Higher order chromatin degradation in glial cells: the role of calcium

Higher order chromatin degradation (HOCD), i.e. the scission of nuclear chr omatin loops at the matrix attachment regions (MARs), is a hallmark of prog rammed cell death. We have previously demonstrated that hydrogen peroxide ( H2O2) induces rapid HOCD in cultured oligodendrocytes generating two subpop ulations of DNA fragments of greater than or equal to 400 and 50-200 kb. In the present study, we examined the involvement of calcium in this process. HOCD was induced in primary rat oligodendrocytes by exposure to 1 mM H2O2 and assessed by field inversion gel electrophoresis with and without S1 end onuclease digestion, to detect single and double stranded fragmentation, re spectively. Chelating intracellular calcium with BAPTA/AM prior to H2O2 exp osure inhibited HOCD in a dose-dependent manner. Complete inhibition of HOC D was attained with 50 muM BAPTA/AM. The pretreatment of cells with desferr oxamine mesylate, which may lower intracellular calcium levels, also result ed in a profound inhibition of HOCD, but the initial chromatin digestion in to greater than or equal to 400 kb single stranded DNA fragments was unaffe cted. Neither removing extracellular calcium nor blocking calcium release f rom intracellular stores with TMB-8 affected HOCD. Moreover, increasing int racellular calcium with A23187 calcium ionophore did not induce HOCD. Subse quent study in nuclei purified from C6 glioma cells revealed that the endon uclease responsible for HOCD is calcium-independent, but is magnesium-depen dent. Magnesium-induced HOCD was not affected by the removal of calcium fro m nuclei with EGTA, but was practically abrogated in nuclei prepared from B APTA/AM-pretreated cells. These results indicate that although H2O2-induced HOCD is not directly mediated by an increase of intracellular calcium conc entration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated endonuclease in an active form. (C) 2001 Elsevier Science Ltd. All rights reserved.