Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.
BACKGROUND
DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation.


METHODS
HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells.


RESULTS
In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis.


CONCLUSIONS
Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.
Huang
Xuan
X
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10532, USA.
Okafuji
Masaki
M
Traganos
Frank
F
Luther
Ed
E
Holden
Elena
E
Darzynkiewicz
Zbigniew
Z
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008942:Mitoxantrone / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D019772:Topotecan / Q000494:pharmacology*
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D003432:Cross-Linking Reagents; C468783:H2AX protein, human; D006657:Histones; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D019772:Topotecan; D004247:DNA; D008942:Mitoxantrone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin