Lysosomal Cystine Storage Augments Apoptosis in Cultured Human Fibroblasts and Renal Tubular Epithelial Cells
Margaret Park,
Amanda Helip-Wooley and
Jess Thoene
Hayward Genetics Center, Tulane University Health Sciences Center, New Orleans, Louisiana.
Correspondence to Dr. Jess Thoene, Hayward Genetics Center, SL-31, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112. Phone: 504-588-5101; Fax: 504-584-1763;E-mail: jthoene{at}tulane.edu
ABSTRACT. Nephropathic cystinosis is a lethal disorder of lysosomalcystine storage due to defective lysosomal cystine transport.How lysosomal cystine causes this multisystemic disorder culminatingin end-stage renal disease is not known, because the cystineis isolated from cellular metabolism by the lysosomal membrane.It is here reported that in both normal and nephropathic cystinoticfibroblasts and cultured renal proximal tubule epithelial cells,increased lysosomal cystine causes an increased rate of apoptosis.In nephropathic cystinotic fibroblasts, the rate of apoptosisis 14.8% after exposure to TNF-versus 7.8% in control normalfibroblasts. Anti-Fas antibodies and UV exposure induced apoptosisin 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively,versus 5.2% and 7.1% in normal fibroblasts when analyzed byCaspACE (P < 0.05). Similar results were found when the cellswere analyzed by TdT-mediated dUTP nick end labeling (TUNEL).When the cystine content of normal fibroblasts is increasedby exposure to cystine dimethylester (CDME), the apoptotic rateis increased to the rate seen in nephropathic cystinotic cells.Decreasing the cystinotic cells’ cystine content by useof cysteamine results in normalization of the apoptotic rate.Renal proximal tubule epithelial (RPTE) cells are much moresensitive to CDME than fibroblasts, reaching 43.8% apoptosis6 h after exposure to CDME alone, compared with 38.2% when exposedto TNF- alone. Serum withdrawal causes an apoptotic rate of8.7% in nephropathic cystinotic fibroblasts, compared with 6.1%in normal fibroblasts. That rate increases to 37.3% in normalfibroblasts after CDME exposure. Fibroblasts from two cystinoticvariants, benign ocular and intermediate cystinosis, do notdisplay increased apoptosis with increased lysosomal cystine.It is concluded that enhanced apoptosis resulting from lysosomalcystine storage may lead to inappropriate cell death and decreasedcell numbers in many tissues and hence contribute to the nephropathiccystinotic phenotype. The variant forms may represent co-segregationor linkage of rare alleles that confer resistance to apoptosis,moderating the cell loss and causing the milder disease expression.
Nephropathic cystinosis results from failure of expression ofCTNS, located at 17p13, which codes for cystinosin, a 367-aminoacid peptide that functions to transport cystine from lysosomes.It is the only lysosomal amino acid transporter yet cloned.The cDNA has 12 exons and is 2.6 kb in length (1). Nephropathiccystinosis has as its major pathophysiologic effects the progressiveloss of renal function beginning with the renal Fanconi syndromeat less than 1 yr of age, followed by the onset of glomerularfailure after approximately age 7 yr, and reaching ESRD by 10yr of age. The children are stunted, rarely achieving a heightgreater than the 50th percentile for a 3-yr-old in the untreatedstate, and they develop hypothyroidism between the ages of 8and 10 yr. Renal rickets may also occur secondary to phosphaturia.They display a pathognomonic salt and pepper retinopathy andcorneal crystals, which lead to photophobia, corneal ulcerations,and severe debility due to pain as well as impaired vision.Diabetes, esophageal dysmotility, and myopathy may be late complications(2).
