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[Cancer Research 64, 8839-8845, December 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Evidence for the Involvement of Double-Strand Breaks in Heat-Induced Cell Killing

Akihisa Takahashi1, Hideki Matsumoto4, Kosuke Nagayama1, Mutsuko Kitano1, Sayako Hirose1, Hidenori Tanaka1, Eiichiro Mori1, Nobuhiro Yamakawa2, Jun-ichi Yasumoto2, Kazue Yuki3, Ken Ohnishi1 and Takeo Ohnishi1

Departments of 1 Biology, 2 Oral and Maxillofacial Surgery, and 3 Otorhinolaryngology, Nara Medical University School of Medicine, Nara, Japan; and 4 Department of Experimental Radiology and Health Physics, Division of International Social and Health Science, Faculty of Medical Science, University of Fukui, Fukui, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify critical events associated with heat-induced cell killing, we examined foci formation of {gamma}H2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of {gamma}H2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5°C to 45.5°C, we observed a linear increase with time in the number of {gamma}H2AX foci. An inflection point at 42.5°C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of {gamma}H2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of {gamma}H2AX foci formation. We also found that thermotolerance was due to a depression in the number of {gamma}H2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperthermia is widely used to treat patients with various cancers. However, the molecular mechanisms involved in heat-induced cellular responses are still unknown. For example, it has not yet been determined whether the critical event in heat-induced cell killing involves double-strand breaks (DSBs) or operates via other mechanisms. One possibility that has been suggested is that proteins and lipids are the major targets for heat-induced cell killing (1) . Thermoresistance (2) and thermotolerance (3) are acquired when heat-labile proteins are protected by heat shock proteins. The enhancement of radiosensitization by heat (4) and the existence of an inflection point in Arrhenius plots of cell killing after exposure to heat (5) may indicate that heat-induced denaturation and damage to proteins are critical in heat-induced cell killing. Heat-induced protein denaturation results in the disruption of centrosome-dependent mitosis (6) and multiple nuclear matrix-dependent functions [e.g., DNA replication, DNA transcription, mRNA processing, and DNA repair (7) ]. Another possible target involved in cell killing is cellular DNA because it has been reported that heat induces structural alterations and strand breaks in chromatin DNA. Many investigators have reported that cellular DNA strand breaks are detected in heat-treated cells using alkaline elution methods (8) , alkaline unwinding methods (9) , in situ nick translation methods (10) , and pulse-field gel electrophoresis methods (11) . However, these conventional physical methods are not sensitive enough to clarify the relationship between cell killing and DSBs after heat treatment. Hence, DNA strand break formation was considered to be just a component or part of the pathway of events leading to heat-induced cell killing. For the technical reasons noted above, it was almost impossible to show differences in the biological effects of heat- and radiation-induced DSBs.

Recently, the measurement of focus formation of {gamma}H2AX (histone H2AX phosphorylated at serine 139) has attracted considerable attention because this method provides quite sensitive and specific signals indicating the existence of DSBs. There are many reports showing a direct correlation between the number of DSBs and {gamma}H2AX foci (12 , 13) . The work reported here was designed to clarify the relationship between DSBs and cell killing (temperature- and cell cycle-dependent cell killing and the effect of thermotolerance) after heat treatment, and we examined foci formation of {gamma}H2AX in heat-treated cells using immunocytochemical analysis with anti-{gamma}H2AX antibody. We discuss possible mechanisms of heat-induced DSB formation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells.
H1299 (human non–small-cell lung carcinoma p53-deficient) cells were provided by Dr. M. Oren (Weizmann Institute of Science, Rehovot, Israel). H1299 cells were cultured in DMEM-10 [Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum, 20 mmol/L 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, penicillin (50 units/mL), streptomycin (50 µg/mL), and kanamycin (50 µg/mL)]. The cells were cultured at 37°C in a conventional humidified CO2 incubator.

Hyperthermia and Irradiation.
Exponentially growing cells were immersed in a water bath (Thermominder EX; Taitec Co., Ltd., Saitama, Japan) maintained at a constant temperature. X-ray irradiation (1.0 Gy/min) was administered with a 150-kVp X-ray generator (Model MBR-1520R; Hitachi, Tokyo, Japan). Under the present experimental conditions, no marked change in pH value was detected in the medium during the treatment.

The Neutral Single-Cell Electrophoresis (Comet) Assay.
Neutral comet assay was performed using the CometAssay kit (Trevigen Inc., Gaithersburg, MD). Briefly, exponentially growing H1299 cells (105) were suspended in 1 mL of DMEM-10 and heated at 45.5°C or exposed to X-rays. The treated cells were resuspended immediately in 1 mL of ice-cold PBS. A 50-µl aliquot of this suspension was mixed with 0.5 mL of prewarmed 1% low melting point agarose, and 75 µL were pipetted immediately onto a CometSlide. The slide was kept at 4°C for 10 minutes. The slides were then immersed in prechilled lysis solution [2.5 mol/L sodium chloride, 100 mmol/L EDTA (pH 10), 10 mmol/L Tris base, 1% sodium lauryl sarcosinate, and 1% Triton X-100] at 4°C for 30 minutes. After washing with neutral Tris-borate EDTA buffer, slides were then subjected to electrophoresis for 20 minutes at 1 V/cm. After electrophoresis, slides were stained with 1:10,000 diluted SYBR Green in SlowFade reagent (Molecular Probes, Eugene, OR). It has been reported that SYBR Green improves the resolution and sensitivity of the comet assay, particularly for measuring DSBs (14) . Nuclei were visualized under a fluorescence microscope (Olympus BX51; Olympus Optical, Tokyo, Japan) with an IB filter (Olympus Optical). The images of nuclei were captured using a charge-coupled device camera (Penguin 150 CL; Pixera Co., Los Gatos, CA) and analyzed with the Youworks Imaging Technology Software trial version (Youworks Co., Tsukuba, Japan). For each sample, 50 randomly chosen cells were scored for tail moment (15) . Relative tail moments in heat-treated cells or X-ray–irradiated cells were calculated with respect to those in untreated cells. Three independent experiments were performed for each point.

