Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.
BACKGROUND
Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis.


METHODS
To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry.


RESULTS
Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells.


CONCLUSIONS
Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.
Huang
Xuan
X
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10532, USA.
Kurose
Akira
A
Tanaka
Toshiki
T
Traganos
Frank
F
Dai
Wei
W
Darzynkiewicz
Zbigniew
Z
D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects / Q000502:physiology*; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008387:Marine Toxins; D008856:Microscopy, Fluorescence; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; D008387:Marine Toxins; D010080:Oxazoles; D025521:Tumor Suppressor Proteins; D012694:Serine; C059041:calyculin A; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases