Research ArticlesSecretion and Increase of Intracellular CuZn Superoxide Dismutase Content in Human Neuroblastoma SK-N-BE Cells Subjected to Oxidative Stress
Introduction
The production of oxygen free radicals (OFR) is a natural metabolic event that is remarkably increased during ischemia and postischemic injury and as a result of radiation exposure, heat shock, neutrophil activation, and hyperoxia. These events stimulate the production of a series of partially reduced oxygen molecules that directly or indirectly damage cellular and nucleur structures. Three antioxidant enzymes protect the cell against oxidant stress: the superoxide dismutase (SOD) isoenzymes, glutathione peroxidase, and catalase. The three forms of SOD found in eukaryotic cells are encoded by three different genes: MnSOD, which is a mitochondrial enzyme located in the inner membrane [27]; dimeric cytosolic CuZn SOD [17], and the tetrameric high molecular weight extracellular CuZn SOD (EC SOD) that has been found in extracellular fluids 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26.
The brain consumes a large amount of oxygen because it derives its energy mainly from the oxidative metabolism of the mitochondrial respiratory chain. The brain is also particularly vulnerable to oxidant damage due to the high levels of polyunsaturated fatty acids that serves as substrates for lipid peroxidation, the high levels of free iron, which generate hydroxyl free radicals and the low levels of catalase, GSH-Px, and SOD isoenzymes 7, 8, 9. Although the antioxidant effect of cytosolic CuZn SOD is well documented, little is known about the response of this enzyme to oxidative stresses. Strälin et al. [25]found various types of oxidative stresses did not induce SOD isoenzymes in human dermal fibroblasts. Similarly, CuZn SOD was unaffected by oxidative stress while the availability of copper has been shown to be a decisive factor controlling CuZn SOD activity in humans and numerous species of animals [8].
The only SOD isoenzyme thought to be secreted by fibroblasts and glial cells but not by epithelial and endothelial cells is EC SOD [15]. Recently, we provided evidence that human fibroblasts and human hepatocarcinoma cells (HepG2) are capable of secreting cytosolic CuZn SOD [19].
The aim of the present study was to investigate whether human neuroblastoma cells (SK-N-BE), as well as human fibroblasts and HepG2 cells, are able to secrete the dimeric CuZn SOD. We also investigated whether the oxidative agent Fe2+/ascorbate, by generating a mismatch between the production of OFR and the ability of cells to defend against them, can affect the amount of intracellular CuZn SOD or its secretion in SK-N-BE cells.
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Cells
The human neuroblastoma cells SK-N-BE (American Type Culture Collection) were grown in monolayers in RPMI 1640 (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% Foetal Calf Serum (FCS, Sigma), 2 mM L-glutamine, 50 μg/ml streptomycin, and 50 IU/ml penicillin and maintained in 5% CO2 at 37°C.
Metabolic Labeling and Immunoprecipitation Experiments
The cells were grown to semiconfluency in 60 mm dishes. SK-N-BE cells were preincubated for at least 120 min with 2 ml of cysteine-free Dulbecco’s Modified Eagle’s Medium (DMEM) (ICN, U.K.)
SOD Secretion and Its Induction by Oxidative Stress
The immunoprecipitation of [35S]cysteine labeled SK-N-BE cells using antihuman CuZn SOD antibodies yields a major protein band both in cell lysates and in the media. This band was practically absent when cells were immunoprecipitated in presence of an excess (100 μg/ml) of unlabeled human CuZn SOD (Fig. 1). These data show that human neuroblastoma cells, analogously to human fibroblasts and HepG2 cells, secrete CuZn SOD in the culture medium. SK-N-BE cells express neuron-specific enolase as
Discussion
Intracellular CuZn SOD appears to be constitutively expressed in some eukaryote cells [1]and behaves as a “housekeeping” protein in mouse and rat tissues [4]. The copper seems to be involved in the regulation of this enzyme [23-24-11]; however the underlying mechanisms that take part in the control of CuZn SOD activity are still poorly understood.
Under our experimental conditions, Fe2+/ascorbate, used at low concentrations to avoid loss of cell protein and DNA, increased the amount of
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