p21^CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

This study provides evidence for the importance of p2^CDKN1A for tne repair Of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21^CDKN1A and p53 enhance camptothecin-induced histone H2AX phosphorylation (γH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, γH2AX reverses after camptothecin removal. By contrast, γH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21^CDKN1A (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced γH2AX formation in p21-/- cells, we conclude that enhanced γH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced γH2AX formation and inhibits camptothecin-induced p21^CDKN1A upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that γH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21^CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.