title: High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons.
abstract: Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 micro M) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 micro M) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (>680 and 50-300 kbp) was observed after treatment with glutamate or A23187. Traces of approximately 50-kbp fragments were detectable after the K(+)/serum-deprivation. The amount of >680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of >680-kbp HMW fragments and reduced cell death after K(+)/serum-deprivation. No such changes were observed with caspase inhibitors after A23187 treatment. AIF redistribution was observed following glutamate treatment and K(+)/serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.
authors.affiliation: Institute of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Hege Holte
authors.initials: HH
authors.lastname: Slagsvold
authors.forename: Carola Maria
authors.initials: CM
authors.lastname: Rosseland
authors.forename: Chris
authors.initials: C
authors.lastname: Jacobs
authors.forename: Erica
authors.initials: E
authors.lastname: Khuong
authors.forename: Nina
authors.initials: N
authors.lastname: Kristoffersen
authors.forename: Mona
authors.initials: M
authors.lastname: Gaarder
authors.forename: Asa Birgitta
authors.initials: AB
authors.lastname: Fallgren
authors.forename: Henrik Sverre
authors.initials: HS
authors.lastname: Huitfeldt
authors.forename: Ragnhild Elisabeth
authors.initials: RE
authors.lastname: Paulsen
mesh_terms: D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D004247:DNA; D020169:Caspases