Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1676-1679, April 1977
Genetics
Increased resistance of cystic fibrosis fibroblasts to ouabain toxicity (cell culture/survival assay/genetic disease/cardiac glycoside)
J. EPSTEIN"1 AND JAN L. BRESLOWt * Clinical Genetics Division, Department of Medicine, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115 and the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115; and t Metabolism Division, Department of Medicine, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115 and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by John F. Enders, January 24, 1977
ABSTRACT Diploid skin fibroblasts derived from several unrelated patients with cystic fibrosis (CF) were tested for resistance to the cytotoxic effects of ouabain in comparison with cells from normal individuals. Cells from CF and normal individuals were plated at low density and exposed to ouabain at different concentrations for 24 hr. Dose-response curves showed that CF cells survived significantly better than did normal cells at all ouabain concentrations from 0.1 nM to 1 IAM in potassium-deficient medium. In medium containing the usual amount of potassium (6.25 mM), survival of the CF cells exposed to ouabain was decreased to that of the normal cells. If the glucose concentration of the potassium-deficient medium was decreased to 10% of the usual amount, the resistance of the CF cells to ouabain disappeared and their survival was identical to that of the normal cells. These findings may be useful in understanding the biochemical basis of CF and its clinical manifestations. Cystic fibrosis (CF) is one of the most common genetic diseases causing early death in children. It occurs with a frequency of approximately 1/1500-2000 live births in Caucasians, and the mean survival for CF patients is only 15 years at present. The disease is thought to have an autosomal recessive pattern of inheritance, and it is believed that 1 in every 20 persons in the United States carries the trait for the disease. Clinically, homozygous persons are usually found to have chronic pulmonary disease, some degree of pancreatic exocrine dysfunction, and abnormally high concentrations of Na+ and Cl- in their sweat. The clinical manifestations and severity of the disease process may vary greatly among affected persons (1-
3).
The metabolic basis for CF is not yet known, and research efforts to identify some biochemical defect in the disease have taken several different approaches. Some of these findings have suggested that the disease may involve an abnormality of ion transport. The sweat, saliva, and seminal fluids of CF patients have been reported to contain factors that interfere with ion transport in the salivary and sweat glands of normal persons (4, 5). However, no alteration of ion transport has yet been proved directly in CF cells. In the present study, CF and normal fibroblasts were tested for their resistance to the cytotoxic effects of ouabain, a cardiac glycoside known to inhibit the transport of ions across the cell membrane (6). METHODS AND MATERIALS Cells and Media. Human diploid skin fibroblasts were derived by percutaneous punch biopsy of the volar surface of the forearm or from foreskin specimens; informed consent was obtained in all cases. Cell stocks were maintained at 36.5° in Abbreviations: CF, cystic fibrosis; EMEM, Eagle's minimum essential medium; EMEM-B, basic medium with additional glucose (total, 1900 mg/liter); EMEM-C, basic medium but without KCl and with fetal calf serum replaced with dialyzed fetal calf serum; EMEM-D, basic medium but with decreased glucose (total, 100 mg (liter)), without KCl, and with dialyzed fetal calf serum. t To whom reprint requests should be addressed.
humidified 7% C02/93% air atmosphere in Falcon plastic tissue culture flasks. The cells were grown in Eagle's minimal essential medium (EMEM) (MBA, Inc.) containing glucose (1000 mg/liter) and 6.25 mM K+ (as KCl). This was supplemented with nonessential amino acids (IBL, Inc.), penicillin (50 units/ ml), streptomycin (50 ,g/ml), and 10% fetal calf serum (MBA, Inc.). In the experiments reported here, this basic medium was modified as follows: for EMEM-B, an additional (900 mg/liter) glucose was added; for EMEM-C, KCl was omitted and 10% dialyzed fetal calf serum was substituted for fetal calf serum; and for EMEM-D, KCl was omitted, the glucose concentration was decreased to 100 mg/liter, and 10% dialyzed fetal calf serum was substituted for fetal calf serum. Three carefully tested lots of fetal calf serum were used in the experiments reported here and were chosen for their ability to yield high plating efficiences and optimal growth of fibroblasts. Dialyzed fetal calf serum was prepared by dialysis against three changes of 20 volumes of 0.15 M NaCl for 24-86 hr at 4°. Flame photometry was used to verify that each batch of dialyzed serum and EMEM-C contained no detectable levels of K+. All cell strains were negative on testing for mycoplasma. Survival Experiments. Strains used in these studies were between the 5th and 25th population doublings in vitro. When different strains were to be compared, they were chosen to be within three population doublings of each other. For survival assays, the appropriate strains were harvested from exponentially growing monolayers with 0.05% trypsin (Difco, 1:250) and 0.5 mM Na2EDTA. Trypsinized cells were rinsed once, resuspended in EMEM-D, and counted with a Coulter electronic cell counter. Various numbers of cells were distributed into three or four replicate 100-mm diameter Falcon plastic tissue culture dishes in 9 ml of EMEM-B, EMEM-C, or EMEM-D. Inocula were chosen so as to provide