Hypoxia induced replication arrest has been

Hypoxia-induced replication arrest has been demonstrated in a variety of organisms in addition to mammals; including Zebrafish , , and (brine shrimp) which have been shown to survive for 4 years and longer in anoxic conditions . Despite this breadth of study, little is known about the mechanism of hypoxia-induced replication arrest and in particular its genetic regulation. To date no gene has been described as having a role to play in hypoxia-induced S-phase arrest, and many have been excluded, including ATM, Hif 1α, p53 and p21 (Hammond and Giaccia, unpublished observations) . Hypoxia-induced replication arrest has been attributed to a depletion of ribonucleotide pools in hypoxic cells. Two oxygen-dependent enzymes are believed to be responsible for this depletion, dihydroorotate dehydrogenase and ribonucleotide reductase . In support of this hypothesis, studies have demonstrated that by adding nucleosides to hypoxic cell cultures, replication can re-start. This finding is however somewhat controversial in part because it is unclear if the effect is on arrested S- or G1-phase cells. Some reports conclude that by adding ribonucleosides, BMS 193885 previously arrested in G1 can now enter S-phase, suggesting that the oxygen-dependent depletion of ribonucleotide pools is one of the mechanisms by which hypoxia-treated G1 cells are prevented from progressing into S-phase . This decrease in ribonucleotide pools along with hypoxia-induced inhibition of translation led to the conclusion that hypoxia causes a shutdown of replicon initiation , . These reports show that while replicons do not fire under hypoxic conditions, replicons that had initiated prior to treatment continue normally. Hypoxia-induced replication arrest can be very rapid (1–2h) or can continue for 6–7h under hypoxic treatment . As previously noted many of these discrepancies arise from variations in oxygen concentrations and cell types. For example, some studies have used Ehrlich ascites cells which are known to be extremely resistant to hypoxia , . Whilst the detailed mechanism of hypoxia-induced S-phase arrest still requires further investigation, the checkpoint response initiated by replication arrest has become clearer. The recent discoveries of Zou and Elledge demonstrating that ATRIP recognizes complexes of RPA and single-stranded DNA (ssDNA) provide a mechanism by which ATR/ATRIP respond to stalled replication. Severe hypoxia results in regions of ssDNA within S-phase nuclei (). This assay is performed by labeling cells with BrdU for one cell division before treatment with hypoxia. BrdU staining is then visualized in the absence of a DNA-denaturation step allowing anti-BrdU antibodies access only to regions of single-stranded DNA, shown schematically in part A. Thus, severe hypoxia causes a stalling of replication forks as single-stranded regions of DNA would not be generated. It seems probable that the decrease in DNA synthesis observed in response to hypoxia is multi-faceted, and results from both a failure of replicons to initiate and the stalling of previously active replication forks. Each of the ATM/ATR substrates shown to be phosphorylated in response to hypoxia has been studied in great depth in response to stresses such as ionizing radiation. In contrast, comparatively little is known about the function of these pathways in response to hypoxia. The p53 tumor suppressor gene is one of, if not the most investigated genes or proteins and yet its action in response to hypoxia is still unclear. In response to hypoxia, p53 is phosphorylated by ATR and as a result accumulates in the nucleus. The co-ordinate decrease in mdm2 levels in human cells may also have a role to play in this accumulation . Once stabilized in hypoxic cells, p53 is able to induce apoptosis through a mitochondrial signaled pathway involving caspase 9 and caspase 3, in sensitive cell backgrounds but seems to have no effect on the cell cycle . The other hypoxia-induced ATR targets are generally believed to respond to DNA damage. However hypoxia is a unique physiologically relevant stress that does not induce DNA damage detectable by comet assay. This is in direct contrast to other stresses known to induce ATR activity such as hydroxyurea, aphidicolin or UV, all of which can induce comet-detectable DNA damage in a dose-dependent manner. The lack of hypoxia-induced DNA damage is an important finding that will impact future studies. Ideally this finding should be verified with alternatives to the comet assay. However when hypoxic cells are re-oxygenated they undergo substantial levels of DNA damage (roughly equivalent to 5Gy ionizing radiation) therefore limiting us to methods that can be carried out under hypoxia .