The proximate cause of cell death in the cystinotic phenotypeis not known, nor is it readily inferred, because the cystineis isolated within lysosomes. The only apparent mode of egressfor cystine from lysosomes lacking a functional cystine transporteris exocytosis. After exocytosis, the lysosomal cystine wouldbe deposited at the external face of the plasma membrane, wherethe amino acid transporter (X-gc) is available to transportcystine directly back into the cytosol. There it would encountera normal cellular concentration of GSH (5 mM) and be reducedto cysteine, with the concomitant oxidation of GSH to GSSG.NADPH-GSSG reductase functions to reduce GSSG back to GSH, effectivelycompleting the transformation of lysosomal cystine to cytosoliccysteine (3). The cysteine so generated is then available forprotein and GSH synthesis (4). Nothing in this sequence suggestsa disadvantage for cystinotic cells nor offers an explanationfor the lethality of this phenotype.
Recent studies on apoptosis have implicated lysosomes as participantsin the critically important process of programmed cell death(5). Lysosomal participation in apoptosis has been documentedin a number of studies, but the extent of involvement is stillbeing determined (6,7). It is clear that permeabilization ofthe lysosomal membrane occurs in this process and that cathepsinsB and D are released. Movement of cathepsins from granular tocytosolic locations during apoptosis has been found in fibroblastsundergoing an increase in oxidative stress. Induction of apoptosisin macrophage-like cells occurs upon exposure to a lysosomotopicdetergent, which causes graded lysosomal leakage (8). Similareffects have been seen after photo-oxidation of lysosomes, leadingto increased membrane permeability and resulting in TdT-mediateddUTP nick end labeling (TUNEL) positivity of the nucleus, anaccepted measure of apoptosis (9). We here report that lysosomalcystine loading alone causes increased apoptosis in culturedRPTE cells and also causes increased apoptosis in cystinoticand normal fibroblasts after standard apoptotic stimuli. Twofibroblast lines derived from variant forms of cystinosis, intermediate,in which renal death occurs in the teens or twenties, and ocular,in which no renal involvement occurs (2), do not show an increasedapoptotic response, even though the lysosomal cystine contentis in the same range as in the nephropathic cells.
Cell Culture
Normal and cystinotic fibroblasts were purchased from The CoriellMutant Cell Repository, and cultured in Coon modification ofHam F12 medium, supplemented with 10% fetal calf serum (FCS).Renal proximal tubule epithelial (RPTE) cells were purchasedfrom Biowhittaker, cultured in renal epithelial basal mediumsupplemented with one Singlequots kit per 500 ml to make renalepithelial growth media (REGM, Biowhittaker). Fibroblasts andRPTE were maintained in a 5% carbon dioxide, 95% air, humidifiedincubator at 37°C (4).
Induction of apoptosis and assays for its detection were performedusing commercially available reagents. Normal and cystinoticfibroblasts were matched for passage number (± 3 passages)and cell density, and then exposed to one of three apoptotictriggers: TNF- (2 ng/ml) with actinomycin D (2.5 µg/mL)for 16 h; anti-Fas antibody (500 ng/ml) with actinomycin D (2.5µg/mL) for 16 h; or UVB light (60 mJ) (10–13). Afterexposure, the cells were maintained in Coon modification ofHam F12 medium for 16 h before analysis. The cells were thenassayed for apoptosis. Serum withdrawal was also used as anapoptotic stimulus (14–17), in which case the cells wereincubated in F12 medium without serum for 24 h and then analyzedfor apoptosis as described. Three commercially available apoptosisassays were employed. CaspACE (Promega) is an FITC-conjugatedcell-permeable form of the pan-caspase inhibitor zVAD-Fmk, whichbinds to activated caspase(s). Cells were incubated in FITC-VAD-Fmk-containingmedium (10 µM for 30 min at 37°C), washed, and thenfixed in 10% buffered formalin (30 min at room temperature)before analysis. TUNEL employs terminal deoxynucleotidyl transferase(TdT) to label the ends of double-stranded DNA breaks, whichoccur in apoptotic cells, with FITC-conjugated dUTP. Cells werefixed in 4% buffered formalin, washed, incubated in permeabilizationsolution (0.1% Triton X-100 in 0.1% Na Citrate) for 2 min onice and then stained with 25 µl of TdT/45 µl oflabeled dNTP mix for 40 min at 37°C. Annexin V-propidiumiodide (PI) staining was performed as described (5 and AnnexinV Fluos kit directions, Roche Laboratories). The cells werevisually enumerated by fluorescence microscopy, scoring a minimumof five fields (250 to 300 cells) for fluorescence in triplicatefollowed by counting all cells in the same field by light microscopy,with a minimum of 750 cells scored per condition. The apoptosisrate is the total number of cells that fluoresce divided bythe total cells in the field.