approximately 75-100 surviving colonies per dish. Twenty-four hours later, 1 ml of the appropriate concentration of ouabain in EMEM-D was added to each dish. After a 24-hr incubation period with the drug, the medium was removed, each dish was rinsed twice with 10 ml of Ca2+- and Mg2+-free Earle's balanced salt solution, and then renewed with 10 ml of EMEM-B. The dishes were renewed with EMEM-B every 6-8 days. After 14-21 days of incubation, the dishes were rinsed with 0.15 M NaCl, fixed with methanol, and stained for colony counting; only those colonies containing a minimum of 50 cells were counted. The survival of the cells exposed to ouabain was expressed as percentage of the number of cells plated, corrected for the actual plating efficiency of the cells in the absence of ouabain for each experiment. The ouabain (Sigma Chemical) was dissolved in distilled water to a final concentration of 0.1 mM, sterilized by filtration through a 0.22-aim membrane filter, stored at -20°, and thawed immediately before use. This stock solution was diluted with EMEM-D to the appropriate concentrations for use in survival experiments. 1676
Genetics: Epstein and Breslow
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Sources of fibroblast strains
Strain
S-127 S-147 S-149 S-203 S-204 EX-25 S-115 S-116 S-206 S-212
Age of patient (years)
Sex
Diagnosis
Site of biopsy
M F M M M M M M F F
CF CF CF CF CF Normal Normal Normal Normal Normal
Forearm Forearm Forearm Forearm Forearm Forearm Foreskin Foreskin Forearm Forearm
16 22 21 23 Newborn 24 Newborn Newborn
Newborn 18
Selection of Normal and CF Patients. Control fibroblasts strains were from healthy donors for whom it could be established that the donor had no previous personal or family history of CF or other undiagnosed medical problems. The CF strains used in these studies were obtained from unrelated patients who were being seen at the Children's Hospital Medical Center, Boston, MA, and who had well-documented, clinically confirmed CF with positive sweat tests and involvement of either the respiratory or gastrointestinal system or both. The sources of all strains are shown in Table 1. RESULTS The results of dose-response studies to determine the cytotoxic effect of ouabain on normal and CF fibroblasts are presented as survival curves in which the number of survivors, after correction for plating efficiency, is plotted against the concentration of drug. Fig. 1A is a typical survival curve from a study in which the ouabain sensitivity of cells (strain S-147) from a CF patient were compared to the ouabain sensitivity of cells (strain EX-25) from a normal person. The experiment was performed in EMEM-C, a medium deficient in K+. After a LU~ ~
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1677
24-hr exposure to ouabain, the survival of S-147 was greater than that of the normal strain, EX-25. The CF cells exhibited little decrease in survival in the presence of 3.3 nM ouabain, whereas the survival of the normal cells at that concentration was decreased to 60%. At greater doses, the slope of the curve for the normal cells was much steeper than that for the CF cells. Fig. 1B shows the results of an experiment similar to that in Fig. 1A except that the cells were plated and exposed to ouabain in EMEM-B, a medium containing 6.25 mM K+. There was no difference in survival between the two strains after a 24-hr exposure to the drug. In EMEM-B the decreased survival of CF cells was similar to that of normal cells. The normal cells showed the same sensitivity to ouabain in EMEM-B and EMEM-C. The increased ouabain resistance of CF cells, when compared to normal cells in EMEMv-C (K+-deficient medium) also was observed in other experiments using different CF and normal fibroblast strains. Survival studies were performed at ouabain concentrations ranging from 0.1 nM to 1 1AM. However, for comparative purposes only, the survival (corrected for plating efficiency) for each strain at 0.1 AM is presented (Table 2). In most experiments, the CF strains showed approximately 7080% survival after exposure to 0.1 zIM ouabain, compared to only 20% survival for the normal strains. In experiments in which the survival for the CF strain was less than 80%, the survival of the normal strain at 0.1 gM ouabain was also lower. Thus, although the actual survival varied somewhat from experiment to experiment, the CF strains were always more resistant than the normals by a factor of 2.7-4.8. In order to determine what effect a decrease in the amount of energy source available to the cells would have on ouabain resistance, survival experiments similar to those shown in Fig. 1 were performed in EMEM-D, a K+-deficient medium in which the normal concentration of glucose was decreased to 10% of the usual level. In the experiment shown in Fig. 2A, the cells were plated in EMEM-C with a normal glucose concentration and, as shown in Fig. 1, the CF strain was more resistant to ouabain. In Fig. 2B, both strains were plated in EMEM-D,
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FIG. 1. Survival of S-147 (0), a CF strain, and EX-25 (0), a normal strain. Each point represents the mean number of colonies in three or four replicate petri dishes in which the survival in each dish was within 7% of the averaged value. (A) Cells were plated and exposed to ouabain in EMEM-C, a K+-deficient medium. For the two separate experiments in which the strains were compared, the plating efficiencies were 2.0 and 1.0% for S-147, and 1.6 and 1.0% for EX-25. (B) Cells were plated and exposed to ouabain in EMEM-B, a medium containing 6.25 mM K+. For the experiment shown, the plating efficiencies were 2.8% for S-147 and 3.4% for EX-25.
