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

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.

Induction of cytotoxic and genotoxic responses by natural and novel quercetin glycosides.

The flavonoids quercetin, and its natural glycosides isoquercetin and rutin, are phytochemicals commonly consumed in plant-derived foods. Semi-synthetic water-soluble isoquercetin and rutin glycosides, maltooligosyl isoquercetin, monoglucosyl rutin and maltooligosyl rutin were developed by synthetic glycosylation to overcome solubility challenges for improved incorporation in food and medicinal applications. Quercetin and its natural glycosides are known to induce genetic instability and decrease cell proliferation. Using a system of Chinese hamster ovary (CHO) cells, this study examined the differences in cytotoxic and genotoxic responses induced by natural and synthetic flavonoids. Bioactivity evaluations using poly(ADP-ribose) polymerase (PARP) ELISA showed that the synthetic flavonoids were less effective in inhibiting PARP than the natural flavonoids, where PARP inhibitory effects decreased with glycosylation of flavonoids. In the genotoxic studies, treatments with flavonoids at a concentration range of 0.2 µM-1 mM induced significant frequencies of sister chromatid exchange (SCE) and micronuclei in CHO cells compared to spontaneous occurrences. The synthetic flavonoids monoglucosyl rutin and maltooligosyl rutin induced less genotoxic effects than the natural flavonoids. However, maltooligosyl isoquercetin induced similar responses as isoquercetin and rutin. The growth inhibition studies showed glycosylation dependent cytotoxicity in natural flavonoids. The quercetin aglycone exhibited the highest toxicity out of all the flavonoids studied. Differences in growth inhibition were not observed between the synthetic flavonoids, maltooligosyl isoquercetin and monoglucosyl rutin, and natural isoquercetin and rutin, respectively. Maltooligosyl rutin induced less cytotoxicity than rutin and monoglucosyl rutin. Our in vitro studies demonstrated that the synthetic flavonoids generally induced less genotoxic responses than their natural counterparts.

Effects of targeted phosphorylation site mutations in the DNA-PKcs phosphorylation domain on low and high LET radiation sensitivity.

The present study investigated the effect of targeted mutations in the DNA-dependent protein kinase catalytic subunit and phosphorylation domains on the survival of cells in response to different qualities of ionizing radiation. Mutated Chinese hamster ovary V3 cells were exposed to 500 MeV/nucleon initial energy and 200 keV/µm monoenergetic Fe ions; 290 MeV/nucleon initial energy and average 50 keV/µm spread-out Bragg peak C ions; 70 MeV/nucleon initial energy and 1 keV/µm monoenergetic protons; and 0.663 MeV initial energy and 0.3 keV/µm Cs137 γ radiation. The results demonstrated that sensitivity to high linear energy transfer radiation is increased when both S2056 and T2609 clusters each contain a point mutation or multiple mutations are present in either cluster, whereas the phosphoinositide 3 kinase cluster only requires a single mutation to induce the sensitized phenotype of V3 cells. Additionally, the present study demonstrated that sensitivity to DNA cross-linking damage by cisplatin only requires a single mutation in one of the three clusters and that additional point mutations do not increase cell sensitivity.

Potentially lethal damage repair in drug arrested G2-phase cells after radiation exposure.

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2.