In Situ DNA Damaging Foci Analysis on Metaphase Chromosomes.

DNA damage foci such as ionizing radiation-induced foci (IRIF) can visually distinguish the location and number of specific types of DNA damages. This method is widely used to detect DNA damage in interphase cells. These DNA damage foci can be also visualized on metaphase chromosomes. The technique has an advantage as it provides an easy method of quantifying chromosomal DNA damage. Radiation-induced DNA double strand breaks can be assessed for gamma-H2AX foci formation on metaphase chromosomes.Gamma-H2AX foci can be observed at the break point of chromosomes and can persist in newly repair chromosomes. Foci observation may be advantageous compared to classical cytogenetic analysis due to less time required for analysis. Metaphase DNA damage analysis can be also used for the estimation of DNA damage persistence in daughter cells and capacity of DNA repair. Not only DNA double strand breaks can be visualized, but also other types of DNA damage and modification such as oxidative damage, crosslinking, and methylation of DNA can be visualized with appropriate antibodies. This IRIF immunostaining technique can be combined with FISH analysis for the immunoFISH method.

Hypersensitivity of BRCA2 deficient cells to rosemary extract explained by weak PARP inhibitory activity.

Rosemary extract is used in food additives and traditional medicine and has been observed to contain anti-tumor activity. In this study, rosemary extract is hypothesized to induce synthetic lethality in BRCA2 deficient cells by PARP inhibition. Chinese hamster lung V79 cells and its mutant cell lines, V-C8 (BRCA2 deficient) and V-C8 with BRCA2 gene correction were used. Rosemary extract and its major constituent chemicals were tested for their cytotoxicity by colony formation assay in cells of different BRCA2 status. The latter chemicals were tested for inhibitory effect of poly (ADP-ribose) polymerase (PARP) activity in vitro and in vivo. Rosemary has shown selective cytotoxicity against V-C8 cells (IC50 17 µg/ml) compared to V79 cells (IC50 26 µg/ml). Among tested chemicals, gallic acid and carnosic acid showed selective cytotoxicity to V-C8 cells along with PARP inhibitory effects. Carnosol showed comparative PARP inhibitory effects at 100 µM compared to carnosic acid and gallic acid, but the selective cytotoxicity was not observed. In conclusion, we predict that within rosemary extract two specific constituent components; gallic acid and carnosic acid were the cause for the synthetic lethality.

PARP Inhibition by Flavonoids Induced Selective Cell Killing to BRCA2-Deficient Cells.

High consumption of dietary flavonoids might contribute to a reduction of cancer risks. Quercetin and its glycosides have PARP inhibitory effects and can induce selective cytotoxicity in BRCA2-deficient cells by synthetic lethality. We hypothesized that common flavonoids in diet naringenin, hesperetin and their glycosides have a similar structure to quercetin, which might have comparable PARP inhibitory effects, and can induce selective cytotoxicity in BRCA2-deficient cells. We utilized Chinese hamster V79 wild type, V-C8 BRCA2-deficient and its gene-complemented cells. In vitro analysis revealed that both naringenin and hesperetin present a PARP inhibitory effect. This inhibitory effect is less specific than for quercetin. Hesperetin was more cytotoxic to V79 cells than quercetin and naringenin based on colony formation assay. Quercetin and naringenin killed V-C8 cells with lower concentrations, and presented selective cytotoxicity to BRCA2-deficient cells. However, the cytotoxicity of hesperetin was similar among all three cell lines. Glycosyl flavonoids, isoquercetin and rutin as well as naringin showed selective cytotoxicity to BRCA2-deficient cells; hesperidin did not. These results suggest that flavonoids with the PARP inhibitory effect can cause synthetic lethality to BRCA2-deficient cells when other pathways are not the primary cause of death.