Spatio-temporal analysis of DNA damage repair using the X-ray microbeam

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

Low-dose hypersensitivity in Chinese hamster V79 cells targeted with counted protons using a charged-particle microbeam

The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-mum. silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-mum-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-mum-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below I Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques. (C) 2001 by Radiation Research Society.

The addition of arsenic trioxide to low-dose Ara-C in older patients with AML does not improve outcome

Most patients with acute myeloid leukaemia (AML) are older, with many unsuitable for conventional chemotherapy. Low-dose Ara-C (LDAC) is superior to best supportive care but is still inadequate. The combination of arsenic trioxide (ATO) and LDAC showed promise in an unrandomised study. We report a randomised trial of LDAC versus LDAC + ATO. Patients with AML according to WHO criteria or myelodysplastic syndrome with > 10% blasts, considered as unfit for conventional chemotherapy, were randomised between subcutaneous Ara-C (20mg b.d. for 10 days) and the same LDAC schedule with ATO (0.25 mg/kg) on days 1-5, 9 and 11, for at least four courses every 4 to 6 weeks. Overall 166 patients were entered; the trial was terminated on the advice of the DMC, as the projected benefit was not observed. Overall 14% of patients achieved complete remission (CR) and 7% CRi. Median survival was 5.5 months and 19 months for responders (CR: not reached; CRi: 14 months; non-responders: 4 months). There were no differences in response or survival between the arms. Grade 3/4 cardiac and liver toxicity, and supportive care requirements were greater in the ATO arm. This randomised comparison demonstrates that adding ATO to LDAC provides no benefit for older patients with AML. Leukemia (2011) 25, 1122-1127; doi:10.1038/leu.2011.59; published online 8 April 2011