Wei Dai

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

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.

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.

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 α‐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.

Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.

Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser‐10 and Ser‐139, respectively, as well as ATM activation through phosphorylation on Ser‐1981, during PCC, and relate these events to cell‐cycle phase and to initiation of apoptosis.

ATM activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis

Abstract.u2002 Damage that engenders DNA double‐strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto‐ or trans‐phosphorylation on Ser1981 and activated ATM is one of the mediators of histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL‐60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S‐phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S‐phase cells immunostained for ATM‐S1981P and γH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM‐S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2‐ to 3‐fold lower for ATM‐S1981P alone, or for ATM‐S1981P and γH2AX IF combined, than for γH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.

Extent of constitutive histone H2AX phosphorylation on Ser-139 varies in cells with different TP53 status

Abstract.u2002 In response to DNA damage by genotoxic agents, histone H2AX is phosphorylated on Ser‐139. However, during the cell cycle, predominantly in S and G2M phase, histone H2AX is also phosphorylated in untreated normal and tumour cells. This constitutive H2AX phosphorylation is markedly reduced by exposure of cells to the reactive oxygen species scavenger N‐acetyl‐L‐cysteine. Therefore, it appears likely that constitutive H2AX phosphorylation reflects the ongoing oxidative DNA damage induced by the reactive oxygen species during progression through the cell cycle. Because the tumour suppressor p53 (tumour protein p53) is known to induce transcription of genes associated with cell response to oxidative stress, we have compared the intensity of constitutive H2AX phosphorylation, and the effect of N‐acetyl‐L‐cysteine on it, in cells with different tumour protein p53 status. These were human lymphoblastoid cell lines derived from WIL2 cells: TK6, a p53 wt line, NH32, a tumour protein p53 knock‐out derived from TK6, and WTK1, a WIL2‐derived line that expresses a homozygous mutant of tumour protein p53. Also tested were the tumour protein p53‐null promyelocytic HL‐60 cells. The degree of constitutive H2AX phosphorylation was distinctly lower in NH32, WTK1 and HL‐60 compared to TK6 cells in all phases of the cell cycle. Also, the degree of attenuation of constitutive H2AX phosphorylation by N‐acetyl‐L‐cysteine was less pronounced in NH32, WTK1, and HL‐60, compared to TK6 cells. However, the level of reactive oxygen species detected by the cells’ ability to oxidize carboxyl‐dichlorodihydrofluorescein diacetate was not significantly different in the cell lines studied, which would suggest that regardless of tumour protein p53 status, the level of oxidative DNA damage was similar. The observed higher level of constitutive H2AX phosphorylation in cells harbouring wt tumour protein p53 may thus indicate that tumour protein p53 plays a role in facilitating histone H2AX phosphorylation, an important step in the mobilization of the DNA repair machinery at the site of DNA double‐strand breaks.