{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T06:47:52Z","timestamp":1789627672698,"version":"build-2803163510"},"reference-count":41,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2006,3,9]],"date-time":"2006-03-09T00:00:00Z","timestamp":1141862400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2006,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background:<\/jats:title><jats:p>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\u2010strand breaks (DSBs). Aphidicolin (APH), an inhibitor of \u03b1\u2010like 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 <jats:italic>139<\/jats:italic> 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 <jats:italic>Ser<\/jats:italic> 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and Methods:<\/jats:title><jats:p>HL\u201060 cells were treated in cultures with 0.1\u20135.0 mM HU or 1\u20134 \u03bcM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to <jats:italic>Ser<\/jats:italic>1981\u2010ATM or <jats:italic>Ser<\/jats:italic> 139\u2010H2AX epitopes, respectively, concurrent with measurement of cellular DNA content.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results:<\/jats:title><jats:p>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\u20131.4) were more affected than late\u2010S phase (DI = 1.6\u20131.9) cells, ATM was not activated by HU. In fact, the level of constitutive (\u201cprogrammed\u201d) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1\u20135.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at <jats:italic>Ser<\/jats:italic>139 with no evidence of ATM activation, and as in the case of HU, the early\u2010S cells were more affected than the late\u2010S 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\u2010<jats:italic>Ser<\/jats:italic>139 and ATM\u2010<jats:italic>Ser<\/jats:italic>1981 phosphorylation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions:<\/jats:title><jats:p>The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and\/or DNA\u2010dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (\u201cprogrammed\u201d) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. \u00a9 2006 International Society for Analytical Cytology<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.a.20241","type":"journal-article","created":{"date-parts":[[2006,3,9]],"date-time":"2006-03-09T20:46:44Z","timestamp":1141937204000},"page":"212-221","source":"Crossref","is-referenced-by-count":50,"title":["Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on <i>Ser<\/i> 1981 and histone H2AX on <i>Ser<\/i> 139 in relation to cell cycle phase and induction of 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