Data on the DNA damaging and mutagenic potential of the BH3-mimetics ABT-263/Navitoclax and TW-37

Unfortunately, the mutagenic activities of chemotherapy and radiotherapy can provoke development of therapy-induced malignancies in cancer survivors. Non-mutagenic anti-cancer therapies may be less likely to trigger subsequent malignant neoplasms. Here we present data regarding the DNA damaging and mutagenic potential of two drugs that antagonize proteins within the Bcl-2 family: ABT-263/Navitoclax and TW-37. Our data reveal that concentrations of these agents that stimulated Bax/Bak-dependent signaling provoked little DNA damage and failed to trigger mutations in surviving cells. The data supplied in this article is related to the research work entitled "Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells" [1].


a b s t r a c t
Unfortunately, the mutagenic activities of chemotherapy and radiotherapy can provoke development of therapy-induced malignancies in cancer survivors. Non-mutagenic anti-cancer therapies may be less likely to trigger subsequent malignant neoplasms. Here we present data regarding the DNA damaging and mutagenic potential of two drugs that antagonize proteins within the Bcl-2 family: ABT-263/Navitoclax and TW-37. Our data reveal that concentrations of these agents that stimulated Bax/Bakdependent signaling provoked little DNA damage and failed to trigger mutations in surviving cells. The data supplied in this article is related to the research work entitled "Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells" [1].

How data was acquired
Clonogenicity assays, HPRT mutagenesis assays, γH2AX flow cytometry quantitation

Data format
Normalized data Experimental factors Murine embryonic fibroblasts from wild type or Bax À / À , Bak À / À mice were treated with various concentrations of ABT-263 or TW-37, prior to the assays listed above. Experimental features Cells were exposed to drugs (or not) for various periods of time, then washed and either stained with anti-γH2AX for flow cytometry, seeded into normal media to quantitate clonogenicity, or seeded into media containing 6thioguanine to count 6-thioguanine-resistant clones (presumably reflecting mutagenesis at the HPRT locus).

Data source location
La Trobe University, Bundoora, Australia Embryonic fibroblasts derived from wildtype or Bax/Bak-deficient mice were treated with ABT-263 ( Fig. 1) or TW-37 (Fig. 2), or incubated in normal medium. We measured the impact of these treatments on survival, DNA damage and mutagenicity at the HPRT locus.

Cell survival assays
The toxicity of the drugs was assessed by comparing the clonogenic survival of treated and untreated cells. Cells were incubated with drugs, then washed and seeded at various densities in 6well plates. After seven days, cells were stained with methylene blue (Sigma Aldrich) 1.25 g/l in 50% methanol, incubated for 5 min and washed twice with water, then the numbers of colonies were counted.

HPRT assay
To evaluate the ability of the drugs to provoke mutations in clonogenically-competent cells, we quantitated the emergence of colonies in media containing 6-thioguanine (6-TG), using a previously published method [1,3]. This purine analog is toxic to cells expressing functional hypoxanthine-guanine phosphoribosyltransferase (HPRT) and growth of colonies in 6-TG following drug treatment usually results from drug-induced loss-of-function mutations at the HPRT locus [4]. Cells were incubated with the drug or normal media, then washed and incubated in normal media for eight days to allow for expression of the HPRT mutant phenotype. Cells were then seeded at 10 5 cells per 150 mm dish (three dishes per treatment) in media containing 6-thioguanine (Sigma Aldrich). Colonies were stained with methylene blue and counted after 13 days. The number of 6-TG-resistant colonies following drug treatment was calculated by subtracting the number of background colonies that arose in untreated samples.

DNA damage assay
Drug-induced DNA damage was examined by measuring the proportion of cells bearing phosphorylated H2AX, which typically occurs in response to double-stranded DNA breaks [5]. Staining of phosphorylated H2AX was based on a published protocol [6]. Cells were exposed to drugs then stained using anti-phosphorylated H2AX and anti-rabbit FITC antibodies. After washing to remove unbound antibodies, the cells were resuspended in PBS containing PI (5 mg/ml) to limit the analysis to permeabilized intact cells (excluding cells with sub-G1 DNA content). The γH2AX signals from these cells were analyzed using a FACS Canto (BD Biosciences).