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Increased cadmium resistance of skin fibroblasts from menkes disease patients
SHORT COMMUNICATION
Increased Cadmium Resistance of Skin Fibroblasts from Menkes Disease Patients
Abko Hayashi, BS, Mituru Sato, MS, Masataka Anma, MD and T oshitsugu Aob, MD
Cultured skin fibroblasts from patients with Menkes disease and Wilson disease were analyzed as to their sensitivities to copper and cadmium by means of a colony-forming ability and cell growth study. All the Menkes strains exhibited about 3-fold higher levels of resistance to cadmium, whereas the cytotoxity of copper did not differ among the Menkes, Wilson and normal fibroblast strains. The resistance to cadmium of Menkes skin fibroblasts may provide a diagnostic marker of Menkes disease and useful or valuable model for the understanding of detoxification system against heavy metals. Hayashi A, Sato M, Arima M, Aoki T. Increased cadmium resistance of skin fibroblasts from Menkes disease patients. Brain Dev 1986;8:73-6
Patients with Menkes disease show a characteristic low copper content in the liver and serum. The copper concentrations of nonhepatic tissues other than the brain are greater than normal. This elevation is reflected in cultured fibroblasts [1]. These phenomena are associated with a greater amount of metalloFrom the Division of Mental Retardation and Birth Defects Research. National Center for Nervous, Mental and Muscular Disorders, Tokyo (AH, MS, MA); Department of Pediatrics, Toho University School of Medicine, Tokyo (TA). Received for publication: September 18. 1985. Accepted for publication: November 5,1985. Key words: Menkes disease, Wilson disease, cadmium, copper, colony-forming ability. Correspondence address: Dr. Masataka Arima, NatIonal Center for Nervous. Mental and Muscular Disorders. 4-1-1. Ogawahigashi-machi. Kodaira, Tokyo, 187, Japan.
thionein which is a sulfhydryl-rich heavy metal-binding protein [2, 3]. The copper concentrations of cultured fibroblasts from Wilson disease patients, another serum copper deficiency disease, tend to be higher compared to those of normal controls. Cell lines resistant to cadmlUm (Cdr) have been obtained from Chinese hamster ovary cells, mouse fibroblasts and human lymphoblastoid cells. In all these cases, the Cdr cells are capable of synthesizing greater levels of metallothionein than the respective sensitive paren tal cell lines [4-6]. These 0 bserva tions prompted us to investigate in detail whether Menkes disease cells am Wilson disease cells, which are reported to have increased metallothionein levels in the cells, are resistant to copper and cadmium or not. Materials and Methods Skin fibroblasts from three patients with the Menkes disease (CRL1230, GM3700, 8501), from five normal donors (INS-NF2, INS-NF 11, INS-NF19, GM0043, GM3440) and from five patients with Wilson disease (8201, 8207, 8401, GM0032, GM4l28) were used in this study. The' cells with the "G M" prefix were from the Institute of Medical Research, Camden, New Jersey, the cells with the "CRL" prefix were from Flow Laboratories, Irvine, and the others were obtained in our laboratory. Cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (GIBCO No 25Nl03l), penicillin G (1 OOID Iml) and streptomycin sulfate (l00 pg/ml). This medium contained copper of 0.05 pg/ml and undetectable cadmium. Cultures were between the 5th and 12th passage, and were maintained at 37°C in a 95% air/5% CO 2 ,atmosphere. For measurement of cadmium sensitivity, cells were seeded in MEM containing various levels of cadmium followed by incubation for 2 weeks. The cells were fixed and stained, and then the number of colonies composed of at least 50 cells was determined. For measurement of copper sensitivity, cells were seeded in MEM and after 24 hours CuC1 2 was added, the CuC1 2 being removed after a further 24h. The cells were washed with phosphate-buffered saline and then incubated for 2 weeks in MEM because the presence of added copper throughout incubation period did not show good reproducibility of results. For the cell growth experiment, 2 x 10 4 cells were seeded into 35 mm dishes and the cells were allowed to attach to them for 24h. Then
