Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase

Abstract

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitors doxorubicin, etoposide and mitoxantrone (MXT) are widely used antitumor drugs. They stabilize otherwise transient ("cleavable") complexes of topol or topo2 with DNA, respectively. Collisions of DNA replication forks (during replication) or progressing RNA polymerase molecules (during transcription) with these complexes convert them into double-strand DNA breaks (DSBs). Formation of DSBs triggers activation of ATM and phosphorylation of histone H2AX, the markers that have been used to correlate DNA damage with cell cycle phase or induction of apoptosis. In the present study we explored a relationship between H2AX phosphorylation and activation of checkpoint kinase 2 (Chk2) in human lung carcinoma A549 cells treated with TPT or with MXT. Activation of Chk2 was detected immunocytochemically using a phospho-specific (Thr68) Ab and measuring Chk2-Thr68Pimmunofluorescence (IF), concurrently with DNA content, by laser scanning cytometry. In the untreated cells, activated Chk2 was present predominantly in centrosomes. Upon treatment with TPT or MTX, the activated Chk2 presented itself in form of either minute or large IF foci in the cell's nucleoplasm. H2AX phosphorylation whether induced by TPT or MXT was rapid, with the maximal rate occurring during the initial 2 h and peaking at 2 h of treatment. TPT or MXT induced Chk2 activation occurred at a distinctly slower pace, peaking at 4 h. While TPT-induced H2AX phosphorylation and Chk2 activation were maximal in S-phase cells, Chk2 activation was also much pronounced in G2M cells; the least affected by TPT were G 1 cells, MTX-induced H2AX phosphorylation was maximal in G 1 cells while Chk2 activation was maximal in G2M and minimal in G1 cells. The pattern of cell-cycle phase specific response to TPT or MXT by H2AX phosphorylation and Chk2 activation was different when measured either as integrated or maximal pixel of γH2AX or Chk2-Thr68P IF, the former reflecting total IF per nucleus the latter stressing the punctate (foci) character of expression of these phospho-modified proteins. © 2008 International Society for Advancement of Cytometry.