Role for hACF1 in the G2/M damage checkpoint.

Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.

Survival of tumor cells after proton irradiation with ultra-high dose rates.

BACKGROUND: Laser acceleration of protons and heavy ions may in the future be used in radiation therapy. Laser-driven particle beams are pulsed and ultra high dose rates of >109 Gy s⁻¹ may be achieved. Here we compare the radiobiological effects of pulsed and continuous proton beams. METHODS: The ion microbeam SNAKE at the Munich tandem accelerator was used to directly compare a pulsed and a continuous 20 MeV proton beam, which delivered a dose of 3 Gy to a HeLa cell monolayer within < 1 ns or 100 ms, respectively. Investigated endpoints were G2 phase cell cycle arrest, apoptosis, and colony formation. RESULTS: At 10 h after pulsed irradiation, the fraction of G2 cells was significantly lower than after irradiation with the continuous beam, while all other endpoints including colony formation were not significantly different. We determined the relative biological effectiveness (RBE) for pulsed and continuous proton beams relative to x-irradiation as 0.91 ± 0.26 and 0.86 ± 0.33 (mean and SD), respectively. CONCLUSIONS: At the dose rates investigated here, which are expected to correspond to those in radiation therapy using laser-driven particles, the RBE of the pulsed and the (conventional) continuous irradiation mode do not differ significantly.

Differences in the kinetics of gamma-H2AX fluorescence decay after exposure to low and high LET radiation.

PURPOSE: In order to obtain more insight into heavy ion tumour therapy, some features of the underlying molecular mechanisms controlling the cellular response to high linear energy transfer (LET) radiation are currently analysed. MATERIALS AND METHODS: We analysed the decay of the integrated fluorescence intensity of gamma-H2AX (phosphorylated histone H2AX) which is thought to reflect the repair kinetics of radiation-induced DNA double-strand breaks (DSB) using Laser-Scanning-Cytometry. Asynchronous human HeLa cells were irradiated with a single dose of either 1.89 Gy of 55 MeV carbon ions or 5 Gy of 70 kV X-rays. RESULTS: Measurements of the gamma-H2AX-intensities from 15-60 min resulted in a 16 % decrease for carbon ions and in a 43 % decrease for X-rays. After 21 h, the decrease was 77 % for carbon ions and 85 % for X-rays. The corresponding time-effect relationship was fitted by a bi-exponential function showing a fast and a slow component with identical half-life values for both radiation qualities being 24 +/- 4 min and 13.9 +/- 0.7 h, respectively. Apparent differences in the kinetics following high and low LET irradiation could completely be attributed to quantitative differences in their contributions, with the slow component being responsible for 47 % of the repair after exposure to X-rays as compared to 80 % after carbon ion irradiation. CONCLUSION: gamma-H2AX loss kinetics follows a bi-exponential decline with two definite decay times independent of LET. The higher contribution of the slow component determined for carbon ion exposure is thought to reflect the increased amount of complex DSB induced by high LET radiation.