Western Blotting Analysis.
Exponentially growing cells were cultured in 25-cm2 flasks. Lysates from heated or irradiated cells were prepared as described previously (16) . Aliquots of 10 µg of proteins were subjected to Western blotting analysis. After electrophoresis on 15% polyacrylamide gels containing 0.1% SDS, the proteins were transferred electrophoretically onto Poly Screen polyvinylidene difluoride membranes (DuPont/Biotechnology Systems, New England Nuclear Research Products, Boston, MA). The membranes were then incubated with anti–phospho-H2AX monoclonal antibody (JBW301; Upstate Biotechnology, Lake Placid, NY) and anti-actin polyclonal antibody (I-19; Santa Cruz Biotechnology, Santa Cruz, CA). For visualization of the bands, horseradish peroxidase-conjugated anti-mouse IgG antibody (Zymed Laboratories, Inc., San Francisco, CA) and horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech Inc., Piscataway, NJ) for phospho-H2AX and actin, respectively, were used with the BLAST:Blotting Amplification System (DuPont/Biotechnology Systems).

Histologic Study of Histone H2AX Phosphorylation.
Exponentially growing cells were grown on slide glass (S-2111; Matsunami Glass Industrial, Ltd.; Osaka, Japan). After treatment, the cell preparations were fixed with cold methanol for 20 minutes and with acetone for 7 seconds, dried, blocked with 3% skim milk in PBS for 10 minutes, and washed in TPBS (PBS containing 0.05% Tween 20). The cells were incubated with anti-phospho-H2AX monoclonal antibody at a 300-fold dilution for 60 minutes at room temperature; washed with TPBS; incubated with a fluorescein isothiocyanate, AlexaFluor 488-conjugated antimouse IgG second antibody (Molecular Probes) at a 400-fold dilution for 60 minutes at room temperature; and washed in TPBS. The slides were stained and mounted with 1 µg/mL propidium iodide (PI) in Antifade (Molecular Probes). Photographs of the cells were taken with a fluorescence microscope (Olympus BX51, Olympus Optical) under a U-MWIB filter (Olympus Optical). To allow direct comparisons, all of the images were captured using the same parameters. We observed {gamma}H2AX foci with anti-{gamma}H2AX antibody (green fluorescence) and the nuclei with PI (red fluorescence). For quantitative analysis, foci were counted by imaging analysis of the microscopic images obtained using a x40 objective lens. For each sample, cell counting was performed until at least 20 cells were registered. Three independent experiments were performed for each point.

Colony Forming Assay.
Cell survival was measured using a standard colony forming assay. Three flasks were used, and three independent experiments were repeated for each survival point. Colonies obtained after 10 days were fixed with methanol and stained with 2% Giemsa solution. Microscopic colonies composed of more than approximately 50 cells were counted as surviving cells. Equation and variable defined and set were fitted by the single-hit multitarget model: S/S0 = 1 – (1 –e–T/T0)n, where S/S0 is the surviving fraction, T is the heating period (in minutes), T0 is the mean lethal heating period (in minutes), and n is the number of targets. The parameters n and T0 were calculated using KaleidaGraph (Synergy Software, Reading, PA).

Cell Synchronization.
After serum starvation for 28 hours, G1-synchronized cells were cultured in fresh medium for 28 hours. Then, the mitotic cells in the supernatant medium were collected by centrifugation. The mitotic cell pellet was dispersed in fresh medium.

Flow Cytometry.
After treatment, the cells were fixed with cold 70% methanol and kept at –20°C for up to 2 weeks before analysis. Cells were centrifuged and rinsed with TPBS. The cells were blocked with rabbit serum for 15 minutes at room temperature and rinsed with TPBS. The cells were incubated with anti–phospho-H2AX monoclonal antibody (JBW301; Upstate Biotechnology) at a 300-fold dilution for 60 minutes at room temperature; rinsed with TPBS; incubated with a fluorescein isothiocyanate, AlexaFluor 488-conjugated anti-mouse IgG second antibody (Molecular Probes) at a 400-fold dilution for 60 minutes at room temperature; and rinsed in TPBS. Cell cycle distribution was assayed by determining DNA content. For determination of DNA content, cells were fixed with cold 70% methanol. The cells were incubated for 30 minutes at room temperature with 1 mg/mL RNase and 50 µg/mL PI. Before flow cytometric analysis, samples were filtered through a 35-µm nylon mesh. Samples were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Heat-Induced Double-Strand Breaks Using the Neutral Comet Assay.
We analyzed chromosomal DSBs using the comet assay under neutral conditions in H1299 cells exposed to heat or X-rays. Typical comet images of untreated, X-ray–irradiated (30 Gy), and heat-treated cells (45.5°C, 180 minutes) are shown in Fig. 1ACitation . In cells irradiated with 30 Gy, comet tail formation was observed clearly. In cells heated at 45.5°C for 180 minutes, the tail appeared longer than that in controls, and more DNA was observed in the tail when compared with untreated cells. The comet tail moments of heat-treated (45.5°C, 60 minutes) and X-ray–irradiated (10 Gy) cells both increased after treatment (Fig. 1B)Citation . The tail moment of X-ray–irradiated cells increased linearly in a dose-dependent manner between 0 and 30 Gy, and the tail moment of heat-treated cells was slightly increased after 60 minutes of incubation at 45.5°C.



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  		Fig. 1. DSB analysis by neutral comet assays and {gamma}H2AX staining. A, typical images of neutral comet assays immediately after exposure to X-rays or heat treatment (45.5°C). B, the tail moments of cells exposed to different heating periods and radiation doses. Tail moments in X-ray–irradiated cells and heat-treated cells relative to those in untreated cells are shown. C, density of {gamma}H2AX bands with Western blot analysis. H1299 cells were treated with X-rays (3 Gy) or heat (45.5°C, 20 minutes) and then fixed at different time periods after treatment. D, photographs of {gamma}H2AX foci. E, the average number of {gamma}H2AX foci after treatment with X-rays (3 Gy) or heat (45.5°C, 20 minutes). F, focus formation of {gamma}H2AX at 30 minutes after treatment. U, untreated. Error bars, SD.

 
Focus Formation of {gamma}H2AX by Heat.
To clarify the effects of heat on the phosphorylation of histone H2AX, Western blot analysis and immunocytochemical analysis were performed in human H1299 non–small-cell lung carcinoma cells. The phosphorylation of H2AX was detected not only in X-ray–irradiated cells but also in heat-treated cells (Fig. 1C)Citation . The phosphorylation of H2AX in X-ray–irradiated cells increased linearly in a dose-dependent manner over the range of 0 to 30 Gy, and the phosphorylation of H2AX in heat-treated cells also increased up to 60 minutes during incubation at 45.5°C and then reached a plateau during additional incubation. These results were quite similar to the results seen with neutral comet analysis. Formation of {gamma}H2AX foci was observed not only in X-ray–irradiated cells but also in heat-treated cells. Typical photographs of radiation- or heat-induced {gamma}H2AX foci are shown in Fig. 1DCitation . When H1299 cells were irradiated with X-rays, a linear increase of {gamma}H2AX foci formation was also observed in a dose-dependent manner at 30 minutes after X-ray irradiation (Fig. 1E)Citation . The slope of the curve of the number of {gamma}H2AX foci as a function of irradiation dose (Gy–1) was 30.8 (r = 0.999). When H1299 cells were irradiated with X-rays, a linear increase of {gamma}H2AX foci formation was also observed in a dose-dependent manner at 30 minutes after X-irradiation (Fig. 1E)Citation . The slope of the curve of the number of {gamma}H2AX foci as a function of irradiation dose (Gy–1) was 30.8 (r + 0.999). When H1299 cells were heated at 45.5°C, a linear increase of {gamma}H2AX foci formation was observed in a heating time-dependent manner when measured at 30 minutes after heat treatment (Fig. 1E)Citation . The slope of {gamma}H2AX foci/heating period (min–1) was 4.825 ± 0.095 (r = 0.998) at 45.5°C. After heat treatment, cells were cultured for different periods. The average number of {gamma}H2AX foci 30 min after X-irradiation (3 Gy) and after heat-treatment (45.5°C, 20 minutes) was 95.8 ± 3.9 and 99.6 ± 1.5, respectively (Fig. 1F)Citation . The number of {gamma}H2AX foci was observed to increase rapidly and reach a maximum at 30 minutes after X-ray irradiation (3 Gy) or at 30 minutes after heat treatment (45.5°C, 20 minutes).

Foci Formation of {gamma}H2AX at Various Temperatures and after Different Heating Periods.
When H1299 cells were heated at 41.5°C to 45.5°C, a linear increase of foci formation of {gamma}H2AX was observed in a heating time-dependent manner (Fig. 2A)Citation . The same incubation period was required to reach maximum foci formation after heat treatment, regardless of the heating temperature (data not shown). The slopes of the curves of {gamma}H2AX foci/heating period (min–1) were 0.082 ± 0.001 (r = 0.999) at 41.5°C, 0.225 ± 0.005 (r = 0.997) at 42°C, 0.465 ± 0.012 (r = 0.997) at 42.5°C, 0.690 ± 0.012 (r = 0.999) at 43°C, 1.083 ± 0.028 (r = 0.996) at 43.5°C, 1.520 ± 0.047 (r = 0.993) at 44°C, 2.372 ± 0.023 (r = 0.999) at 44.5°C, 3.303 ± 0.060 (r = 0.998) at 45°C, and 4.825 ± 0.095 (r = 0.998) at 45.5°C (Fig. 2A)Citation .



