Characterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells

Abstract

Background: Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.

Results: By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced gammaH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.

Conclusion: Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.

Figure 1

Clonogenic sensitivity of PRR defective Δrad6 and Δrad18 yeast cells towards equitoxic doses of ICRF-187 and m-AMSA. A Δrad52 strain is included for comparison. Error-bars represent SEM of at least 3 experiments.

Figure 2

Analysis of gene expression by real time PCR. Real-time PCR was used to determine the expression of the DNA-damage inducible genes RNR3, HUG1, RAD51, and RAD54 by using the 2-^ΔΔCt method. Gene expression was normalized to that of the actin gene ACT1. It can be seen that exposure of yeast cells to m-AMSA results in higher levels of induction of transcription of these four genes than exposure to ICRF-187 when the two drugs are applied at equitoxic concentrations. Error-bars represent SEM of two independent experiments each performed in duplicate.

Figure 3

Assessing the clonogenic sensitivity of HR and NHEJ deficient and proficient hamster cells towards ICRF-187 and m-AMSA. To determine the sensitivity of the four cell lines AA8 (wild-type), irs1SF (recombination defective caused by non-functional XRCC3), CXR3 (recombination proficient due to ectopic expression of human XRCC3), and V3-3 (defective in NHEJ due to non-functional DNA-PK_cs) towards ICRF-187 and m-AMSA, a clonogenic assay with continuous drug exposure was used. Error-bars represent SEM of two independent experiments.

Figure 4

Assessing the effect of equitoxic concentrations of ICRF-187 and m-AMSA on the level of HR in SPD8 hamster cells. The SPD8 cell line has a defective hprt gene that can be repaired by HR. Panel A depicts the induction of HR induced by increasing concentrations of the two drugs. Panel B depicts the relative survival of cells exposed to similar concentrations of the two drugs. The data represented in Panel A and B was used to generate Panel C, where recombination frequency is plotted against the surviving fraction of cells. This data presentation allows a direct comparison of recombination levels at equitoxic concentrations of the two drugs. Representative data from one of three independent experiments is shown.

Figure 5

Assessing the effect of m-AMSA and ICRF-187 on γH2AX induction in human SCLC OC-NYH cells. To assess the level of DNA breaks in human cells after exposure to 10 μM m-AMSA and 1 mM ICRF-187 for increasing time points, total histones were isolated after incubation with the drugs. 10 μg of purified histones was then used in western blotting experiments. Panel A depicts a typical Western blot showing increased γH2AX induction with increasing drug incubation times. Panel B depicts fold γH2AX induction plotted against drug incubation time to analyse the kinetics of induction of DNA double strand breaks by the two drugs. Insert shows γH2AX induction from 0 to 2 hours for better resolution. Error-bars represent SEM of three independent experiments for ICRF-187 treatment and SEM of two independent experiments for m-AMSA treatment.