Lysosomal cystine depletion of cystinotic fibroblasts was accomplishedby treatment with 1 mM cysteamine-HCl (4) (Sigma) in Ham F12medium lacking cystine (Life Technologies), with 10% FCS, for1 h followed by exposure to the apoptotic triggers in cystine-freemedium to inhibit cystine re-accumulation.
Normal fibroblast lysosomes were loaded with cystine by theaddition of 0.5 mM cystine dimethylester (CDME, Sigma) to normalculture medium for 1 h before treatment (18). The cells weretreated with apoptotic triggers as described above, leavingCDME in the medium to prevent lysosomal cystine loss, and thenanalyzed for apoptosis as described. Lysosomal cystine loadingof RPTE cells was accomplished by exposure to 0.1 mM or 0.25CDME in normal REGM for 1 h before treatment with or withoutapoptotic triggers.
Cystine Binding Protein Assay
Intracellular lysosomal free cystine was determined using acystine binding protein (CBP) assay as described (19). CBP wasprocured from Riverside Scientific. The assay has a sensitivityof 0.1 µM.
Total cell protein was determined by a modification of the Lowrymethod (20). Statistical analyses were performed using pairedt test for means with SSPS for Windows. Results are mean ±1.0 SD; error bars are + 1.0 SD (Figure 5).
Figure 5. The effect of CDME on apoptosis in renal proximal tubule epithelial (RPTE) cells. Semi-confluent RPTE cells were treated with either 0.25 mM CDME or TNF-, harvested at 0.5, 1, 3, and 6 h, stained with TUNEL, and enumerated by fluorescence microscopy. Each experiment was done in triplicate. The control (time 0) cystine content was 0.6 nmol/mg protein. After 1-h incubation, the cystine content was 2.2 nmol/mg protein.
Immunhistochemistry of Normal and Cystinotic Fibroblasts
Cells were stained with Lysotracker Red (Molecular Probes) for5 min at room temperature, washed twice in PBS, and then fixedin 4% formalin (1 h). Slides were immersed in blocking buffer(PBS, pH 7.2, 0.5% Carnation dry milk, 0.1% Triton X-100) for20 min at room temperature, washed twice in PBS, and then incubatedwith primary antibody (anti-cathepsin B, Santa Cruz Biotechnologies)diluted 1:100 for 2 h at room temperature, followed by incubationwith secondary antibody (FITC-conjugated rabbit anti-goat, SantaCruz Biotechnologies), at 4°C overnight. Slides were washedtwice with PBS and then sealed with coverslips and viewed usinga Leica DMRX deconvoluting microscope.
The cell lines studied, their genotypes, and nominal cystinecontent are listed in Table 1. The mutations in cell lines GM00008,GM00760, and GM00046 cause typically severe nephropathic cystinosiswith ESRD by 10 yr of age. The cystine content shown in thecystinotic lines in Table 1 varies between 0.8 and 15.7 nmol/mgprotein, which is that typically seen in cultured cystinoticfibroblasts (2).