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OF CdC1 2 (Ij~l) Fig 1 Survival measurement of normal (shaded zone indicates the range of 5 strains). Menkes (0) and Wilson cells (e) during continuous exposure to CdCI 2 • Cells were plated on 100 mm dishes in complete medium containing various concentrations of CdCI 2 • After 14 days incubation. colonies were stained and scored. CONCENTRATIOi~
various concentrations of copper or cadmium were added. Three days and 6 days after the metal addition, the cells were trypsinized and the number of cells was determined with a Coulter Counter. Cellular morphology was regularly checked with a phase contrast microscopy. Results The toxic effect of cadmium was determined by exposing cells to different concentrations of CdCl 2 for 14 days. The colony-forming efficiency of untreated cells ranged from 9 to 31 % in all cell strains. The Menkes strains exhibited about 3-fold higher levels of resistance to exogenous cadmium, on the basis of relative plating efficiency, as compared with normal cells. One of the five strains of Wilson fibroblasts exhibited about 3-fold higher
74 Brain & Development. Vol 8, No 1,1986
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CONCEIJTRATION OF CuC1 2 (_,1)/'111) Fig 2 Survival measurement of normal (shaded zone), Menkes (0) and Wilson cells (e) after exposure to various concentrations of CuCI 2 • Cells were plated on dishes. Twenty-four hours later. the cells were exposed to CuCl 2 for 24h, and then washed and incubated for 14 days. Colonies were stained and scored.
resistance to cadmium than the normal cells, and the other strains of Wilson cells exhibited sensitivities in the normal range (Fig 1). Plating efficiency studies in the presence of copper showed no differences in sensitivity among the Menkes, Wilson and normal cells (Fig 2). In order to confirm the results of the colonyformation experiment, we studied the sensitivities to cadmium and copper by means of a cell growth experiment. As shown in Fig 3, we obtained the same results in the cell growth experiment as in the colony-formation experiment. Three Menkes cell strains and one of the Wilson cell strains exhibited higher resistance to cadmium and the sensitivities of other cell strains were not significantly different from those of normal cells. The sensitivities to copper did not differ Significantly among the Menkes, Wilson and normal strains. MorpholOgical change was also comparable
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with the results of cell growth studies. After three-day-incubation with I 0 ~M CdCI 2 , no morphological change was observed with Menkes cells, but normal cells rounded up and detached. Discussion All the Menkes strains and one of the Wilson strains were about 3-times more resistant to cadmium. Riordan et al reported that Menkes lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to cadmium [3]. We previously reported that a metallothionein-like protein (MLP) was induced by addition of cadmium, and Menkes cells tended to induce MLP most and Wilson cells induced more MLP than normal cells [7]. The greater MLP synthesizing ability of Menkes strains may account for their resistance to cadmium cytotoxity. Moreover, Menkes cells have a high level Cu-MLP without copper addition [7], and the added cadmium may displace copper from MLP already present. This displacement may lessen cadmium toxicity in Menkes cells. One of the Wilson strains exhibited cadmium resistance which may reflect the difference in the degree of induction of MLP. It was reported that Menkes cells were found to be more sensitive to copper and not resistant to cadmium in morphological change studies [8, 9]. In another study, no significant difference in copper sensitivity was found between normal and Menkes lymphoblasts as to their viability after addition of the copper [ 10]. Because there is a discrepancy between these reports, we studied cytotoxity using sensitive colony-forming ability and cell growth tests in the conditions with and without morphological change. In our experiments, consistent results were obtained. In all strains of
Fig 3 The effects of 10 pM CdCl, A) and 20 iJg/ml CuCI, B) on the relative growth rate. Cells were plated on 35 mm dishes. Twenty-four hours later, CdCl, or CuCl, was added, and after 3 days the numbers of cells were determined with a Coulter Counter.
