Letters to the Editor
DNA Double Strand Breaks Do Not Play a
Role in Heat-Induced Cell Killing
To the Editor:
In their recent article, Takahashi et al. (1) suggest that heatinduced DNA double strand breaks contribute to heat-induced cell
killing because heat treatment induces histone gH2AX-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 gH2AX 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
I2005 American Association for Cancer Research.
doi:10.1158/1078-0432.CAN-05-0006
Cancer Res 2005; 65: (22). November 15, 2005
Figure 1. Absence of a correlation between radiation sensitivity and heat
sensitivity. A, a cross-correlation of radiation sensitivity (expressed as the
dose of X-ray required to kill 90% of the cells) with heat sensitivity (expressed
as the equivalent time of heating at 44jC required to kill 90% of the cells) in
30 different mouse cell lines derived from the literature. B, comparison of
heat sensitivity between various radiosensitive mutants (deficient in either
nonhomologous end joining or in homologous recombination) and their isogenic
repair–proficient counterparts. The average sensitivity of the groups (points
with bars ) is not different; there is also no trend for increase or decrease in
heat sensitivity in matched panels (lines ).
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 heatinduced killing.
It is now known that gH2AX-containing foci can also be induced
by other non–double strand breaks–inducing treatments, such as
treatment with the methylating agent N-methyl-NV-nitro-N-nitrosoguanidine (7) or exposure to hypertonic buffers (8), showing that
the gH2AX 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
10632
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DNA Double Strand Breaks and Heat-Induced Cell Killing
Takahashi et al. (1) of induction of gH2AX 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
gH2AX 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.
Harm H. Kampinga
Department of Radiation and
Stress Cell Biology
University of Groningen
Groningen, the Netherlands
Andrei Laszlo
Division of Radiation and
Cancer Biology
Department of Radiation Oncology
Washington University School of
Medicine
St. Louis, Missouri
References
1. Takahashi A, Matsumoto H, Nagayama K, et al. Evidence for the involvement of
double-strand breaks in heat-induced cell killing. Cancer Res 2004;64:8839–45.
2. Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone
guardian of the genome. DNA Repair (Amst) 2004;3:959–67.
3. Kampinga HH, Dikomey E. Hyperthermic radiosensitization: mode of action and
clinical relevance. Int J Radiat Biol 2001;77:399–408.
4. Jorritsma JB, Konings AWT. DNA lesions in hyperthermic killing: effects of
thermotolerance, procaine, and erythritol. Radiat Res 1986;106:89–97.
5. Mirzoeva OK, Petrini JHJ. DNA damage-dependent nuclear dynamics of the Mre11
complex. Mol Cell Biol 2001;21:281–8.
6. Seno JD, Dynlacht JR. Intracellular redistribution and modification of proteins of
the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock.
J Cell Physiol 2004;199:157–70.
7. Stojic L, Mojas N, Cejka P, et al. Mismatch repair-dependent G2 checkpoint induced
by low doses of SN1 type methylating agents requires the ATR kinase. Genes Dev
2004;18:1331–44.
8. Reitsema TJ, Banath JP, MacPhail SH, Olive PL. Hypertonic saline enhances
expression of phosphorylated histone H2AX after irradiation. Radiat Res 2004;161:
402–8.
and chemical (bleomycin, etoposide, and N-methyl-NV-nitro-Nnitrosoguanidine) stresses (1). Although gH2AX foci are also
induced by replication arrest with ataxia-telangiectasia-mutated
and Rad3-related activation, it is well established that the
formation of gH2AX 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 DNAdependent 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 gH2AX focus formation (3); therefore, it
currently cannot be claimed that gH2AX 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. gH2AX 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 gH2AX 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 heatinduced 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.
Akihisa Takahashi
Eiichiro Mori
Takeo Ohnishi
Department of Biology,
Nara Medical University
School of Medicine,
Nara, Japan
A Possible Role of DNA Double
Strand Breaks in Heat-Induced
Cell Killing
In Response:
Recently, we reported that heat exposure led to the observation
of gH2AX 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 gH2AX after heat
treatment in a manner similar to that seen after exposure to
X-rays (data not shown). An immunocytochemical assay recognizing gH2AX 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)
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References
1. Takahashi A, Ohnishi T. Does gH2AX focus formation depend on the presence of
DNA double strand breaks? Cancer Lett. In press 2005.
2. Kaneko H, Igarashi K, Kataoka K, Miura M. Heat shock induces phosphorylation of
histone H2AX in mammalian cells. Biochem Biophys Res Commun 2005;328:1101–6.
3. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular
autophosphorylation and dimer dissociation. Nature 2003;421:499–506.
4. Hunt CR, Dix DJ, Sharma GG, et al. Genomic instability and enhanced
radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice. Mol Cell Biol 2004;24:899–911.
5. Kampinga HH. Hyperthermia, thermotolerance and topoisomerase II inhibitors.
Br J Cancer 1995;72:333–8.
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Cancer Res 2005; 65: (22). November 15, 2005
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer
Research.
DNA Double Strand Breaks Do Not Play a Role in
Heat-Induced Cell Killing
Harm H. Kampinga and Andrei Laszlo
Cancer Res 2005;65:10632-10633.
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