Effects of mild cold shock (25°C) followed by warming up at 37°C on the cellular stress response

Abstract

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX(S/L) ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.

Figure 1. Phase contrast micrographs of WI26 cells.

Cells were continuously cultured at 37°C (A), at 25°C for 5 days (B) or at 25°C for 5 days followed by a rewarming to 37°C for 24h (C). Arrows point to refringent, apoptotic-like cells. Bar: 100µm.

Figure 2. Temperature dependence of cell proliferation.

(A) WI26 cells maintained at 37°C for 1 to 6 days (— - - —) or at 25°C for 1 to 5 days (——) and then warmed at 37°C for 1 to 72 hours (- - - -), and DNA content was measured. The black arrow indicates the transition from 25°C to 37°C. Insert provides an enlarged view of the first 24 hours of rewarming at 37°C. (B) [³H]-thymidine incorporation by WI26 cells maintained for 5 days at 25°C and warmed at 37°C for 1 to 48 h. (C) Representative western blot probed with antibodies specific for phospho ERK 1/2 or total ERK 1/2. (D) Quantification of the western blots. Data are expressed as the mean ratio of P-ERK/ total ERK normalized to ratio in control cells (T0), taken as 1. T0: 1 day at 37°C; 1D and 5D: 1 or 5 days at 25°C; 1h, 2h, 4h, 8h and 24h: 5 days at 25°C followed by 1 to 24h at 37°C.

Figure 3. Cold-shock and rewarming affect heat and cold shock genes expression.

The expression of heat shock (HSP70) and cold shock genes (CIRBP and RBM3) was quantified at the mRNA level by RT-PCR in WI26 cells cultured at 25°C for 1 and 5 days before warming up at 37°C for 1h to 24h. (A) Representative gels showing the RT-PCR amplification products. (B) HSP70, CIRBP and RBM3 mRNA levels are expressed as mean ± SD (n=3) after normalization to the 28S rRNA content used as calibrator. Values at T0 were arbitrary taken as 1. Legend for culture schedule is the same as in Figure 2.

Figure 4. Cold shock and rewarming affect cell viability.

WI26 cells were cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h. Cells kept at 37°C were used as control (T0). FACS analysis was performed after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes). 12.000 to 18.000 events were collected for each experiment. (A) Example of dots graphs (I), contour graphs (II) and annexin V curves (III) of control cells and cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h). Alive cells [A] (double negative staining), cells in early apoptosis [EA] (annexin V positive, PI negative), in late apoptosis [LA] (double positive) and necrotic [N] (annexin V negative, PI positive) are indicated on the graphs. Annexin V curves (III) were used to define the gating allowing to discriminate the populations. (B) Percentage of apoptotic cells as measured by FACS analysis. Cells were cultured at 37°C (T0) or 25°C for 5 days and subsequent warming-up at 37°C for 2 to 24h. (C) Percentage of dead cells as measured by trypan blue exclusion assay. Cells were cultured in duplicate for 5 days at 25°C and then rewarmed for 2 to 24h at 37°C.

Figure 5. Cold shock and rewarming induce autophagy and a cellular stress response.

The level of LC3 I and II (A), phospho-JNK (P-JNK) and total JNK (D) was analyzed by western blot in cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. The levels of ERK1/2 were taken as calibrator and used to monitor protein loading. Results are expressed as the mean ratio of LC3 II/I (B) and of P-JNK/ total JNK (D) taking T0 as 1. (C) Immunostaining of LC3 was performed in WI26 cells maintained at 37°C in the presence or in the absence of serum or at various time points during the cold shock and rewarming. Actin stress fibers appear in red, LC3 in green and nucleus in blue. Bar: 100µm.

Figure 6. Rewarming-induced ROS production and ROS-dependant apoptosis.

(A) ROS were measured in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of 15mM N-Acetylcysteine (NAC). Data are expressed in arbitrary units taking T0 as 1. Significant inhibition by NAC versus condition without NAC is indicated # # # (p<0.001). (B) Cell viability was measured by FACS analysis after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes) of cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h) in the presence or in the absence of 15mM of NAC added at T0. (C) Percentage of apoptotic cells in the indicated culture conditions.

Figure 7. Cold shock and rewarming induce H2AX phosphorylation through ROS production.

The levels of phosphorylated H2AX (γH2AX) were analyzed in WI26 cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blots. (B) γH2AX was quantified by western blotting. Data are expressed in arbitrary units after normalization by ERK1/2, taking T0 as 1. (C) γH2AX in WI26 cells in normal conditions (1D37°C) and during cold shock for 5 days and rewarming for 2 to 24h was evidenced by immunostaining. Actin stress fibers are stained in red and γH2AX in green. Bar: 100µm. (D) γH2AX was measured by western blot in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of NAC 15mM added at T0. After normalization by GAPDH, results are expressed in arbitrary units taking 5D as 1.

Figure 8. Return to normal temperature after a cold shock affects the phosphorylation of p53.

Total and phosphorylated p53 (P-p53) were analyzed in WI26 cells cultured in normal conditions or at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blot. (B) Quantifications of the western-blots are expressed as the mean ratio P-p53/total p53, taking T0 as 1. Total protein loading was monitored by ERK1/2.