1678
Proc. Nati. Acad. Sci., USA 74 (1977)
Genetics: Epstein and Breslow
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OUABAIN CONCENTRATION (M) FIG. 2. Survival of S-149 (o), a CF strain, and EX-25 (0), a normal strain. Each point represents the mean number of colonies in three or four replicate petri dishes in which the survival in each dish was within 7% of the averaged value. (A) Cells were plated and exposed to ouabain in EMEM-C, a K+-deficient medium containing glucose at 1000 mg/liter. The plating efficiencies were 4.8% for S-149 and 3.1% for EX-25. (B) Cells were plated and exposed to ouabain in EMEM-D, a medium deficient in K+ and glucose (100 mg/liter). The plating efficiencies were 4.2% for S-149 and 3.0% for EX-25.
and the survival of S-149, the CF strain, was decreased to that of the normal strain. Both strains showed a survival of approximately 20% at 0.1 AtM ouabain in the low-glucose medium. Because the plating efficiencies were the same in the two media, the effect of decreased glucose concentration on the ouabain resistance of the CF cells is not likely to be due to any effect on the plating efficiency. Because in recent years much attention has been paid to factors produced by CF cells in culture, we tried to evaluate whether such factors might be influencing our results. Therefore, we also performed survival experiments in which medium was exchanged between normal and CF cultures after 24- and 48-hr incubations. There was no change in the ouabain sensitivities of the different fibroblast strains. This would suggest that the different ouabain sensitivities of CF and normal cells cannot be altered by any stable factors that might be released into the medium. DISCUSSION The purpose of this study was to determine whether or not CF and normal cells have the same sensitivity to ouabain, a cardiac glycoside known to inhibit the transport of ions across the cell membrane (6). It was found that CF fibroblasts are more resistant to the cytotoxic effects of ouabain than are normal cells when plated into K+-free medium for 24 hr and then exposed to the drug in the same medium for another 24 hr. However, the increased ouabain resistance of CF cells disappears if the cells are plated in medium containing a normal K+ concentration (6.25 mM) for 24 hr. In addition, the increased resistance of the CF cells to ouabain seen in K+-deficient medium disappears if the glucose concentration in the K+-deficient me-
dium is decreased to 10% of the normal level. It cannot be determined from the data presented here how the higher glucose concentration increases the resistance of the CF cells to ouabain. The mechanism by which ouabain kills cells is uncertain, but it is believed that the drug inhibits the cellular Na+/K+ transport system (6). In studies of cells of normal ouabain sensitivity, it has been shown that the drug binds to the cell surface with high affinity, causing a subsequent rapid dissipation of the normal cellular Na+/K+ gradient (7). The swelling of the cells which results is believed to cause cell death. Mutant mammalian cells resistant to high concentrations of ouabain have recently been isolated. These mutants have been selected from mouse L cells (8), hamster CHO cells (8), human lymphoblastoid lines (9), and human diploid skin fibroblasts (10, 11). In each case the frequency of ouabain resistant mutants could be increased by exposure of the wild-type cells to mutagenic agents (8-11), and the phenotype was stably expressed through many generations of growth in the absence of the drug. These experiments suggest that ouabain resistance arises as the result of genetic change. In some of these studies on ouabainresistant mutants, it was shown that less radiolabeled ouabain bound to the resistant cells than bound to normal cells and that these resistant cells showed less inhibition of 86Rb+ and 42K+ transport by ouabain than did normal cells (9, 11). The Na+/ K+-stimulated ATPase activity in isolated plasma membranes in some of these mutants also showed decreased sensitivity to ouabain when compared to the membranes derived from the wild-type cells (9, 11). The shapes of the survival curves in other reports for cells exposed to ouabain differ from those reported here. The sur-
Genetics: Epstein and Breslow
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 2. Survival of cells in ouabain Exp.
Strain
1
S-149 (CF) EX-25 S-149 (CF) EX-25 S-147 (CF) S-206 S-147 (CF) S-115 S-147 (CF) EX-25 S-147 (CF) EX-25 S-147 (CF) EX-25 S-127 (CF) S-116 S-215 (CF) S-115 S-203 (CF) S-115 S-203 (CF) S-212 S-204 (CF) S-116
2 3 4
5
6 7
8 9
10 11 12
Plating efficiency* Survivalt
5.5 2.7 3.0 1.4 2.9 1.1 2.0 1.6 1.6 2.0 1.5 1.5 1.0 1.8 1.0 2.7 1.4 1.1 2.2 3.3 1.0 1.0 1.2 1.8
0.78 0.18 0.87 0.27 0.75 0.20 0.86 0.18 0.60 0.22 0.78 0.18 0.90 0.25 0.58 0.15 0.70 0.20 0.80 0.28 0.83 0.18 0.80 0.21
1679
Cloning studies must be performed to determine whether or with respect to drug sensitivity exist in these fibroblast strains. It should be noted that the plating efficiencies of both normal and CF cells were similarly low and the increased resistance of CF cells to ouabain was seen in several different fibroblast strains, suggesting that, if they exist, subpopulations of similar sensitivity were present in all of the strains examined. It has been reported (13) that CF fibroblasts exhibit a lower rate of thymidine incorporation into acid-insoluble material than do normal fibroblasts. It also has recently been reported (14) that CF fibroblasts have a 3- to 5-fold higher increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels after isoproterenol stimulation than do normal cells. None of these studies, including those of the present report, however, have shown a specific deficiency in any gene product in CF cells. not variants
RatioT
4.3
3.2 3.8
4.8 2.7
4.3 3.6
3.9 3.5
2.9 4.6 3.8
* Plating efficiency of cells in EMEM-C in the absence of ouabain. t Survival (corrected for plating efficiency) at ouabain dose of 0.1 AM
in EMEM-C. Ratio of survival of CF cells to normal cells in the indicated experiment.
