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J Korean Neurol Assoc. 2001 Jul;19(4):384-392. Korean. Original Article.
Kim BC , Kim MK , Cho KH , Kim YS , Lee KY .
Department of Neurology, Chonnam National University Medical School.
Department of Biochemistry, Chonnam National University Medical School.
Abstract

BACKGROUND: The Mitogen-activated Protein Kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. Cell death is usually mediated through apoptosis regulated by extracellular factors. We investigated the apoptotic processes of PC12 cells induced by hypoxia and the activation of MAPKs in apoptosis. METHODS: PC12 cells were maintained in a RPMI 1640 medium containing 5% fetal bovine serum (FBS), 10% horse serum, and antibiotics. Hypoxia was induced in a humidified 37 degrees C incubator within a BBL GasPac Pouch. The apoptosis of PC12 cells was observed with an electron microscope and DNA laddering on agarose-gel electrophoresis. The phosphorylation of MAPKs was measured by an image analysis system after a Western blot. RESULTS: Hypoxic apoptosis occurred maximally when PC12 cells in 2% FBS and 5mM glucose media were incubated in an anaerobic state for 6 hours and then reoxygenated for 18 hours. The phosphorylation of MAPKs was observed 30 min after hypoxia and sustained for at least 2 hours. Phosphorylations of extracellular signal-regulated kinase (ERK) and p38 kinase were reduced after 4 hours of hypoxia, whereas those of c-Jun N-terminal kinase (JNK) persisted for 6 hrs Nerve Growth Factor (NGF). Significantly inhibited DNA fragmentation in hypoxia-induced apopto-sis of PC12 cells. NGF accentuated phosphorylation of ERK in both normoxia and hypoxia. Nerve growth factor (NGF) reduced the phosphorylation of JNK but did not affect the phosphorylation of p38 kinase. CONCLUSIONS: We established the conditions for PC12 cell apoptosis caused by hypoxia. These results suggest that activation of JNK and p38 kinase might be the apoptotic signals induced by hypoxia and regulated by different pathways. (J Korean Neurol Assoc 19(4):384~392, 2001)

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