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  		Fig. 2. Arrhenius plot analysis of focus formation of {gamma}H2AX and cell killing after heat treatment. H1299 cells were treated at different temperatures (41.5°C to 45.5°C) for different heating periods. A, focus formation of {gamma}H2AX at 30 minutes after heat treatment. B, survival curves. C, Arrhenius plots of the rate constants (min–1; focus formation of {gamma}H2AX and 1/T0) versus inverse temperature 1/K. Error bars, SD.

 
Survival versus Temperature and Heating Period.
Survival curves after heating at various temperatures (41.5°C to 45.5°C) and for different heating periods are shown in Fig. 2BCitation . The heat sensitivity and the T0 value decreased as the heating temperature increased. The values of T0 (in minutes) were 444.4 ± 21.8 (r = 0.999) at 41.5°C, 169.9 ± 16.6 (r = 0.996) at 42°C, 93.4 ± 2.6 (r = 0.999) at 42.5°C, 50.4 ± 1.4 (r = 0.999) at 43°C, 35.5 ± 1.7 (r = 0.999) at 43.5°C, 22.1 ± 2.1 (r = 0.997) at 44°C, 17.5 ± 0.5 (r = 0.999) at 44.5°C, 13.9 ± 1.4 (r = 0.996) at 45°C, and 9.6 ± 0.7 (r = 0.998) at 45.5°C.

Arrhenius Plot Analysis.
For quantitative evaluation, we performed Arrhenius plot analysis by plotting the rate constant (min–1; 1/T0 and foci formation of {gamma}H2AX) versus the inverse of the temperature (1/K; Fig. 2CCitation ). We observed an inflection point at 42.5°C in the Arrhenius plot of {gamma}H2AX foci formation and cell killing. Based on the Arrhenius plots, the activation energies of cell killing were estimated to be 643 ± 13 kJ/mol (r = 0.982) above the inflection point and 1,130 ± 103 kJ/mol (r = 0.994) below the inflection point (42.5°C). The energies of {gamma}H2AX foci formation were estimated to be 655 ± 3 kJ/mol (1,087 ± 5 fJ/focus; r = 0.999) above the inflection point (42.5°C) and 1,301 ± 81 kJ/mol (2,160 ± 134 fJ/focus; r = 0.997) below the inflection point (42.5°C). From these results, the calculated number of events required for heat-induced cell killing was 39.8 ± 4.7 {gamma}H2AX foci in H1299 cells.

Dependence of Heat-Induced Phosphorylation of H2AX on the Cell Cycle.
H1299 cells were exposed to X-rays or heat (45.5°C) and then fixed 30 minutes after treatment. The cell cycle distribution and {gamma}H2AX content were assayed by flow cytometry. Typical histograms of radiation- or heat-induced phosphorylation of H2AX are shown in Fig. 3ACitation . Straight lines fit well to curves for the mean {gamma}H2AX intensity of cells treated with X-rays or heat versus dose or time (Fig. 3B)Citation . When G1, S and G2-M phase cells were analyzed independently for expression of {gamma}H2AX, the slopes of the dose-response curves were 55.7 ± 1.1 (r = 0.998), 70.7 ± 0.2 (r = 0.999), and 83.1 ± 0.5 (r = 0.999), respectively. Phosphorylation of H2AX in X-ray–irradiated cells increased depending on the DNA content in the cell. The slopes of the heating time-response curves in G1, S and G2-M phase cells were 7.70 ± 0.11 (r = 0.999), 11.83 ± 0.47 (r = 0.993), and 10.15 ± 0.11 (r = 0.999), respectively. H2AX was phosphorylated more frequently when cells were heated in S phase than in G1 or G2-M phase.



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  		Fig. 3. Dependence of heat-induced phosphorylation of H2AX on the cell cycle. H1299 cells were treated with X-rays or heat (45.5°C) and then fixed 30 minutes after treatment. Cell cycle distribution and {gamma}H2AX content were assayed by flow cytometry. A, typical distributions of DNA content and {gamma}H2AX content. B, phosphorylation of {gamma}H2AX at 30 minutes after treatment with various heating periods and radiation doses. Error bars, SD.

 
Cell Cycle Dependence of Heat-Induced {gamma}H2AX Foci Formation and Cell Killing.
To clarify the cell cycle dependency of heat-induced {gamma}H2AX foci formation and cell killing, H1299 cells were synchronized at mitosis. The cell cycle phase distributions after plating synchronized mitotic cells are shown in Fig. 4ACitation . When H1299 cells were heated at 45.5°C at different cell cycle phases, a linear increase in {gamma}H2AX foci formation was observed in a heating time-dependent manner (Fig. 4B)Citation . The slopes [{gamma}H2AX foci/heating period (min–1)] after plating mitotic cells were 2.65 ± 0.28 (r = 0.963) at 2.5 hours, 2.80 ± 0.11 (r = 0.995) at 5 hours, 2.92 ± 0.21 (r = 0.979) at 7.5 hours, 3.92 ± 0.20 (r = 0.990) at 10 hours, 5.56 ± 0.41 (r = 0.979) at 12.5 hours, 4.25 ± 0.33 (r = 0.975) at 15 hours, and 3.95 ± 0.39 (r = 0.958) at 17.5 hours. We found that {gamma}H2AX foci were formed more frequently when cells were heated in S phase, rather than in the G1 or G2-M phases. The survival curves of cells that were heat-treated at different cell cycle phases are shown in Fig. 4CCitation . The T0 values (in minutes) after plating mitotic cells were 11.49 ± 0.31 (r = 0.999) at 2.5 hours, 13.10 ± 0.87 (r = 0.997) at 5 hours, 10.76 ± 0.79 (r = 0.997) at 7.5 hours, 9.27 ± 0.45 (r = 0.999) at 10 hours, 7.44 ± 0.21 (r = 0.999) at 12.5 hours, 8.63 ± 0.30 (r = 0.999) at 15 hours, and 9.24 ± 0.31 (r = 0.999) at 17.5 hours. Cell mortality was higher when cells were heated in S phase than when they were heated in G1 or G2-M phase. The rate constants (min–1; {gamma}H2AX foci formation and 1/T0) for cells with different cell cycle distributions are shown in Fig. 5DCitation , and the two curves for the rate constants appear similar. From these results, the calculated number of events required to initiate cell killing in H1299 cells with heat was 36.1 ± 3.7.