vival curves for mouse L cells and hamster CHO cells were obtained with cells plated at low density in K+-containing medium. Ouabain concentrations of 0.1 mM were required to cause any significant decrease in the survival of wild-type cells; a 10-fold increase in drug dose (to 1 mM) decreased survival to 10-4 (8). In our studies, 0.1 nM ouabain significantly decreased survival in normal cells, and a 10,000-fold increase in drug dose (to 1 AM) decreased survival to approximately 10-1. The previously reported survival curves for human fibroblasts exposed to ouabain (11) cannot be compared to our data because the conditions used are different. In that study, human fibroblasts were plated in K+-containing medium with various ouabain concentrations, and cell counts were determined 6-8 days later. In contrast, in the present study, the cells were exposed to ouabain for only 24 hr in K+-deficient medium, and the surviving cells were required to form colonies. In one previous study (12), it was reported that CF cells bound the same amount of radiolabeled ouabain to their cell surface as did normal fibroblasts. However, in that report, the binding studies were performed with cells that had been maintained in medium that contained K+ and the cells were transferred to K+-deficient, low-glucose (100 mg/liter) salt solution just before the addition of the ouabain. Our results under similar conditions (Figs. 1B and 2B) show that there is no detectable difference in the survival of CF and normal cells in the presence of ouabain. Our survival studies have revealed increased ouabain resistance in unselected populations of CF fibroblasts but have not detected subpopulations of normal cells that varied in drug sensitivity; however, the possible existence of such variants cannot be ruled out solely by the use of survival assay methods.
We thank Dr. Richard L. Davidson for his critical reading of this manuscript and for his comments and suggestions during the course of the work. We thank Dr. Harry Shwachman for helping to provide to clinical material. This work was supported by U.S. Public Health Service Grants HL-15895 (J.L.B.), HD-06276 and HD-04807 (R.L.D.), and HD-05072 (J.E.) access
1. Report of the Committee for a Study for Evaluation of Testing for Cystic Fibrosis. The National Academy of Sciences (1976) "Evaluation of testing for cystic fibrosis," J. of Pediat. 88, (no. 4, part 2), pp. 711-750. 2. Cystic Fibrosis Foundation (1975) 1973 Report on Survival Studies of Patients with Cystic Fibrosis, Atlanta, Georgia, January 1975, Tables 3-6. 3. DiSant'Agnese, P. A., Darling, R. C., Perera, G. A. & Shea, E. (1953) "Abnormal electrolyte composition of sweat in cystic fi-
brosis of the pancreas: Clinical significance and relationship to the disease," Pediatrics 12, 549-563. 4. Mangos, J. A., McSherry, N. R. & Benke, P. J. (1967) "A sodium transport inhibitory factor in the saliva of patients with cystic fibrosis of the pancreas," Pediat. Res. 1, 436-442. 5. Taussig, L. M. & Gardner, J. D. (1972) "Effects of saliva and plasma from cystic fibrosis patients on membrane transport," Lancet i, 1368-1369. 6. Schatzmann, J. J. (1953) "Herzglycoside als Hemmstoffe fur den aktiven Kalium und Nutrium Transport durch die Erythrocytenmembran," Helv. Physiol. acta 11, 346-354. 7. Shank, B. B. & Smith, N. E. (1975) "Regulation of cellular sodium pump activity," J. Cell. Physiol. 87, 377-388. 8. Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L. & Till, J. E. (1974) "Ouabain resistant mutants of mouse and hamster cells in culture," Cell 1, 9-20. 9. Lever, J. E. & Seegmiller, J. E. (1976) "Ouabain-resistant human lymphoblastoid lines altered in the (Na+, K+)-dependent ATPase membrane transport system," J. Cell. Physiol 88, 343-352. 10. Corsaro, C. M. & Migeon, B. R. (1975) "Effect of ouabain resistance on human diploid fibroblasts carrying other genetic variants," Exp. Cell Res. 95, 47-53. 11. Mankovitz, R., Buchwald, M. & Baker, R. M. (1974) "Isolation of ouabain-resistant human diploid fibroblasts," Cell 3, 221226. 12. Quissell, D. 0. & Pitot, H. C. (1974) "Number of ouabain binding sites in fibroblasts derived from normal subjects and patients with cystic fibrosis," Nature 247, 115-116. 13. Barranco, S. C., Bolton, W. E., Haenelt, B. R. & Abell, C. W. (1976) "Differences in the incorporation of thymidine into DNA of normal and cystic fibrosis fibroblasts in vitro," J. Cell. Physiol. 88, 33-42. 14. Buchwald, M. (1976) "Abnormal levels of 3'-5'-cyclic AMP in isoproterenol-stimulated fibroblasts from patients with cystic fibrosis," Proc. Natl. Acad. Sci. USA 73, 2899-2903.
