J. tnher. Metab. Dis. 15 (1992) 943-944
© SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
SHORT REPORT Selective killing of cells with oxidative defects in galactose medium: A screening test for affected patient fibroblasts R. Petrova-Benedict 1, J. R. Bunci&, D. C. Wallace 2 and B. H. Robinson 1.
It was shown a number of years ago that Chinese hamster (CH) fibroblasts with defects in mitochondrial respiration were able to keep up the energy charge within the celt and maintain normal growth rates, primarily by doubling the rates of conversion of glucose to lactic acid (Sodeberg et al t980; Day and Schemer t982). We have made similar observations in cultured skin fibrobtasts from patients with defects of the mitochondrial respiratory chain (Robinson et al 1986, 1987a). Substitution of galactose (5 mmol/L) for glucose in the culture medium was shown to be lethal for oxidative-deficient CH fibroblasts (Sodeberg et al 1980). We investigated the effects of galactose on the viability of a variety of cell lines from patients with different oxidative defects by subculturing the skin fibroblasts into ~-MEM containing 5 mmol/L galactose. Cell lines used were as described by Robinson et al (1987b), Robinson et al (1990) and Gterum et al (t988). While control cell lines displayed a pattern of growth and survival no different from those grown in glucose medium, within 24 h of subculture no live cells survived from patients with severe neonatal complex I deficiency, cytochrome oxidase deficiency (Leigh disease), and multiple respiratory chain complex deficiency. Cells from patients with severe pyruvate dehydrogenase complex deficiency, complex I deficiency with Leigh disease, and partial cytochrome oxidase deficiency (Leigh disease) showed extremely poor growth with cell death after 2 weeks in culture. Cell lines from patients with partial deficiency of the pyruvate dehydrogenase complex, pyruvate carboxylase deficiency, liver-specific partial cytochrome oxidase deficiency, Leber hereditary optic neuropathy (homoplasmic for the mtDNA 11 778 mutation), and Kearns-Sayre syndrome showed no adverse effects of culture in galactose-containing medium. When these cell lines were reassessed by culture in a medium containing galactose plus 25 #mol/L azide, the results were the same except that one of two cell lines tested with Leber hereditary optic neuropathy (LHON) grew very poorly and died. The cell lines showing susceptibility to galactose metabolism appear to be those with the more overt or severe oxidative defects, and the extent of the susceptibiity appears to bear some relationship to the nature of the oxidative defect. Thus severe complex I deficiency, multiple respiratory chain deficiency and severe cytochrome oxidase deficiency cells are almost immediately killed in galactose medium, whereas *Correspondence 1Departments of Biochemistry and Paediatrics, University of Toronto and Department of Genetics, The Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario Canada M5G 1X8; 2Department of Biochemistry, Emory University, Atlanta, Georgia, USA 943
944
Short Report
milder oxidative defects in cells allow the cells to survive for a while, though growth is extremely poor. In general, the susceptibility can be predicted for respiratory-chain defects by reference to the redox state as predicted by the lactate-to-pyruvate ratio in cells. If this ratio is significantly elevated, the cells do not survive or grow well in galactose. There are two exceptions to this. The first is severe deficiency of the pyruvate dehydrogenase complex. This is very restrictive to oxidative metabolism without raising the lactate to pyruvate ratio. The second is the m t D N A defect present in L H O N . Two cell lines are used here, both homoplasmic for the mutation present at 11 778, and both survive well in gatactose despite the fact that one exhibits a high L/P ratio. Polymorphisms present in other complex I subunits may affect the penetrance and severity of the L H O N mutations in respect to both biochemical and clinical phenotype (Johns and Berman 1991). Thus, it is interesting that inclusion of 25 ktmol/L azide as a further restriction to oxidative metabolism compromised the L H O N cells with the elevated redox state but not the L H O N cells with normal redox status. We conclude that this type of approach, perhaps with further refinements, may allow for the development of testing procedures by cell culture that can be instituted as a standard test for certain types of oxidative defects. This approach may also facilitate elucidation of the genes responsible for oxidative defects by the use of selective media after cDNA transfection experiments. We thank the National Foundation, the March of Dimes for support of this work. REFERENCES
