Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5642-5646, December 1977 Genetics
Increased dexamethasone resistance of cystic fibrosis fibroblasts (survival assay/cell culture/sterol nucleus/ethacrynic acid/ion transport)
J. EPSTEIN*tI, JAN L. BRESLOW*, AND RICHARD L. DAVIDSONt§ * Metabolism Division, Department of Medicine, Children's Hospital Medical Center, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; t Division of Human Genetics, Department of Medicine, Children's Hospital Medical Center, Boston, Massachusetts 02115; and § Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by John F. Enders, September 26, 1977
ABSTRACT We demonstrated previously that fibroblasts from cystic fibrosis (CF) patients are significantly more resistant to the toxic effects of ouabain than normal human fibroblasts are. Ouabain is generally assumed to act primarily at the level of the cell membrane and to cause killing of cells by inhibiting ion transport. We have therefore examined the ability of normal and CF cells to survive exposure to ethacrynic acid, another inhibitor of ion transport, and to survive colchicine and aminopterin, resistance to which has been associated with membrane alterations in other cells. The effect of dexamethasone, which has a sterol nucleus similar to that of ouabain but is thought to have a different site of cellular action, was also tested. Exposure of the cells to ethacrynic acid, colchicine, or aminopterin did not reveal any differences in survival between normal and CF fibroblasts. However, CF cells survived exposure to dexamethasone significantly better than normal cells did. These results suggest that normal and CF cells do not differ in terms of a generalized resistance to ion transport inhibitors or to drugs that must pass through the membrane to be active. Instead, the results raise the possibility that CF cells have an enhanced resistance to drugs that have the sterol nucleus found in ouabain and dexamethasone.
methasone was chosen because it contains a sterol nucleus similar to that of ouabain but presumably has a different site of cellular action. METHODS AND MATERIALS Cells and Media. Human diploid skin fibroblasts were derived from normal and CF individuals by percutaneous punch biopsy of the volar surface of the forearm or from foreskin specimens after obtaining informed consent in all cases. Cell stocks were maintained at 36.50 in a 7% C02/93% air, humidified incubator in Eagle's minimal essential medium (EMEM) [Microbiological Associates (MBA), Inc.] supplemented with 1X nonessential amino acids (MBA, Inc.), penicillin at 50 units/ml, streptomycin at 50,tg/ml, and 10% fetal calf serum (MBA, Inc.). Two lots of fetal calf serum, selected for their ability to yield high plating efficiencies and rapid growth rates (population doubling times between 24 and 26 hr) were used in the experiments. All the cell strains used in these experiments were found to be free of mycoplasma when tested after growth for 2 weeks in antibiotic-free medium. EMEM contains 6.25 mM KCI and 1000 mg of glucose per liter. For the survival experiments this basic medium was altered as follows: for EMEM-B, an additional 900 mg of glucose per liter was added; for EMEM-C, KCl was omitted and 10% dialyzed (K+-free) fetal calf serum was substituted for fetal calf serum. Dialyzed fetal calf serum was prepared as described previously (1). All preparations of EMEM-C and dialyzed fetal calf serum were shown by flame photometry to contain no detectable K+. For EMEM-E, which contained 6.25 mM K+ and 10% dialyzed fetal calf serum, the glucose concentration of EMEM was reduced from 1000 to 100 mg/liter. Survival Experiments. The cell strains used in these experiments were between 5 and 25 population doublings in vitro, and