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

Abstract

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 a-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 lM 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 5 1.1-1.4) were

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

“…Other deoxycytidine nucleoside analogues with slightly different mechanisms of action, 1-h-D-arabinofuranosylcytosine and troxacitabine, induced similar levels of H2AX phosphorylation. Recent reports have shown that H2AX is phosphorylated on DNA polymerase and ribonucleotide reductase inhibition by aphidicolin and hydroxyurea, respectively (33,50). Together, those investigations and this study suggest that H2AX is phosphorylated in response to agents that inhibit DNA synthesis.…”

“…For instance, the IC 50 . Thus, the evidence indicates that the increased H2AX phosphorylation during checkpoint abrogation after exposure to UCN-01 is due to the inhibition of Chk1.…”

Pol η is required for DNA replication during nucleotide deprivation by hydroxyurea

“…The symmetrical-arrested forks generated in hydroxyurea will have little if any exposed single strand regions, unlike after UV, especially in Pol Z-deficient cells, where the leading strands contain kilobase lengths of exposed single strand regions that may be covered by single strand binding protein, RPA (Cordeiro-stone et al, 1999;Cordonnier et al, 1999). This extensive disruption of the replication fork architecture may provide a signal for the kinases responsible for the pan-staining response, which takes longer to develop after hydroxurea and may represent preapoptotic cells (Feijoo et al, 2001;Zhao and Piwnica-Worms, 2001;Lu et al, 2006;Mukherjee et al, 2006;Kurose et al, 2006a). The greater accumulation of normal cells at the G1/S boundary with greater levels of apoptosis strongly suggest that apoptosis from hydroxyurea occurs at this boundary.…”

“…Common types of damage involve DNA double strand breaks arising directly or through replication fork breakage that transduce phosphorylation of serine-139 of the C-terminus of the variant histone H2AX (gH2AX) and accumulation of immunofluorescent foci containing gH2AX, Mre11/Rad 50/Nbs, Rad 51 and proliferating cell nuclear antigen (PCNA) (Limoli et al, 2000(Limoli et al, , 2002a(Limoli et al, , 2005Thakur et al, 2001;Ward and Chen, 2001;Cleaver et al, 2002;Furuta et al, 2003;O'Driscoll et al, 2003;Halicka et al, 2005;Lowndes and Toh, 2005;Marti et al, 2006). Replication arrest from hydroxyurea and UV is mechanistically different and neither generate double strand breaks directly; hydroxyurea limits deoxyribonucleotide precursors (Kurose et al, 2006a), whereas UV directly blocks replication fork progression with DNA photoproducts.…”

Regulation of DNA damage response and cell cycle in radiation-resistant HL60 myeloid leukemia cells

“…It is noteworthy that the phosphorylation of DNA-PKcs occurred only at 120 min after irradiation. Furthermore, the increase in the level of γ-H2AX in HL60 cells at 120 min may indicate the progression of apoptosis because γ-H2AX occurs in cases of DNA fragmentation due to apoptosis (16). Therefore, these data may indicate that the fate of irradiated-cells could be determined within 120 min of irradiation.…”