Understanding the regulation of endogenous mutagenesis

There are a number of cellular mechanisms conserved across eukaryotic organisms that prevent or reduce spontaneous mutation rates and maintain genomic stability. Inactivation of processes that reduce the spontaneous mutation rate, can lead to carcinogenesis and ageing. DNA mismatch repair (MMR) proteins increase the fidelity of DNA replication by several orders of magnitude by correcting mismatches and frameshift mutations generated during DNA replication. Mutations in MMR genes are associated with Lynch Syndrome, a cancer predisposition syndrome characterized by elevated mutation rates. In order to identify gene deletions which, show additive effect when combined with lower MMR activity (hypomorphic MMR), a genome-wide screen was carried out in S. cerevisiae. The genome-wide screen identified more than 163 deletion strains (3.2% of the total number of strains screened), whose deletion resulted in increased mutation rates in MMR compromised cells which suggests their role in compensating for defective MMR. The 163 genes are enriched in multiple biological processes such as DNA repair, DNA replication etc. The screen also revealed 543 deletion strains (10.6% of the total strains screened), which showed a decrease in mutation rate in hypomorphic MMR cells, suggesting mutation rate can also go down when MMR is not functional in cells. The genes resulting in lower mutation rate were enriched in biological processes, including protein metabolism and transport, general cell metabolism and growth. Interestingly, deletion of genes involved in regulation of transcription coupled repair resulted in lower mutation rate, suggesting their potential role in driving mutagenesis

Gold amides as anticancer drugs: synthesis and activity studies

Modular gold amide chemotherapeutics: Access to modern chemotherapeutics with robust and flexible synthetic routes that are amenable to extensive customisation is a key requirement in drug synthesis and discovery. A class of chiral gold amide complexes featuring amino acid derived ligands is reported herein. They all exhibit in vitro cytotoxicity against two slow growing breast cancer cell lines with limited toxicity towards normal epithelial cells

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

<div><p>Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.</p></div

Variation in the length and number of tracks between individual flat fibroblast cells.

<p>(A) γH2AX foci in 48BR (WT) primary and 2BN (XLF) hTERT cells were enumerated from 0.5–24 h post 1 Gy X-rays. Foci were scored in 2D using a Zeiss Axioplan microscope. (B) 48BR (WT) cells were fixed at 30 min following 1 Gy Fe ions and stained with γH2AX and DAPI. Asterisks represent non-track cells. (C) Distribution in the percentage of γH2AX tracks following Fe irradiation in B. (D) The average number of tracks per cell following 1 Gy Fe is shown. The predicted fluence of particles traversing the nuclease after 1 Gy horizontal Fe irradiation was estimated to be ∼1.1 with a Poisson distribution predicting and ∼70% cells receiving a particle track (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#s2" target="_blank">Materials and Methods</a>). (E) Scatter plots of track length per cell following 1 Gy Fe irradiation are shown in 48BR (WT) and 2BN (XLF) G0/G1 cells. Tracks whose length are <0.025 were excluded from the data, since they were indistinguishable from delta-electron induced foci. ∼250 cells were examined in each analysis (C–E). (F) Clustered γH2AX foci within the tracks is ATM/DNA-PK dependent, indicating that they are DSBs. 48BR (WT) cells were fixed at 30 min post Fe irradiation with/without ATM plus DNA-PK inhibitor. To examine the percentage of track positive cells, >200 cells were scored. Error bars represent the standard deviations (SD) from 2 experiments. The analysis was performed by DeltaVision microscope without deconvolution (B–F). Note that although the cells were exposed to 1 Gy Fe ions, the dose to individual cells can differ due to differing number of particles traversing the cell. In the ensuing analysis, we examine cells which have a single particle traversal.</p

Clustered γH2AX foci arising within the particle tracks represent a signature of high LET particle radiation and are repaired slowly by NHEJ in G1.

<p>(A) 48BR (WT) primary and 2BN (XLF) hTERT G0/G1 cells were irradiated in a horizontal direction with 1 Gy Fe irradiation. Images are taken using the DeltaVision microscope followed by deconvolution. Representative images at 8 and 24 h post Fe irradiation are shown. The nucleus outline is drawn with a dashed line from the DAPI staining. (B, C) Percentage of individual foci within a cluster was analysed from >100 individual clusters at each time point. Similar results were obtained in two independent experiments. Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed.</p

Generation of “simple” γH2AX foci at distances away from the particle track and they are repaired rapidly by NHEJ.