the normal and CF strains being compared in a given experiment were within four doublings of each other. The cells were harvested for survival assays as described previously (1), suspended in EMEM-B, EMEM-C, or EMEM-E, and counted. Various number of cells were distributed into three replicate 100-mm diameter plastic tissue culture dishes (Falcon) containing 9 ml of EMEM-B, EMEM-C, or EMEM-E. The dishes were inoculated with sufficient numbers of cells to yield 75-100 colonies following drug exposure. Twenty-four hours after plating, 1 ml of drug (at 10 times the desired final concentration) was added to each dish. After 24-hr incubation in the presence of the drug, the dishes were rinsed twice with 10 ml of Earle's balanced salt solution and renewed with 10 ml of EMEM-B. Incubation was continued for 14-21 days, during which time the dishes were renewed every 6-8 days with fresh EMEM-B. After colonies containing a minimum of 50-75 cells
We previously reported that fibroblasts from cystic fibrosis (CF) patients were more resistant to the toxic effects of ouabain than were fibroblasts from normal individuals (1). Because ouabain is known to inhibit the transport of ions across cellular membranes by inhibiting the membrane Na+, K+-ATPase (2), it seemed that the ouabain resistance of CF cells could be accounted for by some alteration of either the ion transport systems or membrane properties in CF cells. In this report, we examine the sensitivity of CF and normal cells to other cytotoxic drugs in order to provide information on the specificity of the ouabain effect and provide further insight into the differences between the cells. The drugs tested were ethacrynic acid, a specific inhibitor of ion transport that acts by a mechanism different from that of ouabain (3); colchicine, an inhibitor of microtubule formation that enters the cell by unmediated diffusion across the cell membrane (4); aminopterin, a folic acid antagonist that also enters the cell by unmediated diffusion (5); strophanthidin, a ouabain analogue that inhibits ion transport (6); and dexamethasone, a synthetic glucocorticoid that interacts with cells through a specific cytoplasmic receptor (7). Ethacrynic acid was tested to see if CF cells exhibited a generalized resistance to ion transport inhibitors whose molecular structures were unlike ouabain. Colchicine and aminopterin were tested because resistance to these drugs, like ouabain, has been found to be associated with membrane alterations in other cells (4, 5). Strophanthidin was included as an ion inhibitor whose basic molecular structure is similar to that of ouabain except that it lacks a rhamnose moiety. DexaThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CF, cystic fibrosis; EMEM, Eagle's minimum essential medium. f To whom reprint requests should be addressed.
5642
Genetics:
Epstein et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
5643
Table 1. Survival of cells in various drugs Plating efficienMedium cy,* %
Survival,t
% Drug Strain 76 1.8 Ouabain 1 S-215(CF) EMEM-C 18 1.3 (0.1MuM) S-235 81 1.2 Ouabain 2 S-220(CF) EMEM-C 24 S-316 2.0 (0.1,MM) 10 4.9 Ouabain 3 S-220(CF) EMEM-B 13 (0.1 AM) EX-25 7.3 14 1.5 Ethacrynic 4 S-215(CF) EMEM-C 1.3 acid (10Mug/ml) 12 S-235 20 5 S-215(CF) EMEM-B 9.0 Ethacrynic 9.6 acid (10 Ag/ml) 19 S-235 23 1.3 Colchicine 6 S-215(CF) EMEM-C 1.9 20 S-235 (0.1,MM) 20 7.0 Colchicine 7 S-220(CF) EMEM-B 18 (0.1 MM) 10.5 EX-25 27 1.4 Aminopterin 8 S-215(CF) EMEM-C 1.0 25 (1MUM) S-235 9 S-220(CF) EMEM-B 10.6 Aminopterin 19 7.7 (1 MM) 17 EX-25 79 10 S-127(CF) EMEM-C 3.3 Strophanthidin 2.2 (0.1 AM) 28 S-115 * Plating efficiency of cells in the absence of drug in the indicated medium. t Survival (corrected for plating efficiency) in the concentration of drug indicated.
Exp.