Genetics
Increased resistance of cystic fibrosis fibroblasts to ouabain toxicity (cell culture/survival assay/genetic disease/cardiac glycoside)
J. EPSTEIN"1 AND JAN L. BRESLOWt * Clinical Genetics Division, Department of Medicine, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115 and the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115; and t Metabolism Division, Department of Medicine, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115 and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by John F. Enders, January 24, 1977
ABSTRACT Diploid skin fibroblasts derived from several unrelated patients with cystic fibrosis (CF) were tested for resistance to the cytotoxic effects of ouabain in comparison with cells from normal individuals. Cells from CF and normal individuals were plated at low density and exposed to ouabain at different concentrations for 24 hr. Dose-response curves showed that CF cells survived significantly better than did normal cells at all ouabain concentrations from 0.1 nM to 1 IAM in potassium-deficient medium. In medium containing the usual amount of potassium (6.25 mM), survival of the CF cells exposed to ouabain was decreased to that of the normal cells. If the glucose concentration of the potassium-deficient medium was decreased to 10% of the usual amount, the resistance of the CF cells to ouabain disappeared and their survival was identical to that of the normal cells. These findings may be useful in understanding the biochemical basis of CF and its clinical manifestations. Cystic fibrosis (CF) is one of the most common genetic diseases causing early death in children. It occurs with a frequency of approximately 1/1500-2000 live births in Caucasians, and the mean survival for CF patients is only 15 years at present. The disease is thought to have an autosomal recessive pattern of inheritance, and it is believed that 1 in every 20 persons in the United States carries the trait for the disease. Clinically, homozygous persons are usually found to have chronic pulmonary disease, some degree of pancreatic exocrine dysfunction, and abnormally high concentrations of Na+ and Cl- in their sweat. The clinical manifestations and severity of the disease process may vary greatly among affected persons (1-
3).
The metabolic basis for CF is not yet known, and research efforts to identify some biochemical defect in the disease have taken several different approaches. Some of these findings have suggested that the disease may involve an abnormality of ion transport. The sweat, saliva, and seminal fluids of CF patients have been reported to contain factors that interfere with ion transport in the salivary and sweat glands of normal persons (4, 5). However, no alteration of ion transport has yet been proved directly in CF cells. In the present study, CF and normal fibroblasts were tested for their resistance to the cytotoxic effects of ouabain, a cardiac glycoside known to inhibit the transport of ions across the cell membrane (6). METHODS AND MATERIALS Cells and Media. Human diploid skin fibroblasts were derived by percutaneous punch biopsy of the volar surface of the forearm or from foreskin specimens; informed consent was obtained in all cases. Cell stocks were maintained at 36.5° in Abbreviations: CF, cystic fibrosis; EMEM, Eagle's minimum essential medium; EMEM-B, basic medium with additional glucose (total, 1900 mg/liter); EMEM-C, basic medium but without KCl and with fetal calf serum replaced with dialyzed fetal calf serum; EMEM-D, basic medium but with decreased glucose (total, 100 mg (liter)), without KCl, and with dialyzed fetal calf serum. t To whom reprint requests should be addressed.
humidified 7% C02/93% air atmosphere in Falcon plastic tissue culture flasks. The cells were grown in Eagle's minimal essential medium (EMEM) (MBA, Inc.) containing glucose (1000 mg/liter) and 6.25 mM K+ (as KCl). This was supplemented with nonessential amino acids (IBL, Inc.), penicillin (50 units/ ml), streptomycin (50 ,g/ml), and 10% fetal calf serum (MBA, Inc.). In the experiments reported here, this basic medium was modified as follows: for EMEM-B, an additional (900 mg/liter) glucose was added; for EMEM-C, KCl was omitted and 10% dialyzed fetal calf serum was substituted for fetal calf serum; and for EMEM-D, KCl was omitted, the glucose concentration was decreased to 100 mg/liter, and 10% dialyzed fetal calf serum was substituted for fetal calf serum. Three carefully tested lots of fetal calf serum were used in the experiments reported here and were chosen for their ability to yield high plating efficiences and optimal growth of fibroblasts. Dialyzed fetal calf serum was prepared by dialysis against three changes of 20 volumes of 0.15 M NaCl for 24-86 hr at 4°. Flame photometry was used to verify that each batch of dialyzed serum and EMEM-C contained no detectable levels of K+. All cell strains were negative on testing for mycoplasma. Survival Experiments. Strains used in these studies were between the 5th and 25th population doublings in vitro. When different strains were to be compared, they were chosen to be within three population doublings of each other. For survival assays, the appropriate strains were harvested from exponentially growing monolayers with 0.05% trypsin (Difco, 1:250) and 0.5 mM Na2EDTA. Trypsinized cells were rinsed once, resuspended in EMEM-D, and counted with a Coulter electronic cell counter. Various numbers of cells were distributed into three or four replicate 100-mm diameter Falcon plastic tissue culture dishes in 9 ml of EMEM-B, EMEM-C, or EMEM-D. Inocula were chosen so as to provide approximately 75-100 