Menkes cells we could confirm the resistanee to cadmium. Although the existence of strains with normal sensitivity to cadmium could not be ruled out, we can safely say that resistance to cadmium is a common character in Menkes fibroblasts. There have been several reports of cadmium resistant mutant cells [4-6], but now we report the resistance to cadmium in a genetically inherited disease. These results will be useful for elucidation of the mechanism of the disease and for studies of the cellular detoxification system against cadmium and other heavy metals. References 1. Goka TJ. Stevenson RE, Hefferan PM. Howell RR. Menkes disease: a biochemical abnormality in cultured human fibroblasts. Proc Natl Acad Sci USA 1976;73:604-6. 2. Labadie GU, Hirschhorn K, Katz S. Beratis NG. Increased copper metallothionein in Menkes cultured skin fibroblasts. Pediatr Res 1981; 15: 257-61. 3. Riordan JR, Jolicoeur-Paquet L. Metallothionein accumulation may account for intracellular copper retention in Menkes' disease. J Bioi Chem 1982;257:4639-45. 4. Hildebrand CEo Tobey RA. Campbell EW, Enger MD. A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity. Exp Cell Res 1979;124: 237-46. 5. Maiti I, Mbikay M, Marengo C, Thirion JP. Immunological characterization of metallothioneins in mouse LMTK cells and in a variant resistant to cadmium. J Cell Physiol1982; 112:35-41. 6. Shworak NW. Snyder FF. Gedamu L. Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2). Biochim Biophys Acta 1983;763: 332-8. 7. Sato M. Hayashi A. Ito H. Tojo M. Arima M. Copper level and metallothionein-like Cu-binding protein in cultured skin fibroblasts from patients
Hayashi et al: Menkes fibroblasts
75
with Menkes' disease and Wilson's disease (in Japanese). Brain and Nerve (Tokyo) 1984;36: 1063-8. 8. Chan WY, Garmca AD, Rennert OM. Cell culture studies of Menkes kinky hair disease. Gin Chim Acta 1978;88:495-507. 9. Leone A, Pavlakis GN, Hamer DH. Menkes'
76 Brain & Development, Val 8, No 1,1986
disease: abnormal metallothionein gene regulation in response to copper. Cell 1985 ;40: 301-9. 10. Yazaki M. Study on abnormal copper metabolism in Menkes' kinky hair disease and Wilson's disease. Nagoya Med J (Nagoya) 1981;25:16986.
Increased Cadmium Resistance of Skin Fibroblasts from Menkes Disease Patients
Abko Hayashi, BS, Mituru Sato, MS, Masataka Anma, MD and T oshitsugu Aob, MD
Cultured skin fibroblasts from patients with Menkes disease and Wilson disease were analyzed as to their sensitivities to copper and cadmium by means of a colony-forming ability and cell growth study. All the Menkes strains exhibited about 3-fold higher levels of resistance to cadmium, whereas the cytotoxity of copper did not differ among the Menkes, Wilson and normal fibroblast strains. The resistance to cadmium of Menkes skin fibroblasts may provide a diagnostic marker of Menkes disease and useful or valuable model for the understanding of detoxification system against heavy metals. Hayashi A, Sato M, Arima M, Aoki T. Increased cadmium resistance of skin fibroblasts from Menkes disease patients. Brain Dev 1986;8:73-6
Patients with Menkes disease show a characteristic low copper content in the liver and serum. The copper concentrations of nonhepatic tissues other than the brain are greater than normal. This elevation is reflected in cultured fibroblasts [1]. These phenomena are associated with a greater amount of metalloFrom the Division of Mental Retardation and Birth Defects Research. National Center for Nervous, Mental and Muscular Disorders, Tokyo (AH, MS, MA); Department of Pediatrics, Toho University School of Medicine, Tokyo (TA). Received for publication: September 18. 1985. Accepted for publication: November 5,1985. Key words: Menkes disease, Wilson disease, cadmium, copper, colony-forming ability. Correspondence address: Dr. Masataka Arima, NatIonal Center for Nervous. Mental and Muscular Disorders. 4-1-1. Ogawahigashi-machi. Kodaira, Tokyo, 187, Japan.
