This study shows that sub-lethal heat injury causes ‘bystander’ cells (nearby, but not heated cells) to show evidence of DNA damage and suffer significant lethality. To our knowledge this is previously unreported. The so called ‘bystander effect’ occurred when the inducing cells were moderately heat stressed, but not at higher temperatures causing complete thermal necrosis of such cells. This suggests that an ‘active’ cellular process is involved in the heated, inducing cells, in contrast to a ‘passive’ mechanism such as leakage of cellular content because of thermally induced membrane damage and cellular disintegration. To further investigate this, we performed a ‘washing’ experiment where we exchanged the DMEM medium 5 minutes after heat exposure and compared with ‘non-washing’ (Figure 4). We confirmed for both protocols (wash and no washing) a statistically significant bystander effect at 46°C (washed P=0.009, non-washed P=0.001) and at 50°C (washed P=0.048, non-washed P=0.001). The media exchange did not significantly affect the bystander effect when ‘washed’ and ‘non-washed’ bystander cells were compared at 46°C (P=0.168) and at 50°C (P=0.158). This experimental data can be seen as a strong support that the bystander effect is not induced by rapid release of cellular content or debris as expected after acute membrane damage. These findings rather suggest that the thermal bystander effect is an active process in which viable, heat-injured cells induce a delayed signal cascade and/or mediators that damage or kill surrounding bystander cells. Therefore, we like to call this process active thermal bystander effect (ATBE). The putative mediator(s) of the ATBE remain unknown. Although all data strongly support the notion that the thermal bystander is mainly caused by an active cellular process, at this point it cannot exclude with certainty that at least partially some passive process (for example, nonspecific release of catabolic enzymes from dead cells) might be also involved in the bystander effect for the investigated temperature range......
.....Higher temperatures that produce cell necrosis and/or lysis can cause a passive bystander effect. Dabrowska et al. (2005) described a bystander effect in human cancer cells, after extremely high heat exposure of 75°C for 10 minutes. Direct thermal cell necrosis because of high temperatures might result in the release of cellular debris, including lysosomes into the extracellular matrix, possibly damaging the surrounding cells, comparable with an inflammatory process. This is distinct from the ATBE of our study, which is mediated by thermally damaged, but viable cells. The mediators created with either ABTE or with high, necrotic temperatures, such as those used by Dabrowska et al. remain to be determined.
Mild and moderate heat exposure is well known to cause stress response called heat shock in mammalian cells (Page and Shear, 1988; Miller and Ziskin, 1989). Depending on temperature, heating time, cell type and culture conditions, heated cells can generally follow three different pathways. At a survivable combination of temperature and heating time, heat-shock proteins are activated to protect the cells. Heat-shock protein protection follows complex pathways including stabilization of denaturated cytoplasmic and membrane proteins, nuclear structures, and inhibition of apoptosis. Above a certain combination of temperature and heating time, apoptosis is induced despite the heat-shock response. At still higher temperatures, cells die acutely by thermal necrosis, which releases cell debris. Harmon et al. (1990) detected in murine mastocytoma cell cultures an increase of apoptosis after heating the cells for 30 minutes up to 45°C, whereas higher temperature of 46 and 47°C showed only necrotic cell death. Membrane changes in heat-exposed cells seem to be an important alteration, highly correlated with cell lethality (Calderwood and Hahn, 1983; Konings and Ruifrok, 1985; Majda et al., 1994; Coss and Linnemans, 1996).
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