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Letters to the Editor |
Department of Radiation and Stress Cell Biology, University of Groningen, Groningen, the Netherlands
Division of Radiation and Cancer Biology, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri
To the Editor:
In their recent article, Takahashi et al. (1) suggest that heat-induced DNA double strand breaks contribute to heat-induced cell killing because heat treatment induces histone 
H2AX-containing foci. Such foci have been associated with double strand breaks induced by ionizing radiation, other agents, and other stresses (2). However, the authors disregard the hyperthermia biology literature, which indicates that heat-induced DNA damage is not involved in heat killing.
Heat probably does not induce DNA damage directly. The authors point out, however, that heat may induce DNA base damage indirectly via protein damage (1). For repair of ionizing radiation–induced base damage, it is thought that heat inhibits the excision step without impairing the incision step (3). For clustered base damage, such imbalance in incision and excision may result in conversion of base damage into DNA double strand breaks (3). By analogy, the authors speculate that heat-induced base damage may be converted into double strand breaks. However, the levels of base damage do not correlate with the extent of heat-induced cell killing (4). Moreover, although data using the neutral comet assay is presented (1), the use of a variety of sensitive biochemical and biophysical approaches that are more double strand break specific than the comet assay have failed to detect double strand breaks after heat (3). Moreover, Mre11, a protein directly involved double strand break repair that also forms foci immediately after ionizing radiation (5), is not found in foci after heat shock; rather, Mre11 exits from the nucleus after heat shock (6). This clearly shows that the H2AX foci seen after heat shock are completely different from those seen after ionizing radiation and cannot be taken as evidence for heat-induced double strand break.
Several other observations are also inconsistent with the notion that DNA damage is involved in heat-induced killing. Incorporation of BrdUrd into DNA, known to destabilize DNA and to enhance radiosensitivity, does not enhance heat killing. The potential lethal damage response associated with the ability to repair ionizing radiation–induced DNA damage has not been found for hyperthermia. Finally, if heat shock would induce DNA double strand breaks, either directly or indirectly, chromosomal aberrations, associated with the ionizing radiation–induced DNA double strand breaks, would be expected to occur also in cells heated in various phases of the cell cycle, which is not the case (3).
The authors nevertheless state that a DNA double strand break repair–deficient cell line is also heat sensitive, arguing in favor of a role for double strand break in heat killing (1). However, a literature survey on 30 different mouse cell lines reveals no general correlation between heat sensitivity and radiosensitivity (Fig. 1A). Because the authors suggest that double strand break only partially determine heat killing, one could still explain the latter by assuming cell line–dependent differences in the sensitivity of other targets of heat (i.e., protein damage) that would mask this correlation. Therefore, we also analyzed data from the literature describing a number isogenic panels of radiosensitive cells deficient in DNA double strand break repair and their normal counterparts for their heat sensitivity. It is highly unlikely that DNA repair gene–complemented cell lines would simultaneously acquire an altered capacity to repair protein damage (the presumed main cause of heat-induced cell death). Thus, if heat would indeed induce DNA double strand breaks relevant to its toxicity, then this should be revealed in such pairs. However, as can be seen in Fig. 1B, there is no relation between double strand break repair deficiency and heat sensitivity in these isogenic panels. Thus, on the basis of these data, one must conclude that even if double strand breaks are induced by heat, they do not contribute to heat-induced killing.
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H2AX-containing foci can also be induced by other non–double strand breaks–inducing treatments, such as treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (7) or exposure to hypertonic buffers (8), showing that the H2AX phosphorylation and foci formation is a general stress response, e.g., to changes in the chromatin (2), rather than to double strand breaks only. Heat is also known to induce major alterations in chromatin structure, not by inducing DNA damage but as a consequence of heat-induced protein denaturation or aggregation (3). In fact, the new and interesting observations by Takahashi et al. (1) of induction of H2AX foci by heat shock may be the first in situ evidence for sites of heat-induced chromatin alterations. However, in light of the fact that the literature of hyperthermia biology presents a consensus concerning the lack of a role of DNA damage in heat-induced cell killing, as outlined above, and the fact that the mere formation of foci containing H2AX is not accepted as sole evidence for the presence of DNA double strand breaks (2), the new data presented by Takahashi et al. (1) do not warrant the conclusion that DNA double strand breaks are involved in heat killing. References
Department of Biology, Nara Medical University, School of Medicine, Nara, Japan
In Response:
Recently, we reported that heat exposure led to the observation of H2AX focus formation, not only in the S phase but also in the G1 and G2 phases, and that double strand break–recognizing proteins (Nbs1 and Mre11) colocalized with the H2AX after heat treatment in a manner similar to that seen after exposure to X-rays (data not shown). An immunocytochemical assay recognizing H2AX foci is accepted as being an extremely sensitive and specific indicator for the existence of a double strand break that is induced by physical (X-ray, acidic, and hyperosmotic conditions) and chemical (bleomycin, etoposide, and N-methyl-N'-nitro-N-nitrosoguanidine) stresses (1). Although H2AX foci are also induced by replication arrest with ataxia-telangiectasia-mutated and Rad3-related activation, it is well established that the formation of H2AX foci depends on the formation of double strand breaks and not just on the presence of the S phase (1). A recent article indicates that heat-induced H2AX phosphorylation is mediated by ataxia telangiectasia mutated protein and DNA-dependent protein kinase (2), which are activated by the presence of double strand breaks. Although chromatin-modifying treatments induce ataxia telangiectasia mutated protein autophosphorylation, these treatments failed to induce ataxia telangiectasia mutated protein and H2AX focus formation (3); therefore, it currently cannot be claimed that H2AX foci are detected after these treatments. The frequency of chromosome aberrations induced by heat may be determined not only by double strand breaks but also by the inducible heat-shock protein 70: Heat-shock protein 70–deficient mice display a high frequency of chromosome aberrations when compared with wild-type mice (4). We also reported that heat-induced DNA fragmentation was detected with the comet assay. Moreover, Kampinga (5) detected a slightly increased level of DNA fragmentation in heat-treated cells when compared with untreated cells using pulsed-field gel electrophoresis. These findings provide strong support for the idea that heat induces double strand breaks.
Double strand breaks represent a significant DNA damage event: One double strand break remaining unrepaired in a cell can potentially result in cell death. H2AX foci were clearly observed in heat-treated cells when compared with X-irradiated cells. Other observations should also be noted: A correlation was found between the number of heat-induced H2AX foci observed and the mean lethal heating period. It may not be possible to compare heat sensitivity and radiosensitivity in DNA repair–deficient cells and wild-type cells because DNA repair enzymes might be inactivated by heat treatment even in wild-type cells.
These observations provide support for the concept that heat-induced double strand breaks may contribute to heat-induced cell killing. Further investigations are required to elucidate the exact mechanism leading to heat-induced double strand break formation. Such studies could contribute to new concepts and further understanding of hyperthermic biology and oncology.
References
H2AX focus formation depend on the presence of DNA double strand breaks? Cancer Lett. In press 2005.
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