thionein which is a sulfhydryl-rich heavy metal-binding protein [2, 3]. The copper concentrations of cultured fibroblasts from Wilson disease patients, another serum copper deficiency disease, tend to be higher compared to those of normal controls. Cell lines resistant to cadmlUm (Cdr) have been obtained from Chinese hamster ovary cells, mouse fibroblasts and human lymphoblastoid cells. In all these cases, the Cdr cells are capable of synthesizing greater levels of metallothionein than the respective sensitive paren tal cell lines [4-6]. These 0 bserva tions prompted us to investigate in detail whether Menkes disease cells am Wilson disease cells, which are reported to have increased metallothionein levels in the cells, are resistant to copper and cadmium or not. Materials and Methods Skin fibroblasts from three patients with the Menkes disease (CRL1230, GM3700, 8501), from five normal donors (INS-NF2, INS-NF 11, INS-NF19, GM0043, GM3440) and from five patients with Wilson disease (8201, 8207, 8401, GM0032, GM4l28) were used in this study. The' cells with the "G M" prefix were from the Institute of Medical Research, Camden, New Jersey, the cells with the "CRL" prefix were from Flow Laboratories, Irvine, and the others were obtained in our laboratory. Cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (GIBCO No 25Nl03l), penicillin G (1 OOID Iml) and streptomycin sulfate (l00 pg/ml). This medium contained copper of 0.05 pg/ml and undetectable cadmium. Cultures were between the 5th and 12th passage, and were maintained at 37°C in a 95% air/5% CO 2 ,atmosphere. For measurement of cadmium sensitivity, cells were seeded in MEM containing various levels of cadmium followed by incubation for 2 weeks. The cells were fixed and stained, and then the number of colonies composed of at least 50 cells was determined. For measurement of copper sensitivity, cells were seeded in MEM and after 24 hours CuC1 2 was added, the CuC1 2 being removed after a further 24h. The cells were washed with phosphate-buffered saline and then incubated for 2 weeks in MEM because the presence of added copper throughout incubation period did not show good reproducibility of results. For the cell growth experiment, 2 x 10 4 cells were seeded into 35 mm dishes and the cells were allowed to attach to them for 24h. Then
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OF CdC1 2 (Ij~l) Fig 1 Survival measurement of normal (shaded zone indicates the range of 5 strains). Menkes (0) and Wilson cells (e) during continuous exposure to CdCI 2 • Cells were plated on 100 mm dishes in complete medium containing various concentrations of CdCI 2 • After 14 days incubation. colonies were stained and scored. CONCENTRATIOi~
various concentrations of copper or cadmium were added. Three days and 6 days after the metal addition, the cells were trypsinized and the number of cells was determined with a Coulter Counter. Cellular morphology was regularly checked with a phase contrast microscopy. Results The toxic effect of cadmium was determined by exposing cells to different concentrations of CdCl 2 for 14 days. The colony-forming efficiency of untreated cells ranged from 9 to 31 % in all cell strains. The Menkes strains exhibited about 3-fold higher levels of resistance to exogenous cadmium, on the basis of relative plating efficiency, as compared with normal cells. One of the five strains of Wilson fibroblasts exhibited about 3-fold higher
74 Brain & Development. Vol 8, No 1,1986
0·1 L--_ _. l - -_ _-'--_ _..l..-_ _ 1.0 20 30 10 o
..L-
CONCEIJTRATION OF CuC1 2 (_,1)/'111) Fig 2 Survival measurement of normal (shaded zone), Menkes (0) and Wilson cells (e) after exposure to various concentrations of CuCI 2 • Cells were plated on dishes. Twenty-four hours later. the cells were exposed to CuCl 2 for 24h, and then washed and incubated for 14 days. Colonies were stained and scored.
