Transformation of skin fibroblast cells of a cystinotic patient by simian virus 40: evidence for an establishment of a permanent cell clone which retains the original metabolism defect.

Human skin fibroblast cells derived from a juvenile patient with nephropathic cystinosis were transformed by simian virus 40. Transformed cell clones were isolated and established in tissue culture. In comparison to the parental cystinotic cells, the newly isolated, transformed cell clones had a higher plating efficiency, a modal chromosome number of 68, grew in soft agar, and showed a nuclear immunofluorescence typical for SV 40-specific tumor (T) antigen. The content of intracellular, unbound cystine in the transformed cell clone was of the same level (6.1 nmol 1/2 cystine/mg protein) as in the parental cystinotic cells (7.4 nmol). Control cells (SV 80 and WI-38) contained normal levels of cystine (0.31 and 0.47 nmol 1/2 cystine/mg protein). The growth characteristics make the transformed cystinotic cell clone suitable for large scale preparation of cellular constituents, i.e. lysosomes which seem to be affected in cystinotic patients.     

Decrease of intracellular cystine content in cystinotic fibroblasts by inhibitors of gamma-glutamyl transpeptidase

Cystine content of skin fibroblasts derived from patients with cystinosis was decreased by inhibitors of gamma-glutamyl transpeptidase, the initial enzyme in glutathione catabolism. The addition of maleate or the gamma-glutamyl hydrazone of alpha-ketobutyric acid to culture medium (1-20 mM) resulted in dose-dependent decreases of up to 55% on intracellular cystine content of cystinotic cells in 24 h. L-Serine in sodium borate buffer (40 mM each) produced similar results and further decreased cystine levels to 14% of cystinotic control values after 10 days incubation. Analysis of intracellular amino acids showed that, in general, other amino acids remained unchanged following serine-borate treatment. These results suggest that cystine storage in cystinotic tissues may be related to metabolism of glutathione.     

Increased free-cystine content of fibroblasts cultured from patients with cystinosis

The presence of a significantly increased content of free-cystine in skin fibroblasts from both homozygotes and heterozygotes for cystinosis emphasizes the central role of cystine in this disease, even though the primary defect responsible for cystine accumulation is yet to be determined. The studies described in this communication provide evidence that cystine is compartmentalized in a subcellular location in cystinotic cells. In fact, the very growth of cystinotic fibroblasts in the presence more than 100 times the usual content of free-cystine is evidence that the accumulated cystine is not freely dispersed throughout the cell, since would otherwise inhibit many enzymes requiring free sulfhydryl groups for activity (Patrick, 1965). We have no evidence as to whether the cystine is located in a known subcellular organelle or in a previously unrecognized location. Skin fibroblasts may provide a convenient tool to pursue these questions.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Comparative study of cystine clearance in cystinotic and I-cell fibroblasts upon exposure to cystine dimethyl ester

I-cell fibroblasts can accumulate cystine at levels comparable to those seen in homozygous cystinotic fibroblasts. Cystine accumulation in cystinosis is accounted for cystine clearance defect in situ. To unravel the question whether the same clearance defect or two different mechanisms cause cystine accumulation in I-cell disease, we used the cystine loading technique upon exposure of skin fibroblasts to radioactive cystine dimethyl ester. Normal, cystinotic and I-cell fibroblasts were exposed to radioactive cystine dimethyl ester, and the clearance of the generated radioactive cystine was measured. Cystinotic cells showed a marked defect in cystine clearance in situ, as compared to normal fibroblasts. In I-cell fibroblasts, we observed slow hydrolysis of cystine dimethyl ester to cystine, indicating low esterase activity, but no defect in clearance of the generated cystine. Cysteine production from the exogenous cystine dimethyl ester, presumably by cytoplasmic hydrolysis of the generated cystine, is normal in I-cell fibroblasts. Thus, our results indicate that, unlike cystinosis, there is no cystine clearance defect in situ for cystine in I-cell disease, and probably unrelated mechanisms cause cystine storage in cystinosis and I-cell disease.     