Table 1. Genotype and cystine content of cell linesa
Apoptosis induced in three nephropathic cystinotic, two normal,one intermediate cystinotic, and one ocular cystinotic fibroblastline by three separate inducers is shown in Tables 2 and 3.In Table 2, apoptosis in these lines was assessed by CaspACE.The cystinotic cells on average display about 2 to 3 times theapoptotic rate for the three apoptotic triggers compared withthe normal cell lines. The cystinosis variant lines do not showincreased apoptosis under these conditions. The differencesare statistically significant at P = 0.05 between the averagesfor cystinotic and normal lines and between nephropathic andocular cystinotic lines. There is no significant differencein the rate of apoptosis between intermediate and nephropathiclines, ocular cells and normal cells, or intermediate versusnormal cells (Table 2). Similar results were obtained when thecells were analyzed by TUNEL, except that line GM00008 did notdiffer significantly from the normal lines’ response afteranti-Fas or UV exposure, nor did line GM00046 after TNF- exposure(Table 3). Again, the variant lines did not show augmented apoptosiscompared with the normal fibroblast lines.
Table 3. The apoptosis rate in normal and cystinotic fibroblastsa
Modulation of the apoptotic response by altering the lysosomalcystine content of normal or nephropathic cystinotic fibroblastsis shown in Figure 1. The bar graphs show the apoptosis ratesinduced by exposure of two cystinotic and two normal cell linecells to TNF- or UV radiation before and after correction ofthe cystine content of nephropathic fibroblasts to normal levelswith MEA and before and after increasing the cystine contentof normal fibroblasts to cystinotic levels by pre-incubationwith CDME. The mean rate of apoptosis for the cystinotic cellsbefore cystine depletion was 15.6 ± 2.7%; after cystinedepletion by exposure to MEA, it fell to 6.1 ± 2.8%.This difference is significant at P < 0.001. The averagecontrol apoptotic rate for the normal fibroblast lines was 7.2± 1.3%, which rose to 18.7 ± 5.4% after exposureto CDME (P < .001). The effect holds whether induction wasby TNF- or UV light. Representative fluorescence micrographsdisplaying this effect after UV exposure are shown in Figure 2.The high rate of apoptosis in cystinotic fibroblasts withinitial cystine content of 4.0 nmol/mg protein is shown in Figure 2A.This fell after treatment with cysteamine, which loweredthe cystine content to < 0.1 nmol/mg protein (Figure 2B).The normal rate of apoptosis in normal fibroblasts (cystinecontent <0.1 nmol/mg protein) is shown in Figure 2C, andthis increases to a rate similar to that seen in cystinoticfibroblasts after pre-exposure of the cells to CDME (Figure 2D),which increased the cystine content to 1.95 nmol/mg protein.Representative phase micrographs of fibroblasts treated withTNF-, showing characteristic apoptotic morphology, and the effectof lysosomal cystine on apoptosis are shown in Figure 3, inwhich typical blebs in the plasma membrane are seen after treatmentwith TNF- in both normal and cystinotic cell lines and the modulatingeffects of MEA and CDME on apoptosis are seen. Serum withdrawalin cultured fibroblasts caused less apoptosis above baselinethan the other stimuli employed; however, increasing lysosomalcystine, either naturally due to defective CTNS function orartificially due to CDME loading, again resulted in an increasedapoptotic rate (Table 4).
Figure 1. The effect of MEA or cystine dimethylester (CDME) on apoptosis in semi-confluent nephropathic cystinotic or normal fibroblasts. Nephropathic cystinotic fibroblasts (passage 7–13) were treated with cysteamine 1 h before exposure to apoptotic triggers, depleting them to a normal lysosomal cystine content (0.01 to 0.13 nmol of cystine per mg of protein). Normal fibroblasts also at passage 7–13 were pretreated with 0.5 mM CDME for 1 h before apoptotic triggers, loading their lysosomes with cystine to the cystinotic range (0.47 to 1.95 nmol of cystine per mg of protein). They were then treated with apoptotic stimuli and incubated for 16 h while in cystine-free or CDME-containing medium. Apoptosis was assayed by CaspACE, and scored as before.
