Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1-3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose-survival curves is included.

Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5°C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized ondemand induction of HR deficiency, an approach that we term induced synthetic lethality.anti-cancer treatment | RAD51 | double-strand break M any anti-cancer therapies are based on cytotoxicity of DNA double strand breaks (DSBs) induced by ionizing radiation or, indirectly, by chemical agents. However, efficient DSB repair mechanisms protect cells from the genotoxic effects of DSBs, thereby reducing the effectiveness of the therapies. Two major pathways are involved in DSB repair in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR uses intact homologous DNA sequences, usually the sister chromatid in postreplicative chromatin, to faithfully restore DNA breaks (1), whereas NHEJ operates throughout the entire cell cycle and does not require a DNA template (2). Agents inhibiting DNA repair processes potentiate the cytotoxicity of DSBs in cancer therapy (3). Elevated temperature is one such agent that, via unclear mechanisms, interferes with multiple pathways of DNA repair (4-6) and is clinically applied (7). ResultsTo investigate if HR, among other processes and DSB repair pathways, is influenced by elevated temperature, we used an isogenic set of mouse embryonic stem (ES) cells that are either HR proficient (wild-type) or HR deficient (Rad54 −/− ) due to the disruption of the Rad54 gene, which is important for HR activity (1). We compared radiosensitization of these cells by incubating them at 37°C or 41°C before irradiation. For this and subsequent experiments we chose temperatures below 43°C, because they are relevant in clinical practice (8). Interestingly, we observed that wild-type but not Rad54 −/− cells were radiosensitized by preincubation at 41°C compared with cells incubated at 37°C (Fig. 1A). Similarly, HeLa cells, in which the important HR factors XRCC3 or BRCA2 were down-regulated using siRNA, were refractory to further temperature-mediated radiosensitization (Fig. 1B and Fig. S1). These results suggest that elevated temperature inactivates HR. To directly measure the effect of temperature on HR, we quantitated HR-mediated gene targeting in ES cells (9) and found that the efficiency of gene targeting was significantly reduced by preincubation at 41°C compared with 37°C (Fig. 1C). Similarly, preincubation at 41°C reduced the frequency of spontaneous and mitomycin C-induced sister chromatid exchanges in SW-1573 cells (Fig. S2A), w...

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.