Modification of glycolysis affects cell sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria

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Abstract

The effect of alteration of the glycolytic pathway on cell damage induced by oxidative stress was investigated with dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells that either overexpress cytosolic glycerol-3-phosphate dehydrogenase (CHO/cGPDH cells) or are depleted of the A subunit of lactate dehydrogenase as a result of anti-sense RNA expression (CHO/anti-LDH cells). The extent of oxidative phosphorylation in CHO/anti-LDH and CHO/cGPDH cells was increased and decreased, respectively, relative to that in parental CHO cells, as revealed by measurement of the intracellular content of ATP, the rate of cellular O2 consumption, the mitochondrial membrane potentialΨm), and the generation of reactive oxygen species. The sensitivity of these cell lines to cell death induced by the exogenous oxidant tert-butyl hydroperoxide decreased according to the rank order CHO/anti-LDH > CHO > CHO/cGPDH. Exogenous pyruvate markedly increased the sensitivity of CHO/cGPDH cells to oxidant-induced death. The differences among the three cell lines in susceptibility to oxidant-induced death were reflected in the proportion of oxidant-treated cells with a subdiploid DNA content, with a collapsed ΔΨm, and with cytochrome c in the cytosol, indicating that death was mediated by apoptosis. These results demonstrate that the influx of respiratory substrate into mitochondria is an important determinant of cell sensitivity to oxidant-induced apoptosis.

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Materials and methods

Cell culture and viability assay. The dihydrofolate reductase (dhfr)-deficient CHO (CHO dhfr) cells were maintained under a humidified atmosphere of 5% CO2 at 37 °C in NaHCO3-buffered F-12 medium supplemented with l-glutamine, 10% fetal bovine serum, penicillin (1000 U/ml), and streptomycin (1000 μg/ml). For determination of cell viability, cells (3 × 105) were seeded onto 60-mm dishes, cultured for 12 h, and then incubated for an additional 12 h in fresh medium before exposure to tert-butyl

Effect of modification of the glycolytic pathway on the input of respiratory substrate into mitochondria

In this study, we tried to bypass the glycolytic pathway by overexpressing cGPDH and anti-sense LDH-A RNA. To maximize the effect of anti-sense ldh gene in the cell, we used dihydroforate reductase(dhfr)-deficient CHO cells (CHO dhfr), which are available for an efficient co-amplification system using dhfr gene in the presence of an amplificable marker, methotrexate (MTX). One of the several target proteins to amplify the gene in mammalian cells is DHFR, an enzyme that catabolizes the

Acknowledgements

This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

References (42)

  • J.F. Turrens et al.

    Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria

    Arch. Biochem. Biophys.

    (1985)
  • J.F. Turrens et al.

    The effect of hyperoxia on superoxide production by lung submitochondrial particles

    Arch. Biochem. Biophys.

    (1982)
  • J.E. Biaglow et al.

    Effect of oncogene transformation of rat embryo cells on cellular oxygen consumption and glycolysis

    Biochem. Biophys. Res. Commun.

    (1997)
  • A. Guidarelli et al.

    Stimulation of oxygen consumption promotes mitochondrial calcium accumulation, a process associated with, and causally linked to, enhanced formation of tert-butylhydroperoxide-induced DNA single-strand breaks

    Exp. Cell. Res.

    (1997)
  • X. Liu et al.

    Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c

    Cell

    (1996)
  • C. Du et al.

    Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition

    Cell

    (2000)
  • A.M. Verhagen et al.

    Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins

    Cell

    (2000)
  • P. Bernardi et al.

    Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations

    J. Biol. Chem.

    (1992)
  • A.J. Kowaltowski et al.

    Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition

    Biochim. Biophys. Acta

    (1997)
  • K. Guillemin et al.

    The hypoxic response: huffing and HIFing

    Cell

    (1997)
  • C.V. Dang et al.

    Oncogenic alterations of metabolism

    Trends Biochem. Sci.

    (1999)
  • Cited by (0)

    Abbreviations: ROS, reactive oxygen species; cGPDH, cytosolic glycerol-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; t-BOOH, tert-butyl hydroperoxide; PBS, phosphate-buffered saline; MPT, mitochondrial permeability transition; ΔΨm, mitochondrial membrane potential; Rh123, rhodamine 123; DCFH-DA, 2,7-dichlorofluorescin diacetate; NEM, N-ethylmaleimide.

    1

    These authors contributed equally to this work.

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