Figure 2. (A through D) The effect of lysosomal cystine in normal and nephropathic cystinotic fibroblasts. Nephropathic cystinotic fibroblasts were exposed to UV light (60 mJ) (A) or pretreated with 1.0 mM MEA for 1 h and then exposed to UV light (60 mJ) (B). Normal fibroblasts were exposed to UV light (60 mJ) (C) or pretreated with 0.5 mM CDME for 1 h and then exposed to UV light (60 mJ) (D). Apoptosis was then assessed by CaspACE and photographed by fluorescent microscopy. The cystine content of parallel plates was as follows: A, 4.0 nmol/mg protein; B, 0.01 nmol/mg protein; C, <0.01 nmol/mg protein; D, 1.95 nmol/mg protein.
Figure 3. The effect of CDME or MEA on the morphology of normal or nephropathic cystinotic fibroblasts. Fibroblasts were cultured under the usual conditions and exposed to TNF- for 16 h as described in Materials and Methods. Panels a through c are normal fibroblasts; panels d through f are nephropathic cystinotic fibroblasts. Panels b and e show cells after TNF- exposure alone; panels c and f show the effect of CDME in normal cells (c) or MEA in cystinotic cells (f). Note typical apoptotic morphology in panels b, c, and e, with more apoptotic cells in e (cystinotic) than b (normal). Enhanced apoptosis is seen in normal cells to which CDME is added (c). More normal morphology is seen in nephropathic cystinotic cells after MEA treatment (f). Photographs via phase microscopy.
Table 4. Apoptosis in normal and nephropathic cystinotic fibroblasts exposed to serum withdrawal
To substantiate that lysosomes are permeabilized by TNF- (5)under these conditions, cathepsin B (a lysosomal cysteine protease)was localized by immunohistochemistry in normal and nephropathiccystinotic cells before and after induction of apoptosis. Lysosomeswere identified with Lysotracker red dye. In both normal andcystinotic fibroblasts before TNF- treatment, cathepsin B (green)displays a punctuate pattern that is closely associated withthe red lysosomal dye. Induction of apoptosis by TNF- causesa translocation of cathepsin B from a lysosomal location toa diffuse cytosolic location, with loss of colocalization ofcolor (Figure 4).
Figure 4. Loss of colocalization of cathepsin B in normal and nephropathic cystinotic fibroblasts after TNF- exposure. Fibroblasts were maintained under normal culture conditions, exposed to TNF-, loaded with Lysotracker red, fixed, and stained for cathespin B using an anti-cathepsin B antibody as described in Materials and Methods. Panels a through f are normal fibroblasts; a through c are control cells; d through f are treated with TNF-. Panels g through l are cystinotic fibroblasts; g through i are control; j through l are treated with TNF-. Note loss of granularity and decreased colocalization in c versus f and i versus l. Photographs via a deconvoluting microscope.
Cultured human renal proximal tubule epithelial cells displaymarked sensitivity to apoptosis after CDME exposure. The concentrationemployed in fibroblasts (0.5 mM) was toxic to these cells andwas decreased to 0.1 or 0.25 mM for the RPTE experiments. Therate of apoptosis produced by 0.25 mM CDME alone was equal tothat induced by exposure to TNF- alone (Figure 5). The timecourse was accelerated, with these cells attaining a maximumrate of apoptosis within 6 h, (as opposed to 17 h in fibroblasts)followed by lysis and release from the culture dish. The RPTEcystine content after exposure to 0.25 mM CDME for 1 h was 1.99nmol/mg protein. Representative fluorescence micrographs ateach time point are shown in Figure 6. Exposure of RPTE cellsto TNF- after cystine loading by exposure to 0.1 mM CDME andanalysis by Annexin V and propidium iodide (PI) staining yielded4.3% Annexin V-positive cells and zero PI-positive cells at1 h after exposure, 9.5% and 1.7% at 3 h, and 25.3% and 9.1%at 6 h. The proportion of necrotic (dead) RPTE cells increaseswith duration of exposure to CDME, but remains substantiallyless than the number of those that are apoptotic.
Figure 6. The effect of CDME on apoptosis in RPTE cells. Fluorescent micrographs of the cells in Figure 5 are shown at 0.5, 1, 3, and 6 h (A through D, respectively).
