Abstract
Eukaryotic cells have at least two major pathways to repair DNA double-strand break (DSB), i.e., non-homologous end joining (NHEJ) and homologous recombination (HR). Scid cells and hybrid cells were ideal to study the involvement of DSB repair in heat-induced cell killing, because their genetic backgrounds were identical except the pathway of NHEJ.
Scid cells and hybrid cells were heated at 44'C for different periods. To clarify the relationship between thermotolerance and heat-induced DSB, the cells were conditioned by preheating at 44'C and then incubated at 37'C for various intervals. We analyzed the DNA double-strand breaks induced by heat treatment in cells using rH2AX foci formation assay. After a heat treatment, spots of phospholyrated histone 2AX (rH2AX), so-called foci, appear in the cell nucleus. H2AX surrounding the DSB point phosphorylated at the serine 139 residue within a few minute after the DSB-evoking stimuli. This assay is known to be quite sensitive and a specific indicator for DSB. Photograph of the cells were taken with a fluorescence microscope.
Scid cells was sensitive to a heat treatment. After a heat treatment at 44'C, we observed a linear increase with time in the number of rH2AX foci. The number of rH2AX foci of scid cells was significantly high in comparison with hybrid scid cells. Th number of rH2AX foci was correlated with increasing thermosensitivity of the cells. Furthermore, we found that a preheat treatment led to heat-induced thermotoleranace. It was confirmed that the number of rH2AX foci was reduced in preheated cells later than treated with a challenge heat. These finding suggest that thermosensitivity and thermotolerance of cells might be associated with DNA double-strand break repair.