Day CE, Schemer IE (1982) Mapping of the genes of some components of the electron transport chain (Complex I) on the X chromosome of mammals. Sore Cell Genet 8: 691-707. Gterum M, Yanamura W, Capaldi R, Robinson BH (1988) Characterization of cytochrome oxidase mutants in human fibrobtasts. FEBS Lett 236: 100-104. Johns DR, Berman J (1991) Alternative, simultaneous complex I mitochondrial DNA mutations in Leber's Hereditary Optic Neuropathy. Biochem Biophys Res Commun 174: 1324-1330. Robinson BH, Ward J, Goodyer P, Baudet A (1986) Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia. J Clin Invest 77: 1422-1427. Robinson BH, DeMeifleir L, Glerum M, Sherwood WG, Becker L (1987a) Clinical presentation of patients with mitochondrial respiratory chain defects in NADH CoQ reductase and cytochrome oxidase. Clues to the pathogenesis of Leigh's disease. J Pediatr 110: 216-222. Robinson BH, MacMillan H, Petrova-Benedict R, Sherwood WG (1987b) Variable clinical presentation in patients with deficiency of the pyruvate dehydrogenase complex. A review of 30 cases with a defect in the E 1 component of the complex. J Pediatr 111: 525-533. Robinson BH, Glerum DM, Chow W, Petrova-Benedict R, Lightowlers R, Capaldi R (1990) The use of skin fibroblast cultures in the detection of respiratory chain defects in patients with lacticacidemia. Pediatr Res 28: 549-555. Sodeberg K, Nissinen E, Bakey B, Schemer IE (1980) The energy charge of wild-type and respiration-deficient Chinese Hamster cell mutants. J Cell PhysioI 103: 169-172.
J. Inher. Metab. Dis. t5 (1992)
© SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
SHORT REPORT Selective killing of cells with oxidative defects in galactose medium: A screening test for affected patient fibroblasts R. Petrova-Benedict 1, J. R. Bunci&, D. C. Wallace 2 and B. H. Robinson 1.
It was shown a number of years ago that Chinese hamster (CH) fibroblasts with defects in mitochondrial respiration were able to keep up the energy charge within the celt and maintain normal growth rates, primarily by doubling the rates of conversion of glucose to lactic acid (Sodeberg et al t980; Day and Schemer t982). We have made similar observations in cultured skin fibrobtasts from patients with defects of the mitochondrial respiratory chain (Robinson et al 1986, 1987a). Substitution of galactose (5 mmol/L) for glucose in the culture medium was shown to be lethal for oxidative-deficient CH fibroblasts (Sodeberg et al 1980). We investigated the effects of galactose on the viability of a variety of cell lines from patients with different oxidative defects by subculturing the skin fibroblasts into ~-MEM containing 5 mmol/L galactose. Cell lines used were as described by Robinson et al (1987b), Robinson et al (1990) and Gterum et al (t988). While control cell lines displayed a pattern of growth and survival no different from those grown in glucose medium, within 24 h of subculture no live cells survived from patients with severe neonatal complex I deficiency, cytochrome oxidase deficiency (Leigh disease), and multiple respiratory chain complex deficiency. Cells from patients with severe pyruvate dehydrogenase complex deficiency, complex I deficiency with Leigh disease, and partial cytochrome oxidase deficiency (Leigh disease) showed extremely poor growth with cell death after 2 weeks in culture. Cell lines from patients with partial deficiency of the pyruvate dehydrogenase complex, pyruvate carboxylase deficiency, liver-specific partial cytochrome oxidase deficiency, Leber hereditary optic neuropathy (homoplasmic for the mtDNA 11 778 mutation), and Kearns-Sayre syndrome showed no adverse effects