CYANIDE-INDUCED APOPTOSIS AND OXIDATIVE STRESS IN DIFFERENTIATED PC12CELLS

Terminally differentiated PC12 cells are a useful neuron-like model fo r studying programmed cell death in response to nerve growth factor (N GF) deprivation. This in vitro model was used to investigate the mecha nism by which cyanide-induced histotoxic hypoxia produces neuronal deg eneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN fo r 24 h did not produce cell death. In contrast, treatment of different iated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blu e dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg2 +-dependent endonuclease inhibitor aurintricarboxylic acid and the tra nscriptional inhibitor actinomycin D partially attenuated hypoxic toxi city, suggesting roles for endonuclease activation and transcription i n this model of neuronal death. Extracted DNA from cyanide-treated neu rons demonstrated cleavage into oligonucleosomal fragments on gel elec trophoresis. Transmission electron microscopic analysis showed morphol ogical changes consistent with apoptotic cell death, including membran e blebbing and convolution, as well as chromatin condensation and marg ination to the nuclear membrane. Addition of either ascorbate or catal ase to the cultures partially attenuated the loss of cell viability in duced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The a bility of cyanide to elevate intracellular oxidant species was determi ned by microfluorescence in differentiated PC12 cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin. Exposure of cells to 0 .1 mM KCN produced a rapid generation of oxidants that was blocked sim ilar to 50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undif ferentiated, PC12 cells, and that antioxidants significantly reduce th e incidence of cyanide-induced apoptosis.