Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts.
Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.
Schou
Kenneth Bødtker
KB
Department of Molecular Biology, Section of Cellular and Devolopmental Biology, August Krogh Building, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen OE, Denmark.
Schneider
Linda
L
Christensen
Søren Tvorup
ST
Hoffmann
Else Kay
EK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3 / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism* / Q000473:pathology; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology; D053938:DNA Fragmentation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D007038:Hypotonic Solutions / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D007038:Hypotonic Solutions; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; D003513:Cycloheximide; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D019311:Staurosporine