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.     

Effect of selenium compounds on selenium content, growth and 35S-cystine metabolism of skin fibroblasts from normal and cystinotic individuals.

Kidney samples from children with the inborn metabolic disease cystinosis contain 4 times more selenium (Se) than do kidney samples from normal individuals (p = 0.1). However, when cultured skin fibroblasts from cystinotic patients and normal control individuals are incubated in Se-D,L-methionine, Se-D,L-cystine, Se-cystamine X HCl, Se-urea, selenite or in medium without added selenium, only the cystinotic fibroblasts grown in Se-urea or selenite (SeO3=) contain more selenium than do the corresponding normal cells (p less than 0.05). In both types of cultured fibroblasts, the order of descending toxicity per ppm selenium is: Se-urea greater than Se-cystamine greater than Se-cystine greater than or equal to SeO3= much greater than Se-methionine. High (apparently toxic) concentrations of Se-urea and Se-cystamine lower the elevated intracellular free (nonprotein) cystine content of cystinotic fibroblasts to less than 60% of control values; at lower concentrations, these compounds raise the cystine content of these cells to over 140% of control values. Appropriate concentrations of SeO3=, Se-cystine and Se-methionine also elevate the free cystine content of the cystinotic cells. During a 75 minute incubation in 35S-cystine, the incorporation of 35S into the acid precipitable (protein) fraction of both cell types is significantly inhibited by Se-cystamine (approximately 55% control; p less than 0.05). The incorporation of 35S-cystine into glutathione is inhibited by Se-cystine (approximately 40% control) in both fibroblast types (p less than 0.05). In cystinotic cells, Se-cystamine significantly reduces incorporation of 35S-cystine into the cystine pool (40% control) as does SeO3= (67% control; p less than 0.05). Protein and glutathione synthesis in cystinotic fibroblasts are more strongly inhibited by Se-cystine and SeO3=, respectively, than in normal fibroblasts (p less than 0.05). These studies demonstrate that selenium compounds exhibit a different sequence of toxicity in fibroblasts than in the intact animal and that some previously unreported metabolic effects (i.e. inhibition of glutathione synthesis) may contribute to their toxicity.     

Uptake of cystine by cystine-depleted fibroblasts from patients with cystinosis

[³⁵S]L-cystine uptake was measured in cultured skin fibroblasts from patients with nephropathic cystinosis, pretreated with cysteamine to deplete their cystine pools. The uptake was greater in the cystinotic cells than in normal cells. The data suggest that the enhanced [³⁵S]-cystine uptake observed in cystinotic cells is not a consequence of disulfide exchange with stored cystine and may be related to the underlying abnormality in this enigmatic disorder.     

pH-profile of cystine and glutamate transport in normal and cystinotic human fibroblasts

In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porter's ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct.     

Reversion from deficiency of galactose-1-phosphate uridylytransferase (GALT) in an SV40-transformed human fibroblast line

Control SV40-transformed human fibroblasts can be readily adapted to growth on medium containing galactose as sole hexose source (galactose-MEH). However, most cells from a line of SV40-transformed skin fibroblasts from a patient with galactosemia (galactose-1-phosphate uridylyltransferase (GALT) deficiency) died in galactose-MEM. Surviving cells of this line either grew in completely sugar-free media or had acquired significant amounts of GALT activity. Two presumptive revertant cell lines with GALT activity were characterized in detail. The expression of GALT in these two lines was stable in nonselective conditions. Each had different reaction maximum velocities with respect to uridine diphosphoglucose (UDPg) concentration as compared to residual activity in the parental cell strain or control cells. Both appeared to demonstrate heat-inactivation profiles for GALT than differed from the parental cells or controls. UDPG concentration was found to significantly alter the thermostability of GALT. A competitive radioimmunoassay for GALT showed that these two lines had amounts of the GALT protein comparable to that of the parental cell strain or control cells. The electrophoretic mobility of GALT from the two presumptive revertants was found to differ from control cells. It was concluded that structural gene changes were probably responsible for the apparent reversion in these lines.     