surviving colonies per dish. Twenty-four hours later, 1 ml of the appropriate concentration of ouabain in EMEM-D was added to each dish. After a 24-hr incubation period with the drug, the medium was removed, each dish was rinsed twice with 10 ml of Ca2+- and Mg2+-free Earle's balanced salt solution, and then renewed with 10 ml of EMEM-B. The dishes were renewed with EMEM-B every 6-8 days. After 14-21 days of incubation, the dishes were rinsed with 0.15 M NaCl, fixed with methanol, and stained for colony counting; only those colonies containing a minimum of 50 cells were counted. The survival of the cells exposed to ouabain was expressed as percentage of the number of cells plated, corrected for the actual plating efficiency of the cells in the absence of ouabain for each experiment. The ouabain (Sigma Chemical) was dissolved in distilled water to a final concentration of 0.1 mM, sterilized by filtration through a 0.22-aim membrane filter, stored at -20°, and thawed immediately before use. This stock solution was diluted with EMEM-D to the appropriate concentrations for use in survival experiments. 1676
Genetics: Epstein and Breslow
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Sources of fibroblast strains
Strain
S-127 S-147 S-149 S-203 S-204 EX-25 S-115 S-116 S-206 S-212
Age of patient (years)
Sex
Diagnosis
Site of biopsy
M F M M M M M M F F
CF CF CF CF CF Normal Normal Normal Normal Normal
Forearm Forearm Forearm Forearm Forearm Forearm Foreskin Foreskin Forearm Forearm
16 22 21 23 Newborn 24 Newborn Newborn
Newborn 18
Selection of Normal and CF Patients. Control fibroblasts strains were from healthy donors for whom it could be established that the donor had no previous personal or family history of CF or other undiagnosed medical problems. The CF strains used in these studies were obtained from unrelated patients who were being seen at the Children's Hospital Medical Center, Boston, MA, and who had well-documented, clinically confirmed CF with positive sweat tests and involvement of either the respiratory or gastrointestinal system or both. The sources of all strains are shown in Table 1. RESULTS The results of dose-response studies to determine the cytotoxic effect of ouabain on normal and CF fibroblasts are presented as survival curves in which the number of survivors, after correction for plating efficiency, is plotted against the concentration of drug. Fig. 1A is a typical survival curve from a study in which the ouabain sensitivity of cells (strain S-147) from a CF patient were compared to the ouabain sensitivity of cells (strain EX-25) from a normal person. The experiment was performed in EMEM-C, a medium deficient in K+. After a LU~ ~
~
~
1677
24-hr exposure to ouabain, the survival of S-147 was greater than that of the normal strain, EX-25. The CF cells exhibited little decrease in survival in the presence of 3.3 nM ouabain, whereas the survival of the normal cells at that concentration was decreased to 60%. At greater doses, the slope of the curve for the normal cells was much steeper than that for the CF cells. Fig. 1B shows the results of an experiment similar to that in Fig. 1A except that the cells were plated and exposed to ouabain in EMEM-B, a medium containing 6.25 mM K+. There was no difference in survival between the two strains after a 24-hr exposure to the drug. In EMEM-B the decreased survival of CF cells was similar to that of normal cells. The normal cells showed the same sensitivity to ouabain in EMEM-B and EMEM-C. The increased ouabain resistance of CF cells, when compared to normal cells in EMEMv-C (K+-deficient medium) also was observed in other experiments using different CF and normal fibroblast strains. Survival studies were performed at ouabain concentrations ranging from 0.1 nM to 1 1AM. However, for comparative purposes only, the survival (corrected for plating efficiency) for each strain at 0.1 AM is presented (Table 2). In most experiments, the CF strains showed approximately 7080% survival after exposure to 0.1 zIM ouabain, compared to only 20% survival for the normal strains. In experiments in which the survival for the CF strain was less than 80%, the survival of the normal strain at 0.1 gM ouabain was also lower. Thus, although the actual survival varied somewhat from experiment to experiment, the CF strains were always more resistant than the normals by a factor of 2.7-4.8. In order to determine what effect a decrease in the amount of energy source available to the cells would have on ouabain resistance, survival experiments similar to those shown in Fig. 1 were performed in EMEM-D, a K+-deficient medium in which the normal concentration of glucose was decreased to 10% of the usual level. In the experiment shown in Fig. 2A, the cells were plated in EMEM-C with a normal glucose concentration and, as shown in Fig. 1, the CF strain was more resistant to ouabain. In Fig. 2B, both strains were plated in EMEM-D,
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OUABAIN CONCENTRATION (M)
FIG. 1. Survival of S-147 (0), a CF strain, and EX-25 (0), a normal strain. Each point represents the mean number of colonies in three or four replicate petri dishes in which the survival in each dish was within 7% of the averaged value. (A) Cells were plated and exposed to ouabain in EMEM-C, a K+-deficient medium. For the two separate experiments in which the strains were compared, the plating efficiencies were 2.0 and 1.0% for S-147, and 1.6 and 1.0% for EX-25. (B) Cells were plated and exposed to ouabain in EMEM-B, a medium containing 6.25 mM K+. For the experiment shown, the plating efficiencies were 2.8% for S-147 and 3.4% for EX-25.