of culture in galactose-containing medium. When these cell lines were reassessed by culture in a medium containing galactose plus 25 #mol/L azide, the results were the same except that one of two cell lines tested with Leber hereditary optic neuropathy (LHON) grew very poorly and died. The cell lines showing susceptibility to galactose metabolism appear to be those with the more overt or severe oxidative defects, and the extent of the susceptibiity appears to bear some relationship to the nature of the oxidative defect. Thus severe complex I deficiency, multiple respiratory chain deficiency and severe cytochrome oxidase deficiency cells are almost immediately killed in galactose medium, whereas *Correspondence 1Departments of Biochemistry and Paediatrics, University of Toronto and Department of Genetics, The Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario Canada M5G 1X8; 2Department of Biochemistry, Emory University, Atlanta, Georgia, USA 943
944
Short Report
milder oxidative defects in cells allow the cells to survive for a while, though growth is extremely poor. In general, the susceptibility can be predicted for respiratory-chain defects by reference to the redox state as predicted by the lactate-to-pyruvate ratio in cells. If this ratio is significantly elevated, the cells do not survive or grow well in galactose. There are two exceptions to this. The first is severe deficiency of the pyruvate dehydrogenase complex. This is very restrictive to oxidative metabolism without raising the lactate to pyruvate ratio. The second is the m t D N A defect present in L H O N . Two cell lines are used here, both homoplasmic for the mutation present at 11 778, and both survive well in gatactose despite the fact that one exhibits a high L/P ratio. Polymorphisms present in other complex I subunits may affect the penetrance and severity of the L H O N mutations in respect to both biochemical and clinical phenotype (Johns and Berman 1991). Thus, it is interesting that inclusion of 25 ktmol/L azide as a further restriction to oxidative metabolism compromised the L H O N cells with the elevated redox state but not the L H O N cells with normal redox status. We conclude that this type of approach, perhaps with further refinements, may allow for the development of testing procedures by cell culture that can be instituted as a standard test for certain types of oxidative defects. This approach may also facilitate elucidation of the genes responsible for oxidative defects by the use of selective media after cDNA transfection experiments. We thank the National Foundation, the March of Dimes for support of this work. REFERENCES
Day CE, Schemer IE (1982) Mapping of the genes of some components of the electron transport chain (Complex I) on the X chromosome of mammals. Sore Cell Genet 8: 691-707. Gterum M, Yanamura W, Capaldi R, Robinson BH (1988) Characterization of cytochrome oxidase mutants in human fibrobtasts. FEBS Lett 236: 100-104. Johns DR, Berman J (1991) Alternative, simultaneous complex I mitochondrial DNA mutations in Leber's Hereditary Optic Neuropathy. Biochem Biophys Res Commun 174: 1324-1330. Robinson BH, Ward J, Goodyer P, Baudet A (1986) Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia. J Clin Invest 77: 1422-1427. Robinson BH, DeMeifleir L, Glerum M, Sherwood WG, Becker L (1987a) Clinical presentation of patients with mitochondrial respiratory chain defects in NADH CoQ reductase and cytochrome oxidase. Clues to the pathogenesis of Leigh's disease. J Pediatr 110: 216-222. Robinson BH, MacMillan H, Petrova-Benedict R, Sherwood WG (1987b) Variable clinical presentation in patients with deficiency of the pyruvate dehydrogenase complex. A review of 30 cases with a defect in the E 1 component of the complex. J Pediatr 111: 525-533. Robinson BH, Glerum DM, Chow W, Petrova-Benedict R, Lightowlers R, Capaldi R (1990) The use of skin fibroblast cultures in the detection of respiratory chain defects in patients with lacticacidemia. Pediatr Res 28: 549-555. Sodeberg K, Nissinen E, Bakey B, Schemer IE (1980) The energy charge of wild-type and respiration-deficient Chinese Hamster cell mutants. J Cell PhysioI 103: 169-172.
J. Inher. Metab. Dis. t5 (1992)
