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Synchrotrons are capable of producing intense low-energy X-rays that enable the photoactivation of high-Z elements. Photoactivation therapy (PAT) consists of loading tumors with photoactivatable drugs and thereafter irradiating them at an energy, generally close to the K-edge of the element, that enhances the photoelectric effect. To date, three major photoactivatable elements are used in PAT: platinum (cisplatin and carboplatin), iodine (iodinated contrast agents and iododeoxyuridine) and gadolinium (motexafin gadolinium). However, the molecular and cellular events specific to PAT and the radiobiological properties of these photoactivatable drugs are still misknown. Here, it is examined how standard and synchrotron X-rays combined with photoactivatable drugs impact on the cellular response of human endothelial cells. These findings suggest that the radiolysis products of the photoactivatable drugs may participate in the synergetic effects of PAT by increasing the severity of radiation-induced DNA double-strand breaks. Interestingly, subpopulation of highly damaged cells was found to be a cellular pattern specific to PAT. The data show that the efficiency of emerging anti-cancer modalities involving synchrotron photoactivation strongly depends on the choice of photoactivatable drugs, and important series of experiments are required to secure their clinical transfer before applying to humans.

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations., (©2010 AACR.)

Severe hypoxia has been demonstrated to induce a replication arrest which is associated with decreased levels of nucleotides. Chk1 is rapidly phosphorylated in response to severe hypoxia and in turn deactivates TLK1 through phosphorylation. Loss of Chk1 has been shown to sensitize cells to hypoxia/reoxygenation. After short (acute) exposure to hypoxia this is due to an increased rate of reoxygenation-induced replication restart and subsequent p53-dependent apoptosis. After longer (chronic) exposure to hypoxia S phase cells do not undergo reoxygenation-induced replication restart. Cells exposed to these levels of hypoxia however are sensitive to loss of Chk1. This suggests a new role for Chk1 in the cell cycle response to reoxygenation., (© 2010 Landes Bioscience)

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia.

The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.

Synchrotrons are opening new paths in innovative anti-cancer radiotherapy strategies. Indeed, the fluence of X-rays induced by synchrotrons is so high (10(6) times higher than standard medical irradiators) that it enables the production of X-ray beams tunable in energy (monochromatic beams) and in size (micrometric beams). Monochromatic synchrotron X-ray beams theoretically permit photoactivate high-Z elements to be introduced in or close to tumours in order to increase the yield of damage by enhanced energy photoabsorption. This is notably the case of attempts with iodinated contrast agents used in tumour imaging (the computed tomography therapy approach) and with platinated agents used in chemotherapy (the PAT-Plat approach). Micrometric synchrotron X-ray beams theoretically permit very high radiation doses to accumulate in tumours by using arrays of parallel microplanar beams that spare the surrounding tissues (the microbeam radiation therapy approach). These anti-cancer applications of synchrotron radiation have been developed at the European Synchrotron Radiation Facility to be applied to glioma, one of the tumour tissues most refractory to standard treatments. In the present paper the molecular and cellular mechanisms involved in these three approaches are reviewed, in the context of recent advances in radiobiology. Furthermore, by considering the unavoidable biases, an attempt to propose a comparison of the different results obtained in preclinical trials dealing with rats bearing tumours is given.