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.

Comparative study of radioresistance between feline cells and human cells.

Radioresistance of cats has been seen in animal radiotherapy. Feline radioresistance and its underlying mechanism(s) were investigated in fibroblast cells and lymphocytes. We hypothesized that radioresistance was attributable to an increase in the cells ability to repair DNA damage. To investigate this hypothesis, fibroblast cells were exposed to various doses of X rays and then colony formation assays were performed. Survival curves showed that potential lethal damage repair (PLDR) for feline cells were greater than that for human cells. γ-H2AX foci assays were performed to evaluate DNA double-strand breaks (DSBs) formation and repair kinetics. After PLDR, feline cells displayed a decreased residual amount of γ-H2AX foci. Formation of chromosome aberrations (dicentrics) after PLDR as an indicator of radiation-induced DNA damage and repair; human, feline and canine lymphocytes were evaluated. Human and canine lymphocytes showed two to three times the number of dicentrics compared to feline lymphocytes. Finally, micronuclei assays were performed to further confirm the radioresistant nature of feline lymphocytes. In concordance with the results of the chromosome aberration assay, the number of micronuclei in feline lymphocytes was less than observed in human and canine lymphocytes. Taken together, these results show that DNA and chromosome damage induced by X irradiation is more effectively repaired in feline cells, resulting in less residual damage. Our results suggest that both feline fibroblasts and lymphocytes are more radioresistant compared to human cells of similar tissues, and this resistance can be contributed, at least in part, to greater ability for PLDR.

Influence of track directions on the biological consequences in cells irradiated with high LET heavy ions.

PURPOSE: The impact of the damage distribution to cellular survival and chromosomal aberrations following high Linear Energy Transfer (LET) radiation was investigated. MATERIALS AND METHODS: High LET iron-ions (500 MeV/n, LET 200 keV/µm) were delivered to G1-phase synchronized Chinese Hamster Ovary (CHO) cells located at a vertical or horizontal angle relative to the ion beam in order to give same dose but different fluence and damage distribution. RESULTS: Horizontal irradiation produced DNA double-strand break (DSB) along each ion track represented as clustered lines, and vertical irradiation produced a greater fluence. The initial damages measured by premature chromosome condensation were equal per dose in both irradiation types. Horizontal irradiation proved to be less effective in cell killing than vertical at doses of more than 3 Gy. Vertical irradiation produced a higher number of metaphase chromosomal aberrations compared to horizontally irradiated samples. In particular, formation of exchange-type aberrations was the same, but that of deletion-type aberrations were significantly higher after vertical irradiation than horizontal irradiation. CONCLUSIONS: Therefore, we concluded that high fluence per dose is more effective than low fluence per dose to produce radiation-induced chromosomal aberrations and to kill exposed cells following high LET heavy-ion exposure.

Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time- and temperature-dependent manner.

Hyperthermia has long been known as a radio-sensitizing agent that displays anti-tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia-induced radio-sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild-type and non-homologous end joining repair mutants, cells in late S phase showed double the amount heat-induced radio-sensitization effects of G1-phase cells. Both radiation-induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2-phase cells than G0/G1 cells. Additionally, G2-phase cells took approximately two times as long to repair DNA damage over time than G0/G1-phase cells. To supplement these findings, radiation-induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re-formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia-induced radio-sensitization.

Comparison of cellular lethality in DNA repair-proficient or -deficient cell lines resulting from exposure to 70 MeV/n protons or 290 MeV/n carbon ions.

Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

Genomic instability and telomere fusion of canine osteosarcoma cells.

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.