1678
Proc. Nati. Acad. Sci., USA 74 (1977)
Genetics: Epstein and Breslow
0
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OUABAIN CONCENTRATION (M) FIG. 2. Survival of S-149 (o), a CF strain, and EX-25 (0), a normal strain. Each point represents the mean number of colonies in three or four replicate petri dishes in which the survival in each dish was within 7% of the averaged value. (A) Cells were plated and exposed to ouabain in EMEM-C, a K+-deficient medium containing glucose at 1000 mg/liter. The plating efficiencies were 4.8% for S-149 and 3.1% for EX-25. (B) Cells were plated and exposed to ouabain in EMEM-D, a medium deficient in K+ and glucose (100 mg/liter). The plating efficiencies were 4.2% for S-149 and 3.0% for EX-25.
and the survival of S-149, the CF strain, was decreased to that of the normal strain. Both strains showed a survival of approximately 20% at 0.1 AtM ouabain in the low-glucose medium. Because the plating efficiencies were the same in the two media, the effect of decreased glucose concentration on the ouabain resistance of the CF cells is not likely to be due to any effect on the plating efficiency. Because in recent years much attention has been paid to factors produced by CF cells in culture, we tried to evaluate whether such factors might be influencing our results. Therefore, we also performed survival experiments in which medium was exchanged between normal and CF cultures after 24- and 48-hr incubations. There was no change in the ouabain sensitivities of the different fibroblast strains. This would suggest that the different ouabain sensitivities of CF and normal cells cannot be altered by any stable factors that might be released into the medium. DISCUSSION The purpose of this study was to determine whether or not CF and normal cells have the same sensitivity to ouabain, a cardiac glycoside known to inhibit the transport of ions across the cell membrane (6). It was found that CF fibroblasts are more resistant to the cytotoxic effects of ouabain than are normal cells when plated into K+-free medium for 24 hr and then exposed to the drug in the same medium for another 24 hr. However, the increased ouabain resistance of CF cells disappears if the cells are plated in medium containing a normal K+ concentration (6.25 mM) for 24 hr. In addition, the increased resistance of the CF cells to ouabain seen in K+-deficient medium disappears if the glucose concentration in the K+-deficient me-
dium is decreased to 10% of the normal level. It cannot be determined from the data presented here how the higher glucose concentration increases the resistance of the CF cells to ouabain. The mechanism by which ouabain kills cells is uncertain, but it is believed that the drug inhibits the cellular Na+/K+ transport system (6). In studies of cells of normal ouabain sensitivity, it has been shown that the drug binds to the cell surface with high affinity, causing a subsequent rapid dissipation of the normal cellular Na+/K+ gradient (7). The swelling of the cells which results is believed to cause cell death. Mutant mammalian cells resistant to high concentrations of ouabain have recently been isolated. These mutants have been selected from mouse L cells (8), hamster CHO cells (8), human lymphoblastoid lines (9), and human diploid skin fibroblasts (10, 11). In each case the frequency of ouabain resistant mutants could be increased by exposure of the wild-type cells to mutagenic agents (8-11), and the phenotype was stably expressed through many generations of growth in the absence of the drug. These experiments suggest that ouabain resistance arises as the result of genetic change. In some of these studies on ouabainresistant mutants, it was shown that less radiolabeled ouabain bound to the resistant cells than bound to normal cells and that these resistant cells showed less inhibition of 86Rb+ and 42K+ transport by ouabain than did normal cells (9, 11). The Na+/ K+-stimulated ATPase activity in isolated plasma membranes in some of these mutants also showed decreased sensitivity to ouabain when compared to the membranes derived from the wild-type cells (9, 11). The shapes of the survival curves in other reports for cells exposed to ouabain differ from those reported here. The sur-
Genetics: Epstein and Breslow
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 2. Survival of cells in ouabain Exp.
Strain
1
S-149 (CF) EX-25 S-149 (CF) EX-25 S-147 (CF) S-206 S-147 (CF) S-115 S-147 (CF) EX-25 S-147 (CF) EX-25 S-147 (CF) EX-25 S-127 (CF) S-116 S-215 (CF) S-115 S-203 (CF) S-115 S-203 (CF) S-212 S-204 (CF) S-116
2 3 4
5
6 7
8 9
10 11 12
Plating efficiency* Survivalt
5.5 2.7 3.0 1.4 2.9 1.1 2.0 1.6 1.6 2.0 1.5 1.5 1.0 1.8 1.0 2.7 1.4 1.1 2.2 3.3 1.0 1.0 1.2 1.8
0.78 0.18 0.87 0.27 0.75 0.20 0.86 0.18 0.60 0.22 0.78 0.18 0.90 0.25 0.58 0.15 0.70 0.20 0.80 0.28 0.83 0.18 0.80 0.21
1679
Cloning studies must be performed to determine whether or with respect to drug sensitivity exist in these fibroblast strains. It should be noted that the plating efficiencies of both normal and CF cells were similarly low and the increased resistance of CF cells to ouabain was seen in several different fibroblast strains, suggesting that, if they exist, subpopulations of similar sensitivity were present in all of the strains examined. It has been reported (13) that CF fibroblasts exhibit a lower rate of thymidine incorporation into acid-insoluble material than do normal fibroblasts. It also has recently been reported (14) that CF fibroblasts have a 3- to 5-fold higher increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels after isoproterenol stimulation than do normal cells. None of these studies, including those of the present report, however, have shown a specific deficiency in any gene product in CF cells. not variants
RatioT
4.3
3.2 3.8
4.8 2.7
4.3 3.6
3.9 3.5
2.9 4.6 3.8
* Plating efficiency of cells in EMEM-C in the absence of ouabain. t Survival (corrected for plating efficiency) at ouabain dose of 0.1 AM
in EMEM-C. Ratio of survival of CF cells to normal cells in the indicated experiment.