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  		Fig. 4. Dependence of heat-induced {gamma}H2AX foci formation and cell killing on the cell cycle. H1299 cells synchronized at various cell cycle phases were heated (45.5°C). A, phase percentages after plating mitotic cells. B, {gamma}H2AX foci formation 30 minutes after heat treatment. C, survival curves. D, the rate constants (min–1; focus formation of {gamma}H2AXs and 1/T0) for cells with different cell cycle distributions. Error bars, SD.

 


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  		Fig. 5. Relationship between thermotolerance and heat-induced {gamma}H2AX foci formation. H1299 cells were heated (45.5°C) at various intervals after preheating (45.5°C, 5 minutes). A, {gamma}H2AX foci formation 30 minutes after heat treatment. B, survival curves. Dotted lines show survival curves at 45.5°C for different heating periods without preheating. C, rate constants (min–1; {gamma}H2AX foci formation and 1/T0) for cells heated at different times after preheating. Error bars, SD.

 
Relationship between Thermotolerance and Heat-Induced {gamma}H2AX Foci Formation.
To clarify the relationship between thermotolerance and heat-induced {gamma}H2AX foci formation, H1299 cells were conditioned by preheating at 45.5°C for 5 minutes and then incubated at 37°C for various intervals. After H1299 cells were heated at 45.5°C for different periods, a linear rate of {gamma}H2AX foci formation was observed in a heating time-dependent manner (Fig. 5A)Citation . The slopes [{gamma}H2AX foci/heating period (min–1)] after preheating were 2.21 ± 0.02 (r = 1.000) at 1.5 hours, 1.54 ± 0.02 (r = 1.000) at 3 hours, 1.58 ± 0.01 (r = 1.000) at 6 hours, 2.42 ± 0.16 (r = 0.980) at 12 hours, and 3.34 ± 0.23 (r = 0.982) at 24 hours. The formation of {gamma}H2AX foci was suppressed when cells were heated at 3 to 6 hours after a preheating treatment. The survival curves after the challenging heat treatment are shown in Fig. 5BCitation . The T0 values (in minutes) after preheating were 15.63 ± 1.04 (r = 0.997) at 1.5 hours, 28.54 ± 2.74 (r = 0.993) at 3 hours, 28.82 ± 4.36 (r = 0.990) at 6 hours, 18.47 ± 1.60 (r = 0.997) at 12 hours, and 12.70 ± 0.85 (r = 0.997) at 24 hours. Cell killing was efficiently depressed when cells were incubated at 37°C for 3 to 6 hours after preheating. The rate constants (min–1; {gamma}H2AX focus formation and 1/T0) for cells with different periods of incubation between the preheat and challenging heat treatments are shown in Fig. 5CCitation , and the two rate constant curves appear similar.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Heat-Induced {gamma}H2AX Focus Formation Is Dependent on Double-Strand Breaks.
In this study, heat-induced DSB formation was observed using the neutral comet assay with SYBR Green (Fig. 1)Citation . Heat-treated (45.5°C, 60 minutes) cells and X-ray–irradiated (10 Gy) cells showed the same comet tail moments. Using conventional physical methods, many investigators have also demonstrated that heat induces DNA strand breaks (8, 9, 10) . Because these methods indicated that the efficiency of DSB detection was low, it was necessary to use high-dose radiation or prolonged heat treatment to detect DSBs. On the other hand, it was reported that the formation of {gamma}H2AX foci was a sensitive and specific marker for the existence of a DSB (12 , 13) . Using an immunofluorescence technique with anti-{gamma}H2AX antibody, ionizing radiation-induced {gamma}H2AX foci formation and heat-induced {gamma}H2AX foci formation were observed (Fig. 1D)Citation . The number of {gamma}H2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment and 30 minutes after X-ray irradiation (Fig. 1F)Citation . A linear increase in the number of {gamma}H2AX foci was observed to form in a heating time-dependent manner (Fig. 1E)Citation . The heating time-dependent phosphorylation of H2AX was also confirmed by Western blotting (Fig. 1C)Citation and flow cytometry (Fig. 3B)Citation . The relationship between tail moment and {gamma}H2AX foci induced by heat treatment or X-ray irradiation was also in good accord with a previous report showing that heat-treated (45.5°C, 30 minutes) cells and X-ray–irradiated (5 Gy) cells showed the same extent of DSB formation (11) . One possibility is that heat-induced {gamma}H2AX foci formation results from changes in the structure of chromatin. However, it has been reported that chromatin-modifying treatments (hypotonic conditions, exposure to chloroquine or tricostatin A) failed to induce {gamma}H2AX foci formation (17) . These results suggest that {gamma}H2AX foci formation may be dependent on the induction of a DSB by heat treatment or X-ray irradiation. Although Dikomey (9) detected DNA strand breaks only at temperatures above 45°C, the data reported here show it is possible to detect DSBs generated by heating above 41.5°C using a highly sensitive immunofluorescence assay (Fig. 2A)Citation .