resistance to cadmium than the normal cells, and the other strains of Wilson cells exhibited sensitivities in the normal range (Fig 1). Plating efficiency studies in the presence of copper showed no differences in sensitivity among the Menkes, Wilson and normal cells (Fig 2). In order to confirm the results of the colonyformation experiment, we studied the sensitivities to cadmium and copper by means of a cell growth experiment. As shown in Fig 3, we obtained the same results in the cell growth experiment as in the colony-formation experiment. Three Menkes cell strains and one of the Wilson cell strains exhibited higher resistance to cadmium and the sensitivities of other cell strains were not significantly different from those of normal cells. The sensitivities to copper did not differ Significantly among the Menkes, Wilson and normal strains. MorpholOgical change was also comparable
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with the results of cell growth studies. After three-day-incubation with I 0 ~M CdCI 2 , no morphological change was observed with Menkes cells, but normal cells rounded up and detached. Discussion All the Menkes strains and one of the Wilson strains were about 3-times more resistant to cadmium. Riordan et al reported that Menkes lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to cadmium [3]. We previously reported that a metallothionein-like protein (MLP) was induced by addition of cadmium, and Menkes cells tended to induce MLP most and Wilson cells induced more MLP than normal cells [7]. The greater MLP synthesizing ability of Menkes strains may account for their resistance to cadmium cytotoxity. Moreover, Menkes cells have a high level Cu-MLP without copper addition [7], and the added cadmium may displace copper from MLP already present. This displacement may lessen cadmium toxicity in Menkes cells. One of the Wilson strains exhibited cadmium resistance which may reflect the difference in the degree of induction of MLP. It was reported that Menkes cells were found to be more sensitive to copper and not resistant to cadmium in morphological change studies [8, 9]. In another study, no significant difference in copper sensitivity was found between normal and Menkes lymphoblasts as to their viability after addition of the copper [ 10]. Because there is a discrepancy between these reports, we studied cytotoxity using sensitive colony-forming ability and cell growth tests in the conditions with and without morphological change. In our experiments, consistent results were obtained. In all strains of
Fig 3 The effects of 10 pM CdCl, A) and 20 iJg/ml CuCI, B) on the relative growth rate. Cells were plated on 35 mm dishes. Twenty-four hours later, CdCl, or CuCl, was added, and after 3 days the numbers of cells were determined with a Coulter Counter.
Menkes cells we could confirm the resistanee to cadmium. Although the existence of strains with normal sensitivity to cadmium could not be ruled out, we can safely say that resistance to cadmium is a common character in Menkes fibroblasts. There have been several reports of cadmium resistant mutant cells [4-6], but now we report the resistance to cadmium in a genetically inherited disease. These results will be useful for elucidation of the mechanism of the disease and for studies of the cellular detoxification system against cadmium and other heavy metals. References 1. Goka TJ. Stevenson RE, Hefferan PM. Howell RR. Menkes disease: a biochemical abnormality in cultured human fibroblasts. Proc Natl Acad Sci USA 1976;73:604-6. 2. Labadie GU, Hirschhorn K, Katz S. Beratis NG. Increased copper metallothionein in Menkes cultured skin fibroblasts. Pediatr Res 1981; 15: 257-61. 3. Riordan JR, Jolicoeur-Paquet L. Metallothionein accumulation may account for intracellular copper retention in Menkes' disease. J Bioi Chem 1982;257:4639-45. 4. Hildebrand CEo Tobey RA. Campbell EW, Enger MD. A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity. Exp Cell Res 1979;124: 237-46. 5. Maiti I, Mbikay M, Marengo C, Thirion JP. Immunological characterization of metallothioneins in mouse LMTK cells and in a variant resistant to cadmium. J Cell Physiol1982; 112:35-41. 6. Shworak NW. Snyder FF. Gedamu L. Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2). Biochim Biophys Acta 1983;763: 332-8. 7. Sato M. Hayashi A. Ito H. Tojo M. Arima M. Copper level and metallothionein-like Cu-binding protein in cultured skin fibroblasts from patients
Hayashi et al: Menkes fibroblasts
75
with Menkes' disease and Wilson's disease (in Japanese). Brain and Nerve (Tokyo) 1984;36: 1063-8. 8. Chan WY, Garmca AD, Rennert OM. Cell culture studies of Menkes kinky hair disease. Gin Chim Acta 1978;88:495-507. 9. Leone A, Pavlakis GN, Hamer DH. Menkes'
76 Brain & Development, Val 8, No 1,1986
disease: abnormal metallothionein gene regulation in response to copper. Cell 1985 ;40: 301-9. 10. Yazaki M. Study on abnormal copper metabolism in Menkes' kinky hair disease and Wilson's disease. Nagoya Med J (Nagoya) 1981;25:16986.