had formed, the dishes were rinsed, fixed with methanol, and stained with Giemsa for counting. Cell survival is expressed as the number of colonies developing at a given drug dose divided by the total number of cells plated, after correction for the actual plating efficiency of each strain in the absence of drug. Stock solutions of aminopterin, colchicine, and strophanthidin (Sigma Chemical) were prepared by dissolving the drugs in distilled water at concentrations of 100 AM, 5 AtM, and 100 ,M, respectively. The solutions were sterilized by filtration and stored at -20°. Dexamethasone (Sigma Chemical) was dissolved in absolute ethanol at a concentration of 1 mM and stored at -20°. Just before use, the dexamethasone was diluted in EMEM-B, EMEM-C, or EMEM-E and filter sterilized. Control experiments showed that ethanol alone at the concentrations (
Increased dexamethasone resistance of cystic fibrosis fibroblasts (survival assay/cell culture/sterol nucleus/ethacrynic acid/ion transport)
J. EPSTEIN*tI, JAN L. BRESLOW*, AND RICHARD L. DAVIDSONt§ * Metabolism Division, Department of Medicine, Children's Hospital Medical Center, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; t Division of Human Genetics, Department of Medicine, Children's Hospital Medical Center, Boston, Massachusetts 02115; and § Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by John F. Enders, September 26, 1977
ABSTRACT We demonstrated previously that fibroblasts from cystic fibrosis (CF) patients are significantly more resistant to the toxic effects of ouabain than normal human fibroblasts are. Ouabain is generally assumed to act primarily at the level of the cell membrane and to cause killing of cells by inhibiting ion transport. We have therefore examined the ability of normal and CF cells to survive exposure to ethacrynic acid, another inhibitor of ion transport, and to survive colchicine and aminopterin, resistance to which has been associated with membrane alterations in other cells. The effect of dexamethasone, which has a sterol nucleus similar to that of ouabain but is thought to have a different site of cellular action, was also tested. Exposure of the cells to ethacrynic acid, colchicine, or aminopterin did not reveal any differences in survival between normal and CF fibroblasts. However, CF cells survived exposure to dexamethasone significantly better than normal cells did. These results suggest that normal and CF cells do not differ in terms of a generalized resistance to ion transport inhibitors or to drugs that must pass through the membrane to be active. Instead, the results raise the possibility that CF cells have an enhanced resistance to drugs that have the sterol nucleus found in ouabain and dexamethasone.
methasone was chosen because it contains a sterol nucleus similar to that of ouabain but presumably has a different site of cellular action. METHODS AND MATERIALS Cells and Media. Human diploid skin fibroblasts were derived from normal and CF individuals by percutaneous punch biopsy of the volar surface of the forearm or from foreskin specimens after obtaining informed consent in all cases. Cell stocks were maintained at 36.50 in a 7% C02/93% air, humidified incubator in Eagle's minimal essential medium (EMEM) [Microbiological Associates (MBA), Inc.] supplemented with 1X nonessential amino acids (MBA, Inc.), penicillin at 50 units/ml, streptomycin at 50,tg/ml, and 10% fetal calf serum (MBA, Inc.). Two lots of fetal calf serum, selected for their ability to yield high plating efficiencies and rapid growth rates (population doubling times between 24 and 26 hr) were used in the experiments. All the cell strains used in these experiments were found to be free of mycoplasma when tested after growth for 2 weeks in antibiotic-free medium. EMEM contains 6.25 mM KCI and 1000 mg of glucose per liter. For the survival experiments this basic medium was altered as follows: for EMEM-B, an additional 900 mg of glucose per liter was added; for EMEM-C, KCl was omitted and 10% dialyzed (K+-free) fetal calf serum was substituted for fetal calf serum. Dialyzed fetal calf serum was prepared as described previously (1). All preparations of EMEM-C and dialyzed fetal calf serum were shown by flame photometry to contain no detectable K+. For EMEM-E, which contained 6.25 mM K+ and 10% dialyzed fetal calf serum, the glucose concentration of EMEM was reduced from 1000 to 100 mg/liter. Survival Experiments. The cell strains used in these experiments were between 5 and 25 population doublings in vitro, and the normal and CF strains being compared in