The possibility that heat could induce DSB formation has also been supported by previous studies, in which heat and low pH, as well as genotoxic stresses (ionizing radiation and DNA-damaging agents), up-regulated a tumor suppressor protein (p53) in human glioblastoma cells (16 , 18) . Recently, low pH has been reported to induce DSBs through topoisomerase II activation (19) .

Heat-Induced Cell Killing Is Associated with Double-Strand Break Formation.
It has been accepted that heat-induced cell killing is not dependent on the formation of DSBs, although X-ray–induced cell killing is dependent on DSBs. This conclusion is based on results showing that there was no correlation between thermosensitivity and radiosensitivity (11) or between cell cycle-dependent heat– and X-ray–induced cell killing (20) .

If heat and X-rays induced the same numbers of DSBs, heat– and X-ray–induced cell killing would be expected to show the same kinetics. However, heat-induced cell killing was more efficient than X-ray–induced cell killing. These observations were considered to be evidence to indicate that heat-induced cell killing was caused by mechanisms other than DSBs, the mechanism by which X-rays induce cell killing (11) . Therefore, DSBs were not regarded as an important factor in heat-induced cell killing. In H1299 cells, it was also found that heat-induced cell killing was about two times more effective than X-rays (data not shown). A possibility was that this difference in cell killing kinetics might have been caused by cellular repair of DSBs. It is known that repair enzymes for DSBs are heat sensitive. The residual {gamma}H2AX foci are considered to reflect persistent unrepaired DNA damage. The observations reported here indicate that more {gamma}H2AX foci remain in heat-treated cells than in X-ray–irradiated cells (Fig. 1F)Citation .

Because DSB formation from prolonged heat treatment did not occur in a time-dependent manner (Fig. 1B)Citation , it was difficult to correlate cell death with an event responsible for heat-induced cell killing; possibly because of the high rate of cell death, DSB formation did not occur or was not observed to occur in a time-dependent manner. In contrast, heat-induced cell killing may be caused by DSBs during a shorter period of heat treatment (when there is a time-dependent formation of {gamma}H2AX foci). Interestingly, there was an inflection point at 42.5°C in the Arrhenius plot of cell killing and {gamma}H2AX foci formation (Fig. 2C)Citation . Also the thermal activation energies of both cell killing and foci formation were almost the same above and below the inflection point. From this result, it was inferred that the number of {gamma}H2AX foci determines temperature-dependent cell killing. The activation enthalpy for cell killing was also reported to be similar to that of protein denaturation (5) , such as DNA polymerase ß (21) . Therefore, it is possible that heat may induce DSBs through protein denaturation. Collectively, these results strongly support the notion that DSBs might contribute to temperature-dependent cell killing.

As shown in previous reports (20) , more cells were killed when cells were heated at S phase than at G1 or G2 phase (Fig. 4B)Citation . On the other hand, more cells were killed when cells were irradiated during G1 to early S phase than during late S phase to G2 phase (20) . Such a relatively flat response of radiosensitivity throughout the cell cycle was also reported to be observed in a mammalian cell line deficient in homologous recombination (HR) repair (22 , 23) . Thus, the mechanisms of cell cycle-dependent cell killing by ionizing irradiation might be due to the activity of DSB repair because the HR repair pathway functions after a pair of sister chromatids are generated by DNA replication (23) . However, the mechanisms of cell cycle-dependent cell killing by heat are still unknown. It was reported that there was heat-induced chromosomal damage (24) and DSB formation (11) only at S phase. These phenomena were understood to indicate that heat-induced cell killing was caused by mechanisms other than DSB formation. Using the highly sensitive detection system of immunofluorescence techniques with anti-{gamma}H2AX antibody to detect DSBs, we found {gamma}H2AX foci formed not only at S phase but also at G1 and G2 phase and that more {gamma}H2AX foci were formed when cells were heated at S phase than at G1 or G2 phase (Figs. 3Citation and 4A)Citation . We found that the cell cycle-dependent patterns of the rate constant (min–1) between 1/T0 and foci formation of {gamma}H2AX induced by heat were quite similar (Fig. 4D)Citation . This result strongly supports the notion that DSB formation might contribute to cell cycle-dependent cell killing by heat.