vival curves for mouse L cells and hamster CHO cells were obtained with cells plated at low density in K+-containing medium. Ouabain concentrations of 0.1 mM were required to cause any significant decrease in the survival of wild-type cells; a 10-fold increase in drug dose (to 1 mM) decreased survival to 10-4 (8). In our studies, 0.1 nM ouabain significantly decreased survival in normal cells, and a 10,000-fold increase in drug dose (to 1 AM) decreased survival to approximately 10-1. The previously reported survival curves for human fibroblasts exposed to ouabain (11) cannot be compared to our data because the conditions used are different. In that study, human fibroblasts were plated in K+-containing medium with various ouabain concentrations, and cell counts were determined 6-8 days later. In contrast, in the present study, the cells were exposed to ouabain for only 24 hr in K+-deficient medium, and the surviving cells were required to form colonies. In one previous study (12), it was reported that CF cells bound the same amount of radiolabeled ouabain to their cell surface as did normal fibroblasts. However, in that report, the binding studies were performed with cells that had been maintained in medium that contained K+ and the cells were transferred to K+-deficient, low-glucose (100 mg/liter) salt solution just before the addition of the ouabain. Our results under similar conditions (Figs. 1B and 2B) show that there is no detectable difference in the survival of CF and normal cells in the presence of ouabain. Our survival studies have revealed increased ouabain resistance in unselected populations of CF fibroblasts but have not detected subpopulations of normal cells that varied in drug sensitivity; however, the possible existence of such variants cannot be ruled out solely by the use of survival assay methods.
We thank Dr. Richard L. Davidson for his critical reading of this manuscript and for his comments and suggestions during the course of the work. We thank Dr. Harry Shwachman for helping to provide to clinical material. This work was supported by U.S. Public Health Service Grants HL-15895 (J.L.B.), HD-06276 and HD-04807 (R.L.D.), and HD-05072 (J.E.) access
1. Report of the Committee for a Study for Evaluation of Testing for Cystic Fibrosis. The National Academy of Sciences (1976) "Evaluation of testing for cystic fibrosis," J. of Pediat. 88, (no. 4, part 2), pp. 711-750. 2. Cystic Fibrosis Foundation (1975) 1973 Report on Survival Studies of Patients with Cystic Fibrosis, Atlanta, Georgia, January 1975, Tables 3-6. 3. DiSant'Agnese, P. A., Darling, R. C., Perera, G. A. & Shea, E. (1953) "Abnormal electrolyte composition of sweat in cystic fi-
brosis of the pancreas: Clinical significance and relationship to the disease," Pediatrics 12, 549-563. 4. Mangos, J. A., McSherry, N. R. & Benke, P. J. (1967) "A sodium transport inhibitory factor in the saliva of patients with cystic fibrosis of the pancreas," Pediat. Res. 1, 436-442. 5. Taussig, L. M. & Gardner, J. D. (1972) "Effects of saliva and plasma from cystic fibrosis patients on membrane transport," Lancet i, 1368-1369. 6. Schatzmann, J. J. (1953) "Herzglycoside als Hemmstoffe fur den aktiven Kalium und Nutrium Transport durch die Erythrocytenmembran," Helv. Physiol. acta 11, 346-354. 7. Shank, B. B. & Smith, N. E. (1975) "Regulation of cellular sodium pump activity," J. Cell. Physiol. 87, 377-388. 8. Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L. & Till, J. E. (1974) "Ouabain resistant mutants of mouse and hamster cells in culture," Cell 1, 9-20. 9. Lever, J. E. & Seegmiller, J. E. (1976) "Ouabain-resistant human lymphoblastoid lines altered in the (Na+, K+)-dependent ATPase membrane transport system," J. Cell. Physiol 88, 343-352. 10. Corsaro, C. M. & Migeon, B. R. (1975) "Effect of ouabain resistance on human diploid fibroblasts carrying other genetic variants," Exp. Cell Res. 95, 47-53. 11. Mankovitz, R., Buchwald, M. & Baker, R. M. (1974) "Isolation of ouabain-resistant human diploid fibroblasts," Cell 3, 221226. 12. Quissell, D. 0. & Pitot, H. C. (1974) "Number of ouabain binding sites in fibroblasts derived from normal subjects and patients with cystic fibrosis," Nature 247, 115-116. 13. Barranco, S. C., Bolton, W. E., Haenelt, B. R. & Abell, C. W. (1976) "Differences in the incorporation of thymidine into DNA of normal and cystic fibrosis fibroblasts in vitro," J. Cell. Physiol. 88, 33-42. 14. Buchwald, M. (1976) "Abnormal levels of 3'-5'-cyclic AMP in isoproterenol-stimulated fibroblasts from patients with cystic fibrosis," Proc. Natl. Acad. Sci. USA 73, 2899-2903.
