Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis.
BACKGROUND
The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-à-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor.


METHODS
Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry.


RESULTS
Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells.


CONCLUSIONS
The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the "cleavable complexes" stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells.
Kurose
Akira
A
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10532, USA.
Tanaka
Toshiki
T
Huang
Xuan
X
Halicka
H Dorota
HD
Traganos
Frank
F
Dai
Wei
W
Darzynkiewicz
Zbigniew
Z
D017209:Apoptosis / Q000502:physiology*; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D018922:HL-60 Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007399:Interphase / Q000502:physiology; D007700:Kinetics; D008942:Mitoxantrone / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D059003:Topoisomerase Inhibitors; D019772:Topotecan / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D059003:Topoisomerase Inhibitors; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D012694:Serine; D019772:Topotecan; D008942:Mitoxantrone; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases