br Loss of Chk sensitizes cells to
Loss of Chk 1 sensitizes NT157 to hypoxia/reoxygenation In contrast to Chk 2, studies have identified Chk 1 as being phosphorylated in an ATR-dependent manner at residue serine 345 during hypoxia . More recently, siRNA mediated inhibition of Chk 1 signaling was found to sensitize cells exposed to hypoxia/reoxygenation . This has also been confirmed by using the specific Chk 1 inhibitor, CEP-3891 (Cephalon Inc.). Cells treated with this inhibitor during hypoxia/reoxygenation are significantly more sensitive than untreated cells, Fig. 1. We have attributed this sensitivity to the finding that even partial loss of ATR, which activates Chk 1, sensitizes cells to hypoxia/reoxygenation. This sensitivity was associated with an increased accumulation of DNA-damage in the S-phase cells treated with hypoxia when ATR levels were reduced . It is significant that this increase in damage was observed in the absence of any reoxygenation i.e. hypoxia alone, in the presence of unperturbed ATR protein levels we see no DNA-damage during hypoxia. These data and those of others strongly indicate a role for both ATR and Chk 1 in the preservation/stabilization of stalled replication forks to maintain genome instability , , .
Chk 1 and Chk 2 as therapeutic targets Recently the inhibition of the G2 checkpoint has become the focus of drug discovery (reviewed in , ). This is largely a result of the finding that the majority of cancerous cells have mutations in the G1 checkpoint and are heavily reliant on the G2 checkpoint during replication. Small molecules such as CEP-3891 or CEP-6367, which inhibit both Chk 1 and Chk 2, have yet to be tested in clinical trials, but may well prove to be effective inhibitors of the G2 checkpoint. The results of such activity are, however, difficult to predict and may be complicated by the non-specific activities of the drugs and additional functions of Chk 1 and Chk 2. As previously mentioned, inhibition of Chk 1 sensitizes cells to hypoxia/reoxygenation, in accordance with other studies which show inhibition of Chk 1 sensitizes cells to ionizing radiation with CEP-3891 , . Recently, Chk 1 has been demonstrated to have alternate roles to its checkpoint function, including a role during normal S phase and homologous recombination , . Using CEP-3891 Syljuasen et al., demonstrated that Chk 1 was required to prevent aberrant initiation of DNA replication, thus preventing the accumulation of DNA-damage . Both ATR and Chk 1 have also been demonstrated to prevent replicon initiation after exposure to UVC induced DNA-damage . We have investigated a role for Chk 1 in the rapid and robust S-phase arrest induced by severe hypoxia and found that cells with siRNA depleted levels of Chk 1 arrested with the same kinetics as untreated cells . It is our hypothesis that the S-phase arrest induced during hypoxia occurs during both the initiation and elongation phases of replication . It is therefore possible that experiments designed to investigate the inhibition of initiation specifically during hypoxia/reoxygenation may reveal a role for Chk 1 (Fig. 2).
Chk 1, homologous recombination and replication re-start Homologous recombination and in particular Rad 51 has been demonstrated to be required for resolving stalled and collapsed replication forks . This finding has led to the hypothesis that the process of homologous recombination is required for replications re-start in response to reoxygenation in hypoxia-arrested cells. This hypothesis is reliant, of course, on the hypoxia-arrested S-phase cells actually undergoing replication re-start and not entering a quiescent/senescent (S0) state or indeed undergoing apoptosis. Experiments aimed at investigating this have been in part complicated by the contribution of G1 cells which begin to enter S-phase approximately 3h after reoxygenation . In general, the rate of homologous recombination increases in response to treatments that induce an S-phase arrest, for example hydroxyurea . However, it was recently demonstrated that both Rad 51 and homologous recombination were decreased in hypoxic cancer cells , . The down-regulation of Rad 51 was found to be mediated through repression of the Rad 51 promoter and was independent of both the cell cycle and HIF-1 status . Interestingly, the levels of Rad 51 were found to remain low for considerable time periods after reoxygenation (up to 72h), indicating that, if arrested S-phase cells undergo replication re-start it is not dependent on Rad 51. We have also observed a rapid loss of Rad 51 protein during exposure to severe hypoxia (0.02% O2). This is of particular relevance to the role of Chk 1 in hypoxia/reoxygenation, as Chk 1 was recently found to complex with Rad 51 and to be required for homologous recombination induced by hydroxyurea . Therefore, while Chk 1 may provide the link between ATR and Rad 51 in response to treatment with DNA-damaging drugs it does not seem to be the case during the physiological stress of hypoxia. We have also determined that that the levels of Brca 1 decrease during hypoxia, indicating that multiple components of the homologous recombination pathway are affected by hypoxia (unpublished data). However, the Nbs 1 protein, which has also been demonstrated to be essential for homologous recombination, remains at normoxic levels in hypoxia treated cells and is robustly phosphorylated , .