The effects of decreased growth temperature on the cystine content of cystinotic fibroblasts

Cystinotic fibroblasts transferred from 37 degrees C to 28 degrees C accumulated additional cystine over the period from 4 to 7 days of incubation at 28 degrees C, after which the additional cystine was lost; warming (to 37 degrees C) of cells with elevated cystine stores led to rapid cystine loss. These results, taken together with previously published data showing cystine release from cystinotic fibroblasts incubated at above-normal temperature, are interpreted as indicating the presence in the cystinotic fibroblast lysosome membrane of a cystine-porter whose efficacy is increased by an increase in membrane fluidity. This porter may be the residual activity of the cystine porter that is known to be deficient in cystinosis, or it may be a second as yet unrecognized porter. It is further proposed that this porter is responsible for the presumed efflux of cystine from cystinotic lysosomes.     

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.     

A potential new prodrug for the treatment of cystinosis: design, synthesis and in-vitro evaluation

Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised lysosomal levels of cystine in the cells of most organs. The disorder is treated by regular administration of the aminothiol, cysteamine, an odiferous and unpleasant tasting compound that along with its metabolites is excreted in breath and sweat, leading to poor patient compliance. In an attempt to improve patient compliance a series of novel prodrugs has been designed and evaluated as a potential new treatment for nephropathic cystinosis. The first of the prodrugs tested, 3a, was found to decrease the levels of intracellular cystine in cystinotic fibroblasts. This is the first report of a potential new therapeutic treatment for nephropathic cystinosis since the advent of cysteamine bitartrate.     

The effect of chloroquine on the metabolism of [35S]cystine in normal and cystinotic human skin fibroblasts.

The present study concerns the effect of the lysosomotropic drug chloroquine on the uptake and metabolism of [35S]cystine in vitro by normal human fibroblasts and those from patients suffering from the lysosomal storage disease cystinosis. When the cells were cultured with [35S]cystine for periods in excess of 4 h, it was found that chloroquine considerably increased (up to 30-fold) the labelling of the intracellular cystine pool in cystinotic cells, with no increase or a much smaller increase in normal cells. For this effect chloroquine had an optimum concentration of 20 microM, with a small effect still being noticeable at 1 microM. A quinoline analogue, 4-(dimethylaminoethylamino)-7-iodoquinoline, had a similar effect to chloroquine. However, NH4Cl at concentrations of between 100 microM and 50 mM showed either no effect (at the lower concentrations) or a depression of intracellular cystine labelling (at the higher concentrations). The differences between the effects of the quinolines on cystinotic acid normal cells were not due to differences in total cell uptake of drug.     

Renal cell culture using autopsy material from children with cystinosis

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.     

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts.     

Renal cell culture using autopsy material from children with cystinosis

Summary Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE₁, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations. This work was supported by Grants AM 01074-01, AM 18434, and GM 17702 from the National Institutes of Health, Bethesda, MD.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Levels of gamma-glutamyltranspeptidase in cultured skin fibroblasts from cystinotics and normals

The activity of gamma-glutamyltranspeptidase (GGTP) was determined in cultured skin fibroblasts initiated from normals and cystinotics. For the most part, fibroblasts cell lines from patients with nephropathic cystinosis had elevated levels of gamma-glutamyltranspeptidase averaging 37.01 ± 5.88 (16 determinations) as compared with levels in cells from normals of 14.53 ± 1.43 (17 determinations) nanomoles p-nitroaniline released/hr/mg protein with glycylglycine as acceptor substrate. Enzymatic activities also were elevated in affected cells when cystine was the receptor substrate. Increased GGTP is not secondary to the abnormal amounts of intracellular free cystine since the depletion of the cystine pool did not affect the elevated transpeptidase levels. Whether the increased transpeptidase is closely related to the genetic basis for cystinosis, however, remains to be elucidated.