Four apoptotic triggers (TNF-, Anti-Fas, UV light, and serumwithdrawal) and three standard measures of the apoptotic response(TUNEL, CaspAse activation, and Annexin V) were employed toascertain the effect of lysosomal cystine on apoptosis in culturedcells. RPTE cells display increased rates of TUNEL reactivityafter lysosomal cystine loading without exposure to apoptotictriggers (Figures 5 and 6). CDME is known to traverse both plasmaand lysosomal membranes, but it is only concentrated withinthe lysosomes, where resident hydrolases remove the methylestergroups, producing free cystine. The acidic environment producesa net positive charge, inhibiting exit of cystine except viathe lysosomal cystine transporter cystinosin (18,21). We speculatethat under normal circumstances in normal cells, the influxof cystine into lysosomes via proteolysis of disulfide-containingproteins (22,23) proceeds at a rate less than the normal egressrate of cystine via cystinosin. This would yield a small andrelatively constant rate of release of cystine into the cytosol,which could be readily reduced to cysteine by glutathione. Incystinotic fibroblasts, which lack functional cystinosin, cystineaccumulates to very high levels (2). Apoptotic stimuli leadto lysosomal permeabilization and translocation of cathepsinsB and D into the cytosol (5–9) (Figure 4); it is thereforepossible that rapid release of cystine into the cytosol occursduring this permeabilization phase and results in a locallyaltered redox potential, and/or reaction with critical proteinthiols, that may sensitize the cells to, or trigger, the apoptoticresponse (24). In contrast, extracellular cystine deprivationis pro-apoptotic (25–27), and cells can be rescued fromapoptosis by extracellular cystine supplementation via the ectomembraneenzyme, glutathione tranpeptidase, which is localized to theplasma membrane and which functions in the synthesis of reducedglutathione (28). Accessibility of extracellular cystine tothis plasma membrane complex leads to enhanced GSH synthesis,which may not be the case in cystine originating from lysosomes.
Apoptosis is known to play a role in the renal tubular dysfunctionseen in transplantation rejection phenomenon and also in certainforms of retinopathy. The renal tubule and retina are highlysensitive to apoptosis (29,30). These two tissues are the firstto be affected in nephropathic cystinosis (2), and it is feasiblethat the order of tissues involved in the disease reflects theintrinsic sensitivity of each to apoptosis. CDME is known todisrupt renal tubule cell function in animal models and culturedcells (31–33), and it could be that exposure to CDME alonealters the redox potential sufficiently to trigger the apoptoticresponse (Figures 5 and 6). CDME also causes inhibition of Na+-dependenttransporters (33). Free cystine has been known to be nephrotoxicin experimental animals since 1925 (34); however, the mechanismremains to be determined. Cystinosis patients display a typicalswan neck deformity of the proximal tubule (35,36), concomitantwith development of the Fanconi syndrome and consistent withhypocellularity of that structure.
Enhanced sensitivity to apoptosis due to increased lysosomalcystine loading offers another insight into the interplay oflysosomes and apoptosis as well a new perspective in understandingthe pathophysiology of cystinosis. Increased lysosomal cystinein fibroblasts or RPTE cells either as the result of cystinosindeficiency, or induced by incubating normal cells in cystinedimethylester, causes increased apoptosis. This finding is importantbecause it excludes other effects of defective CTNS expressionand alternative effects of CDME exposure, because the only commonelement in the two conditions is elevated lysosomal cystine.Enhanced sensitivity to apoptosis is observed after triggersthat stimulate both the intrinsic apoptotic pathway (UV light)and the extrinsic pathway (TNF-, anti-Fas antibody, or serumwithdrawal). The intrinsic and extrinsic pathways converge atthe BID/Bcl2 locus and then ultimately activate caspases (37).It is somewhat counterintuitive that lysosomal cystine releaseincreases apoptosis, because the executioner caspases are thiolproteases, which should be inhibited by the disulfide cystine.
