Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.
BACKGROUND
DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM.


METHODS
HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content.


RESULTS
While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation.


CONCLUSIONS
The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.
Kurose
Akira
A
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Tanaka
Toshiki
T
Huang
Xuan
X
Traganos
Frank
F
Dai
Wei
W
Darzynkiewicz
Zbigniew
Z
D016590:Aphidicolin / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; 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; D006918:Hydroxyurea / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors / 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; C468783:H2AX protein, human; D006657:Histones; D019384:Nucleic Acid Synthesis Inhibitors; D025521:Tumor Suppressor Proteins; D016590:Aphidicolin; D012694:Serine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea