Cytometry A. 2006 Apr;69(4):212-21
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.
Kurose A, Tanaka T, Huang X, Traganos F, Dai W, Darzynkiewicz
Z.
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. MATERIALS AND 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.
PMID: 16528719
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