Although ATM-mutated cells were more sensitive to killing by ionizing radiation, both normal and ATM-mutated cells showed similar cell killing efficiencies in response to heat alone (25) . On the other hand, it was reported that low concentrations of wortmannin (10 µmol/L) sensitized cells to hyperthermia (26) , and DNA-PKcs–deficient scid mouse cells were highly sensitive to killing by heat (27) as well as by X-rays. These contradictory results could be explained if the relevant repair pathway in heated cells was not HR repair, but nonhomologous end joining repair. The {gamma}H2AX foci colocalize in the nucleus with DNA repair and checkpoint proteins, including 53BP1, Nbs1, Mre11, Rad50, Rad51, and BRCA1 (28, 29, 30) . H2AX-deficient cells are hypersensitive to ionizing radiation due to the failure to rejoin DSBs and lack formation of 53BP1, BRCA1, and Nbs1 foci (31 , 32) . Recently, Bassing et al. (33) reported that H2AX was not required for nonhomologous end joining repair but was required for HR. In addition, in a DNA damage-sensing supercomplex (BRCA1-associated genome surveillance complex) containing HR-related proteins (34) , heat was reported to cause translocation of Mre11 proteins from the nucleus to the cytoplasm (35) and a rapid disappearance of BRCA1 proteins (36) . Those reports also support the hypothetical explanation proposed above.

Possible Mechanisms of Heat-Induced Double-Strand Break Formation.
It is possible to conclude that heat-induced {gamma}H2AX foci formation is the result of DSB formation through a cellular response. Although few DSBs were detected even at high temperatures (>60°C) with polymerase chain reaction methods, a linearity of DSB formation dependent on the heating period above or below 42.5°C was demonstrated in the present experiments. In the case of severe heat treatment, however, DSB formation reached a plateau at 45.5°C (Fig. 1B)Citation , as shown in a previous report (11) . It is possible that because of the high rate of cell death, DSB formation did not occur in a time-dependent manner. When the cells were heated at 45.5°C for >30 minutes, heat-induced cell killing may not have been caused by DSBs. These results mean that heat might induce DSBs in in vivo cultured cells under physiologic conditions, but not during in vitro enzymatic reactions. In fact, it was reported that heat induces base modifications such as oxidative base damage (37) , abasic DNA sites (38) , deamination of cytosine (39) , and other types of damage through free radical species such as oxygen species (37) and nitrogen species (40) . It was also reported that DNA synthesis enzymes such as DNA polymerase ß are heat sensitive compared with incision enzymes for base excision repair and/or nucleotide excision repair (21) . Therefore, it might be possible that heat indirectly induces nick formation through enzymatic repair processes. DSBs could then develop or appear when nicks form in close proximity to each other on both strands (1) . In the present study, the existence of an inflection point in heat-induced focus formation of {gamma}H2AXs (Fig. 2C)Citation was shown. In addition, a preheat treatment led to heat-induced thermotoleranace (Fig. 5B)Citation . It was confirmed that the number of {gamma}H2AX foci was reduced in preheated cells later treated with a challenging heat exposure (Fig. 5)Citation . From these results, it is suggested that heat induces DSB formation through the denaturation and dysfunction of heat-labile repair proteins such as DNA polymerases. In fact, previous reports indicated a positive correlation between thermal tolerance for heat killing and heat-induced loss of DNA polymerase ß activity in the cells (41 , 42) . It is also hypothesized that a nick is converted to a DSB at a DNA replication fork. Thus, {gamma}H2AX foci might accumulate more readily when cells are heated at S phase than when cells are heated at G1 or G2 phase (Figs. 3Citation and 4)Citation .

In conclusion, it is proposed that heat-induced cell killing might be dependent or associated with DSB formation in mammalian cells.


   FOOTNOTES
 
Grant support: Grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Takeo Ohnishi, Department of Biology, Nara Medical University School of Medicine, Shijo-cho 840, Kashihara, Nara 634-8521, Japan. Phone: 81-744-22-3051, ext. 2264; Fax: 81-744-25-3345; E-mail: tohnishi{at}naramed-u.ac.jp

