Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells.

Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5-10bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant gamma-ray dose of 5Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to gamma-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1-3h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6-24h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.

Detection of complex DNA damage in gamma-irradiated acute lymphoblastic leukemia Pre-b NALM-6 cells.

Bistranded complex DNA damage, i.e., double-strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions, is hypothesized to challenge the repair mechanisms of the cell and consequently the genomic integrity. The oxidative clustered DNA lesions may be persistent and may accumulate in human cancer cells for long times after irradiation. To evaluate the detection and possible accumulation of oxidative clustered DNA lesions in leukemia cells exposed to doses equivalent to those used in radiotherapy, we measured the induction of DSBs and three different types of oxidative clustered DNA lesions in NALM-6 cells, a human acute lymphoblastic leukemia (ALL) pre-B cell line, after exposure to (137)Cs gamma rays. For the detection and measurement of DSBs and oxidative clustered DNA lesions, we used an adaptation of the neutral comet assay (single-cell gel electrophoresis) using E. coli repair enzymes (Endo IV, Fpg and Endo III) as enzymatic probes. We found a linear dose response for the induction of DSBs and oxidative clustered DNA lesions. Clustered DNA lesions were more prevalent than prompt DSBs. For each DSB induced by radiation, approximately 2.5 oxidative clustered DNA lesions were detected. To our knowledge, this is the first study to demonstrate the detection and linear induction of oxidative clustered DNA lesions with radiation dose in an ALL cell line. These results point to the biological significance of clustered DNA lesions.

Osmotic effects on arginine kinase function in living muscle of the blue crab Callinectes sapidus.

Flux was examined through the reaction catalyzed by arginine kinase in intact blue crab (Callinectes sapidus) muscle during simulated changes in salinity. Isolated dark levator muscles from the swimming leg were superfused with a saline solution that had an osmolarity equivalent to that of the hemolymph under different salinity regimes. Animals were acclimated for 7 days to a salinity of 5, 17 or 35 per thousand, which corresponds to a hemolymph osmolarity of 640, 720 or 960 mosmoll(-1), respectively. Experiments were conducted under control conditions, in which the osmolarity of the superfusion medium matched that of the acclimated hemolymph, as well as under hypo- and hyperosmotic conditions. These latter treatments were meant to simulate a rapid change in environmental salinity. Pseudo-first-order unidirectional rate constants and flux rates were measured for arginine kinase in the forward and reverse directions using a (31)P-nuclear magnetic resonance saturation transfer method. There were no differences in the rate constants or flux rates among the controls, indicating that arginine kinase function is not modulated by salinity if the animal has had sufficient acclimation time. However, the rate constants and flux rates of arginine kinase varied over a modest 1.7-fold range across the three types of osmotic treatments, although the range for the flux data was reduced when cell volume changes were taken into account. The hyperosmotic treatments led to a reduction in arginine kinase flux, while the hypo-osmotic treatments led to an enhanced arginine kinase flux. We propose that this effect is mediated by an increase in the concentration of perturbing inorganic ions under hyperosmotic conditions and a decrease in the concentration of such ions during the hypo-osmotic treatments.