a given experiment were within four doublings of each other. The cells were harvested for survival assays as described previously (1), suspended in EMEM-B, EMEM-C, or EMEM-E, and counted. Various number of cells were distributed into three replicate 100-mm diameter plastic tissue culture dishes (Falcon) containing 9 ml of EMEM-B, EMEM-C, or EMEM-E. The dishes were inoculated with sufficient numbers of cells to yield 75-100 colonies following drug exposure. Twenty-four hours after plating, 1 ml of drug (at 10 times the desired final concentration) was added to each dish. After 24-hr incubation in the presence of the drug, the dishes were rinsed twice with 10 ml of Earle's balanced salt solution and renewed with 10 ml of EMEM-B. Incubation was continued for 14-21 days, during which time the dishes were renewed every 6-8 days with fresh EMEM-B. After colonies containing a minimum of 50-75 cells
We previously reported that fibroblasts from cystic fibrosis (CF) patients were more resistant to the toxic effects of ouabain than were fibroblasts from normal individuals (1). Because ouabain is known to inhibit the transport of ions across cellular membranes by inhibiting the membrane Na+, K+-ATPase (2), it seemed that the ouabain resistance of CF cells could be accounted for by some alteration of either the ion transport systems or membrane properties in CF cells. In this report, we examine the sensitivity of CF and normal cells to other cytotoxic drugs in order to provide information on the specificity of the ouabain effect and provide further insight into the differences between the cells. The drugs tested were ethacrynic acid, a specific inhibitor of ion transport that acts by a mechanism different from that of ouabain (3); colchicine, an inhibitor of microtubule formation that enters the cell by unmediated diffusion across the cell membrane (4); aminopterin, a folic acid antagonist that also enters the cell by unmediated diffusion (5); strophanthidin, a ouabain analogue that inhibits ion transport (6); and dexamethasone, a synthetic glucocorticoid that interacts with cells through a specific cytoplasmic receptor (7). Ethacrynic acid was tested to see if CF cells exhibited a generalized resistance to ion transport inhibitors whose molecular structures were unlike ouabain. Colchicine and aminopterin were tested because resistance to these drugs, like ouabain, has been found to be associated with membrane alterations in other cells (4, 5). Strophanthidin was included as an ion inhibitor whose basic molecular structure is similar to that of ouabain except that it lacks a rhamnose moiety. DexaThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CF, cystic fibrosis; EMEM, Eagle's minimum essential medium. f To whom reprint requests should be addressed.
5642
Genetics:
Epstein et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
5643
Table 1. Survival of cells in various drugs Plating efficienMedium cy,* %
Survival,t
% Drug Strain 76 1.8 Ouabain 1 S-215(CF) EMEM-C 18 1.3 (0.1MuM) S-235 81 1.2 Ouabain 2 S-220(CF) EMEM-C 24 S-316 2.0 (0.1,MM) 10 4.9 Ouabain 3 S-220(CF) EMEM-B 13 (0.1 AM) EX-25 7.3 14 1.5 Ethacrynic 4 S-215(CF) EMEM-C 1.3 acid (10Mug/ml) 12 S-235 20 5 S-215(CF) EMEM-B 9.0 Ethacrynic 9.6 acid (10 Ag/ml) 19 S-235 23 1.3 Colchicine 6 S-215(CF) EMEM-C 1.9 20 S-235 (0.1,MM) 20 7.0 Colchicine 7 S-220(CF) EMEM-B 18 (0.1 MM) 10.5 EX-25 27 1.4 Aminopterin 8 S-215(CF) EMEM-C 1.0 25 (1MUM) S-235 9 S-220(CF) EMEM-B 10.6 Aminopterin 19 7.7 (1 MM) 17 EX-25 79 10 S-127(CF) EMEM-C 3.3 Strophanthidin 2.2 (0.1 AM) 28 S-115 * Plating efficiency of cells in the absence of drug in the indicated medium. t Survival (corrected for plating efficiency) in the concentration of drug indicated.
Exp.
had formed, the dishes were rinsed, fixed with methanol, and stained with Giemsa for counting. Cell survival is expressed as the number of colonies developing at a given drug dose divided by the total number of cells plated, after correction for the actual plating efficiency of each strain in the absence of drug. Stock solutions of aminopterin, colchicine, and strophanthidin (Sigma Chemical) were prepared by dissolving the drugs in distilled water at concentrations of 100 AM, 5 AtM, and 100 ,M, respectively. The solutions were sterilized by filtration and stored at -20°. Dexamethasone (Sigma Chemical) was dissolved in absolute ethanol at a concentration of 1 mM and stored at -20°. Just before use, the dexamethasone was diluted in EMEM-B, EMEM-C, or EMEM-E and filter sterilized. Control experiments showed that ethanol alone at the concentrations (
