p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

Abstract

This study provides evidence for the importance of p21 CDKN1A for the 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 (cH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, cH2AX reverses after camptothecin removal. By contrast, cH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53À/À) or p21 CDKN1A (p21À/À) as the cells reach the late-S and G2 phases. Since p21À/À cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced cH2AX formation in p21À/À cells, we conclude that enhanced cH2AX formation in p21À/À cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced cH2AX 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 cH2AX 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 p21CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

“…5D). An earlier study also showed increased H2AX phosphorylation after exposure to UCN-01 of cells previously treated with the topoisomerase I inhibitor camptothecin (42). Together, these results confirm the importance of an intact S-phase checkpoint for replication fork stabilization and cellular recovery from DNA-targeted therapeutics.…”

Dose–response transition from cell cycle arrest to apoptosis with selective degradation of Mdm2 and p21WAF1/CIP1 in response to the novel anticancer agent, aminoflavone (NSC 686288)

“…We also find (Figure 4) that under those conditions, Akt is activated, which may in turn phosphorylate and stabilize Mdm2 and possibly also stabilize p21 CIP1/WAF1 . Accumulation of p21 CIP1/WAF1 , which acts as a cyclin-dependent kinase (CDK) inhibitor, would then lead to cell cycle arrest, DNA repair and inhibit apoptosis (Furuta et al, 2006). Accumulation of Mdm2 may also inhibit p53-mediated transcriptional activation of the proapoptotic genes.…”

“…Altogether, these results suggest that, as the AF concentration and DNA damage increase, inactivation of Akt and subsequent reduction of Mdm2 phosphorylation may destabilize Mdm2. p21 CIP1/WAF1 belongs to the CIP/KIP family of CDK inhibitors and mediates cell cycle arrest in response to stimuli such as DNA damage (Dotto, 2000;Takimoto and El-Deiry, 2001;Furuta et al, 2006). p21 CIP1/WAF1 is transcriptionally activated by p53 and its accumulation is a key component of the p53-mediated G1/S arrest in response to DNA damage.…”

Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

“…52 Our results agree with many others showing that CHK1 inhibition sensitizes cells to DNA damaging agents, including cisplatin in non-small cell lung carcinoma cells 53 and camptothecin in colon cancer cells. 54 An attractive idea has been that some cancer cells are selectively sensitive to ATR/CHK1 inhibition in combination with DNA damaging agents, perhaps because of loss of the p53-dependent G 1 checkpoint. 55 Although our results indicate that loss of ATR/ CHK1 or inhibition of CHK1 does not increase UV-induced mutagenesis, it remains to be determined if this would hold true for other DNA-damaging chemotherapeutics.…”