope at an accelerating voltage of 80 kV (Philips, Einhoven, Holland). Western blot analysis of RPE51 cells demonstrated that the majority of the CatD immunoreactive signal originated from active CatD appearing around 34 kDa (Fig. 1A, lane 1). In these, and several other primary human RPE cells (data not shown), only a small amount of proCatD (~52 kDa) was present (Fig. 1A, lane 1). When an equivalent number of cells (4 × 103 cells) from a low CatD producing RPE cell line, D407, was analyzed, there was only a weak signal present (Fig. 1A, lane 2). However, the presence of active CatD (34 kDa) and inactive precursors (52 kDa) became visible when a higher number of cells (4 × 104 cells) was loaded (Fig. 1A, lane 3). Transduction of D407 cells with Ad.proCatD resulted in a significant increase in CatD production (Fig. 1A, lane 4). Western blot analysis demonstrated that the majority of the signal was derived from proCatD that appeared at around 52 kDa. There was also an increase in the active CatD-related signal (34 kDa). Transduction of D407 cells with Ad.LacZ did not induce any change in proCatD or active CatD production (Fig. 1A, lane 5). Cotransduction of D407 cells with Ad.proCatD (m.o.i. of 100) and increasing amounts of Ad.LacZ (m.o.i. of 50, 100, 150 and 200) did not effect the relative amounts of proCatD or active CatD expressed (Fig. 1B, lanes 1–5, respectively). The amount of active CatD was quantified by an assay measuring aspartic protease activity. Aspartic protease activity in the low CatD producing D407 cells was approximately a third of the activity measured in RPE51 (Table 1). Aspartic protease activity was not affected by transduction of D407 cells with Ad.LacZ. In contrast, aspartic protease activity almost tripled when D407 cells were transduced with Ad.proCatD and approached the level measured in RPE51 cells (Table 1).TABLE 1Cathepsin D Activity in Control and Recombinant Adenovirus-Transduced Cells and in Recombinant Adenovirus-Injected Rat EyesSampleUnits of CatD activity/mg total proteinaOne milligram of pure bovine catd protein represents 10 units specific catd activity.RPE51 cells1.171 ± 0.289D407 cells0.365 ± 0.025Ad.proCatD-transduced D407 cells1.036 ± 0.238Ad. LacZ-transduced D407 cells0.364 ± 0.034Saline-injected choroid/RPE0.885 ± 0.120Ad.proCatD-injected choroid/RPE1.403 ± 0.167Ad. LacZ-injected choroid/RPE0.697 ± 0.083Saline-injected retina0.268 ± 0.015Ad.proCatD-injected retina0.242 ± 0.070Ad. LacZ-injected retina0.218 ± 0.068a One milligram of pure bovine catd protein represents 10 units specific catd activity. Open table in a new tab Ad.proCatD-transduced D407 cells retained their POS phagocytosing ability. After the challenge of D407 cells (n = 3) and Ad.proCatD-transduced D407 cells (n = 3) with FITC-POS, there was an increase in fluorescence signal of 19 and 24 times above background, respectively (Table 2). An improved POS digestion ability was detected in Ad.proCatD-transduced RPE cells. The accumulation of autofluorescent signal, correlating to undigested POS (4Rakoczy P.E. Baines M. Kennedy C. Constable I.J. Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.Exp. Eye Res. 1996; 63: 159-167Crossref PubMed Scopus (39) Google Scholar, 23Kennedy C.J. Rakoczy P.E. Robertson T.A. Papadimitriou J.M. Constable I.J. Kinetic studies on phagocytosis and lysosomal digestion of rod outer segments by human retinal pigment epithelial cells in vitro.Exp. Cell Res. 1994; 210: 209-214Crossref PubMed Scopus (42) Google Scholar, 24Rakoczy P.E. Mann K. Cavaney" @default.
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• W2025977442 date "2000-11-01" @default.
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• W2025977442 title "Controlled Production of Active Cathepsin D in Retinal Pigment Epithelial Cells Following Adenovirus-Mediated Gene Delivery" @default.
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• W2025977442 doi "https://doi.org/10.1006/mthe.2000.0195" @default.
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