Received 5/28/04. Revised 9/25/04. Accepted 10/19/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Roti Roti JL Heat-induced cell death and radiosensitization: molecular mechanisms. Natl Cancer Inst Monogr 1982;61:3-10.
  2. Angelidis CE, Lazaridis I, Pagoulatos GN Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance. Eur J Biochem 1991;199:35-9.[Medline]
  3. Burdon RH Thermotolerance and the heat shock proteins. Symp Soc Exp Biol 1987;41:269-83.[Medline]
  4. Kampinga HH, Dikomey E Hyperthermic radiosensitization: mode of action and clinical relevance. Int J Radiat Biol 2001;77:399-408.[CrossRef][Medline]
  5. Dewey WC Arrhenius relationships from the molecule and cell to the clinic. Int J Hyperthermia 1994;10:457-83.[Medline]
  6. Nakahata K, Miyakoda M, Suzuki K, Kodama S, Watanabe M Heat shock induces centrosomal dysfunction, and causes non-apoptotic mitotic catastrophe in human tumour cells. Int J Hyperthermia 2002;18:332-43.[CrossRef][Medline]
  7. Roti Roti JL, Kampinga HH, Malyapa RS, et al Nuclear matrix as a target for hyperthermic killing of cancer cells. Cell Stress Chaperones 1998;3:245-55.[CrossRef][Medline]
  8. Warters RL, Henle KJ DNA degradation in Chinese hamster ovary cells after exposure to hyperthermia. Cancer Res 1982;42:4427-32.[Abstract/Free Full Text]
  9. Dikomey E Effect of hyperthermia at 42°C and 45°C on repair of radiation-induced DNA strand breaks in CHO cells. Int J Radiat Biol Relat Stud Phys Chem Med 1982;41:603-14.[Medline]
  10. Anai H, Maehara Y, Sugimachi K In situ nick translation method reveals DNA strand scission in HeLa cells following heat treatment. Cancer Lett 1988;40:33-8.[CrossRef][Medline]
  11. Wong RS, Dynlacht JR, Cedervall B, Dewey WC Analysis by pulsed-field gel electrophoresis of DNA double-strand breaks induced by heat and/or X-irradiation in bulk and replicating DNA of CHO cells. Int J Radiat Biol 1995;68:141-52.[Medline]
  12. Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM Quantitative detection of 125IdU-induced DNA double-strand breaks with gamma-H2AX antibody. Radiat Res 2002;158:486-92.[CrossRef][Medline]
  13. Rothkamm K, Löbrich M Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc Natl Acad Sci USA 2003;100:5057-62.[Abstract/Free Full Text]
  14. Ward TH, Marples B Technical report: SYBR Green I and the improved sensitivity of the single-cell electrophoresis assay. Int J Radiat Biol 2000;76:61-5.[CrossRef][Medline]
  15. Olive PL, Banath JP, Durand RE Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res 1990;122:86-94.[Medline]
  16. Ohnishi T, Wang X, Ohnishi K, Matsumoto H, Takahashi A p53-dependent induction of WAF1 by heat treatment in human glioblastoma cells. J Biol Chem 1996;271:14510-3.[Abstract/Free Full Text]
  17. Bakkenist CJ, Kastan MB DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature (Lond) 2003;421:499-506.[CrossRef][Medline]
  18. Ohtsubo T, Wang X, Takahashi A, et al p53-dependent induction of WAF1 by low pH culture condition in human glioblastoma cells. Cancer Res 1997;57:3910-3.[Abstract/Free Full Text]
  19. Xiao H, Li TK, Yang JM, Liu LF Acidic pH induces topoisomerase II-mediated DNA damage. Proc Natl Acad Sci USA 2003;100:205-10.[Abstract/Free Full Text]
  20. Westra A, Dewey WC Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int J Radiat Biol 1971;19:467-77.
  21. Spiro IJ, Denman DL, Dewey WC Effect of hyperthermia on CHO DNA polymerases alpha and beta. Radiat Res 1982;89:134-49.[CrossRef][Medline]
  22. Cheong N, Wang X, Wang Y, Iliakis G Loss of S-phase-dependent radioresistance in irs-1 cells exposed to X-rays. Mutat Res 1994;314:77-85.[Medline]
  23. Takata M, Sasaki MS, Sonoda E, et al Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 1998;17:5497-508.[CrossRef][Medline]
  24. Wong RS, Dewey WC Molecular mechanisms for the induction of chromosomal aberrations in CHO cells heated in S phase. Environ Mol Mutagen 1993;22:257-63.[Medline]
  25. Mitchel RE, Chan A, Smith BP, Child SD, Paterson MC The effects of hyperthermia and ionizing radiation in normal and ataxia telangiectasia human fibroblast lines. Radiat Res 1984;99:627-35.[Medline]
  26. Tomita M, Suzuki N, Matsumoto Y, et al Sensitization by wortmannin of heat- or X-ray induced cell death in cultured Chinese hamster V79 cells. J Radiat Res 2000;41:93-102.
  27. Ohnishi T, Komatsu K, Tauchi H, et al Heat-induced accumulation of p53 and hsp72 is suppressed in lung fibroblasts from the SCID mouse. Int J Radiat Biol 2000;76:711-5.[CrossRef][Medline]
  28. Paull TT, Rogakou EP, Yamazaki V, et al A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000;10:886-95.[CrossRef][Medline]
  29. Schultz LB, Chehab NH, Malikzay A, Halazonetis TD p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 2000;151:1381-90.[Abstract/Free Full Text]
  30. Tauchi H, Kobayashi J, Morishima K, et al The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50-hMRE11-NBS1 complex DNA repair activity. J Biol Chem 2001;276:12-5.[Abstract/Free Full Text]
  31. Celeste A, Petersen S, Romanienko PJ, et al Genomic instability in mice lacking histone H2AX. Science (Wash DC) 2002;296:922-7.[Abstract/Free Full Text]
  32. Celeste A, Fernandez-Capetillo O, Kruhlak MJ, et al Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 2003;5:675-9.[CrossRef][Medline]
  33. Bassing CH, Chua KF, Sekiguchi J, et al Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci USA 2002;99:8173-8.[Abstract/Free Full Text]
  34. Wang Y, Cortez D, Yazdi P, et al BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 2000;14:927-39.[Abstract/Free Full Text]
  35. Zhu WG, Seno JD, Beck BD, Dynlacht JR Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat. Radiat Res 2001;156:95-102.[CrossRef][Medline]
  36. Xian Ma Y, Fan S, Xiong J, et al Role of BRCA1 in heat shock response. Oncogene 2003;22:10-27.[CrossRef][Medline]
  37. Bruskov VI, Malakhova LV, