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 topo1 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-Thr68(P)immunofluorescence (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 G(2)M 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 G(2)M and minimal in G(1) 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 gammaH2AX or Chk2-Thr68(P) IF, the former reflecting total IF per nucleus the latter stressing the punctate (foci) character of expression of these phospho-modified
proteins.