Validation of 64Cu-ATSM damaging DNA via high-LET Auger electron emission

Abstract

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a (64)Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of (64)Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from (64)Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair-deficient xrs5 cells were exposed to (64)Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D(10) values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that (64)Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/μm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from (64)Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after (64)Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that (64)Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of (64)Cu-ATSM as a cancer treatment modality for hypoxic tumors.

Keywords: 64Cu-ATSM; CHO; DNA double strand break; auger electron; high LET.

Fig. 1.

⁶⁴Cu-ATSM uptake and survival curves. (A) ⁶⁴Cu-ATSM uptake in CHO wild-type and xrs5 cells. (B) Survival curves of CHO wild-type and xrs5 cells exposed to radiation of varying LETs. (C) Cell-survival curves of CHO wild-type and xrs5 cells exposed to ⁶⁴Cu-ATSM and incubated in hypoxia. Error bars indicate standard errors of the mean of at least three independent experiments.

Fig. 2.

γH2AX foci formation after ⁶⁴Cu-ATSM exposures. (A) Dose response of γH2AX foci formation after ⁶⁴Cu-ATSM exposures. Foci response of cells incubated with varied activities normalized to background. Data points are the mean of at least three experiments. Error bars represent the standard error of the mean. (B) Examples of γH2AX foci formation in CHO wild-type and xrs5 cells without irradiation and after 0.84 and 0.83 Bq/cell of ⁶⁴Cu-ATSM exposure. Arrows indicate cluster foci.

Fig. 3.

Representative chromosome spreads for CHO wild-type and xrs5 cells in control group and exposed to 0.84 Bq/cell and 0.83 Bq/cell of ⁶⁴Cu-ATSM, respectively. Arrows indicate chromatid-type aberrations in CHO wild-type cells after exposure to ⁶⁴Cu-ATSM. On the other hand, damage in xrs5 was extensive enough to prohibit evaluation of definitive chromosomal aberrations after exposure to 0.83 Bq/cell of ⁶⁴Cu-ATSM.