Inappropriate apoptotic cell death may occur in cystinotic tissuesafter extrinsic or intrinsic apoptotic stimuli are presented,which would fail to be executed in normal tissues due to dampingof the apoptotic cascade. This aberrant sensitivity could accountfor the cystinotic phenotype by causing inappropriate cell deaththroughout embryogenesis and the life of the individual, leadingto a hypocellular state in many tissues. The mechanisms causingthis augmented rate of cell death remain to be determined. Itis possible that the cells may proceed to necrosis in some casesunder these circumstances, as suggested by the increase in PIpositivity seen in RPTE cells treated with both TNF- (38) andCDME.
Patients with variant forms of cystinosis are either heterozygousfor CTNS mutations, which, in the homozygous state, cause thenephropathic form of the disease, or they are homozygous forpredicted milder mutations (39,40). The leukocyte cystine contentoverlaps among the various clinical forms of cystinosis, rangingfrom 1.3 to 11.6 nmol/mg protein in nephropathic, 1.7 to 2.5in the intermediate form, and between 0.5 and 1.8 in the ocularform. A similar overlap is shown in cultured fibroblasts inTable 1. Distinction among the varieties is based on the ageat onset of symptoms and clinical severity (2,39–41).
Two variant cystinotic cell lines did not show enhanced ratesof apoptosis despite increased lysosomal cystine (Tables 2 and 3).The combination of a severe nonsense mutation and a splice-sitemutation allows for some residual cystine transport activityand, because the splice site mutation in cell line GM00379 causestruncation of cystinosin just distal to the second lysosomalrecognition site, permits some lysosomal localization of cystinosinand cystine transport activity (39,42). We speculate however,that these phenotypic cystinosis variants are not due to residualcystinosin activity, as the lysosomal cystine content overlapsthat of the nephropathic form; rather, they are due to linkageor to co-segregation of a gene or genes, which confers relativeresistance to apoptosis. The cystinosis mutation itself is rare;if a rare allele is also responsible for resistance to apoptosis,then the extreme scarcity of these variants is explained, asis the milder phenotype. The phenotype could also be causedby diminished expression of a pro-apoptotic gene such as APC,as has recently been described in familial adenomatous polyposis(43). This hypothesis raises the possibility of increased neoplastictransformation in these patients, however none has yet beenrecognized.
We conclude that nephropathic cystinotic fibroblasts enter thepathway for programmed cell death more readily than do normalfibroblasts and that normal fibroblasts and RPTE cells displayaugmented entry into that pathway after their lysosomal cystinecontent is artificially increased. Study of the pathway(s) bywhich cellular perturbations result in modulation of the apoptoticcascade may lead to a clearer understanding of the regulationof apoptosis, and a better understanding of the developmentof the cystinotic phenotype.
Acknowledgments
We gratefully acknowledge the support of the Cystinosis Foundationand the Cystinosis Research Network, the expert secretarialassistance of Frances Martin, and helpful discussions with Dr.Tyler Curiel. This study was presented in part at the AmericanSociety of Human Genetics, October 2001, San Diego, CA.
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Received for publication February 15, 2002.
Accepted for publication August 16, 2002.
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Abstract
Introduction
versus 7.8% in control normal fibroblasts. Anti-Fas antibodies and UV exposure induced apoptosis in 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively, versus 5.2% and 7.1% in normal fibroblasts when analyzed by CaspACE (P < 0.05). Similar results were found when the cells were analyzed by TdT-mediated dUTP nick end labeling (TUNEL). When the cystine content of normal fibroblasts is increased by exposure to cystine dimethylester (CDME), the apoptotic rate is increased to the rate seen in nephropathic cystinotic cells. Decreasing the cystinotic cells’ cystine content by use of cysteamine results in normalization of the apoptotic rate. Renal proximal tubule epithelial (RPTE) cells are much more sensitive to CDME than fibroblasts, reaching 43.8% apoptosis 6 h after exposure to CDME alone, compared with 38.2% when exposed to TNF-
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