Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

Abstract

Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.

Fig. 1.

BJ-hTERT cell growth and survival in hypothermic conditions. (A) BJ-hTERT cells remain attached and flattened in hypothermic conditions but grow more slowly compared to 37 °C. Representative images of the same fields at 24 and 72 h of incubation at 13, 20, 30, and 37 °C were obtained by phase contrast transmitted light microscopy. Magnification, 100×. (B) FACS analysis of cell cycle distribution in BJ-hTERT fibroblasts under normal and hypothermic conditions. Cells were plated at 37 °C for 6 h, grown at low temperatures for 12 h, and then transferred to 37 °C. Cell growth is inhibited under hypothermia but resumed when the cells are returned to 37 °C. The numbers are the S-phase fractions. (C) Clonogenic survival experiment at different temperatures. Experimental scheme and the plating efficiency (PE) at the different growth temperatures are shown. Graph shows clonogenic survival of BJ-hTERT cells after exposure to IR (0, 0.6, 2, 5, 7.5, and 10 Gy) and incubation at different temperatures (37, 30, 20, and 13 °C). Representative images of colonies in unirradiated and 7.5-Gy irradiated samples are shown (magnification, 200×). (D) Neutral comet assay for DSBs at 1 h before 10-Gy IR (white), and 0 (light gray), 1 (medium gray), 4 (dark gray) and 24 (black) h post-IR at the noted temperatures. Error bars denote 95% confidence intervals.

Fig. 2.

Analysis of γ-H2AX focus formation and 53bp1 accumulation in primary human lymphocytes. (A) γ-H2AX focus formation time course after lymphocyte IR exposure to 0.6 and 2 Gy. Intensity (top and bottom graphs) and the numbers (middle graph) of γ-H2AX foci were determined at 13 (filled circles) and 37 °C (open squares) for 24 h. At 8 h post-IR (black arrow), some samples were transferred from 13 to 37 °C (filled squares, dotted line) and incubated for further 16 h. Error bars are the standard deviations of 3 donor samples. (B) γ-H2AX focus formation (left columns) and 53BP1 accumulation at those foci (middle and right columns) at 37 (left panel) and 13 °C (right panel). Human lymphocytes were irradiated with 2 Gy and incubated for the noted times. Samples were fixed using 2% PFA and processed for γ-H2AX foci and 53BP1 analysis. Representative images show lymphocytes stained for γ-H2AX (green) and 53bp1 (red). Colocalized fluorochromes appear yellow in the merged images. The two proteins completely colocalized by 30 min post-IR at 37 °C, but did not colocalize at 13 °C until the cells are transferred to 37 °C. Magnification, 1000×. (C) γ-H2AX focus size was determined for least 100 cells per point in images from the experiment shown in the panels using Adobe Photoshop software. At 13 °C, γ-H2AX foci appear but remain small while at 37 °C, they continue to enlarge.

Fig. 3.

Analysis of oxidative clustered DNA lesions (OCDLs) in primary human lymphocytes. Lymphocytes of three normal donors (A, red; B, blue; C, black symbols) were irradiated ex vivo with 2 Gy IR and incubated at either 37 (open squares) or 13 °C (open circles). Samples were taken at the noted times and the DNA digested to determine the number of (left) oxypyrimidine (EndoIII) (middle) oxypurine (OGG1), and (right) abasic sites (APE1) per Gbp. Some samples were transferred from 13 to 37 °C after 8 h (filled diamonds, dashed line). The dot-dashed line represents the value of unirradiated control samples. Data plotted individually for each donor.