thiostrepton As previously mentioned when hypoxia treated
As previously mentioned when hypoxia-treated cells become re-oxygenated they sustain a significant amount of DNA damage which has been attributed to the formation of reactive oxygen species . This finding represents more than just an interesting in vitro phenomenon as within a tumor, cells have been shown to cycle between hypoxic and oxic states. This has been elegantly demonstrated by Minchinton and colleagues through the use of a dye-based mismatch technique , . In brief, a tumor-bearing animal was initially injected with Hoechst 33342 and then 20min later with DiOC. Both dyes are fluorescent and can be easily visualized after tumor extraction and sectioning. The short-lived dyes diffuse from blood vessels and remain bound in the neighboring cells therefore marking the position of open blood vessels. shows that when the second dye was injected, a blood vessel had opened that was not functional 20min previously. Comet assay data have shown that cells that experience re-oxygenation from 0.02% oxygen to 20% oxygen sustain DNA damage. What remains to be elucidated is whether re-oxygenation within the physiological range of oxygen concentrations also induces a DNA-damaging effect. Given the significant level of damage seen in response to re-oxygenation in vitro it is not surprising to find ATM involved in the re-oxygenation response. Following re-oxygenation, p53 remained phosphorylated at serine 15 for 60–90min in the presence of ATM but quickly diminished in A-T cells. We would therefore predict that ATM undergoes auto-phosphorylation in response to re-oxygenation-induced DNA damage. The recent availability of phosphorylation-specific ATM thiostrepton will aid the investigation of ATM activity in tumor sections and in particular in regions surrounding recently re-opened vessels as shown in . Recent advances in ATM biology have uncovered a strong link between ATM and oxidative stress . Of particular interest here is the finding that ATM null cells are more susceptible to oxidative damage and accumulate more reactive oxygen intermediates in response to stress than wild-type cells . At present it is unclear whether the DNA damage observed in response to re-oxygenation occurs at random sites through the action of reactive oxygen species on DNA or, if damage occurs specifically at stalled replication forks. Future studies will determine if the DNA-damaging effects of re-oxygenation after hypoxia are made worse by the absence of ATM. We would predict that once activated in response to re-oxygenation damage ATM would mediate a cell-cycle arrest in order to ensure sufficient time to repair the damage.
The study of the ATM and ATR kinases is complicated by many factors including the absolute requirement for many of the key players and the high degree of redundancy between the two pathways. Due to this redundancy the doses of both DNA-damaging drugs and ionizing radiation used have become an important consideration . Despite the overlap between ATM- and ATR-mediated signaling, the critical fact remains that ATM is not sufficient to rescue ATR deficient cells . The finding that ATM does not compensate for a lack of ATR during embryogenesis suggests that during normal development cells are exposed to replication stress that activates ATR and not ATM. Severe hypoxia is a unique stress as cells undergo a rapid replication arrest but do not accumulate DNA damage even after extended periods of time. Hypoxia also represents an attractive area of study as it is physiologically relevant, occurring during both normal embryogenesis and tumor progression. Combined hypoxia, used as an initiating stress, provides a valuable tool with which to dissect ATR-mediated signaling.
Ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3 related) are protein kinases with partially overlapping functions that are key components of DNA damage response pathways . In response to DNA double strand breaks (DSBs) ATM becomes an active kinase that phosphorylates proteins involved in cell cycle arrest and DNA repair . In response to other types of DNA damage including UV damage and single strand breaks during DNA replication, ATR appears to play a similar role, phosphorylating a number of the same targets . ATM is mutated in the disease Ataxia-telangiectasia (A-T) , and A-T patients and mice display a variety of symptoms including chromosomal breaks and translocations, and a predisposition to cancer , , , , , , . Targeted disruption in mice leads to chromosomal fragmentation and early embryonic lethality . Consistent with their roles in DNA damage checkpoints, - and -deficient cells are sensitive to genotoxic challenges .