<p>(A) Representative image of γH2AX foci formation at non-track regions. 48BR (WT) primary and 2BN (XLF) hTERT G0/G1 cells were irradiated with 1 Gy pencil Fe ions and stained with γH2AX and DAPI. (B) γH2AX foci at non-track regions in cells which have a single track per nucleus were enumerated following 1 Gy Fe ion irradiation. (C) To investigate whether non-track induced γH2AX foci formation is due to DSBs, the number of γH2AX foci at non-track regions was enumerated with/without ATM plus DNA-PK inhibitor. Since ATM/DNA-PK inhibitor treated cells do not form γH2AX tracks, foci number was enumerated without any bias in these cells. (D) Distribution in the percentage of γH2AX tracks following 0.1 Gy Fe irradiation. (E, F) γH2AX foci at non-track regions with a single track per nucleus was examined following 0.1 Gy Fe irradiation. A region which is located greater than 2 µm from the track was excluded from the analysis (B and F). Images were taken by Olympus BX51 microscope without deconvolution (A and E). γH2AX foci were analysed using Olympus BX51 or Zeiss Axioplan microscope by 2D (B, C, D and F). Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed in B and F.</p

High resolution microscope analysis revealed clustered γH2AX foci formation within the tracks following Fe irradiation.

<p>(A, B) Clustered γH2AX foci formation in 48BR (WT) primary G0/G1 cells is visualised using deconvolution. Images were captured with an Applied Precision DeltaVision RT Olympus IX70 microscope with deconvolution (right). The image resolution using the DeltaVision is compared with Zeiss Axioplan microscope without deonvolution (left). Enlarged individual clustered foci are shown with grey scale in B.</p

Cells with clustered γH2AX foci can enter mitosis despite prolonged checkpoint arrest following exposure to Fe ion irradiation.

<p>A) Mitotic entry was examined in 1BR (WT) hTERT cells at 1 h post X-rays or vertical Fe irradiation. Cells, >400, were scored for mitotic index (MI). Mitotic cells were identified by p-histone H3 Ser10 and cellular morphology by DAPI. Results represent the MI relative to untreated cells. 1BR hTERT cells shows similar G2/M checkpoint responses to that in 48BR primary cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#pone.0070107-Brunton1" target="_blank">[40]</a>. The size and number of γH2AX foci clusters between 48BR primary and 1BR hTERT cells were similar (data not shown). (B) The time of mitotic entry was examined following exposure to 1 Gy X-rays, Fe or Carbon ion irradiation. (C) The number of clustered γH2AX foci in G2 cells was enumerated by 2D analysis using normal microscopy following 1 Gy X-rays, Fe or Carbon ion irradiation. G2 cells were identified by CENP-F staining. (D) Since irradiated G2 cells restart cell cycle progression from 16 h post heavy ion irradiation, the number of DSBs in mitotic cells at 16 and 24 post IR was examined by scoring γH2AX foci in p-histone H3 Ser10 positive M phase cells. (E) Typical image of γH2AX foci in metaphase 1BR hTERT cells were taken by DeltaVision 3D imaging. (F) The maintenance of checkpoint arrest was examined following 0.5, 2 Gy Carbon or 2, 3 Gy X-rays. (G) γH2AX foci in G2 cells were enumerated following 2 Gy Carbon or X-rays. Mitotic index and γH2AX foci in G2 or M cells were examined by 2D analysis using Olympus BX51 or Zeiss Axioplan microscope.</p

Track structure simulation and spatial distribution of DSBs arising from an Fe ion track.

<p>A Simulation of the structure of damage arising from a single Fe ion particle traversal. The black marks and the red marks represent the tracks of Fe ions and the position of DSBs, respectively. The blue ellipsoid shape shows the size of the nucleus. The slighter wider band of the damage within the tracks observed in these experiments is likely explained by DNA movement after radiation exposure. The number of electrons generated decreases as the electron energy decreases. For 416 MeV/n Fe ions, ∼90% of electrons generated are estimated to have energies less than 100 eV. The range of such low-energy electrons is less than a few nm. The majority of other electrons have a range from a nm to 5 µm. The highest energy electrons with a range of a few hundred µm can also arise but at a low frequency (<0.1%).</p