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 BclXS/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.
Highlights
While heat shock has been intensively investigated, cold shock has retained relatively less attention
To support the induction of apoptosis suggested by the morphological alterations illustrated in Figure 1, the expression of several pro- and anti-apoptotic factors was investigated at the mRNA level and the phosphorylation of Akt was evaluated by western blot
We observed that hypothermia induces a re-programming of gene expression, RNA processing and intracellular signaling, and affects cell shape, growth and survival
Summary
While heat shock has been intensively investigated, cold shock has retained relatively less attention. Among a significant number of described cold shock proteins (CSP), only CIRBP (cold-inducible RNA binding protein) and RBM3 (RNA binding motif protein 3), two highly homologous proteins, have been thoroughly characterized Their expression is rapidly and markedly increased during mild hypothermia, and they appear to be key determinants in cold-stress adaptation and to stimulate translation of cold-specific transcripts through various mechanisms [7,11,18]. We thought to investigate the phenotype of cells during storage at 25°C followed by rewarming at 37°C These data bring new cellular and molecular mechanisms that might benefit to research and therapeutical fields using hypothermia. They will further allow to set up a more appropriate experimental design for future space experiments
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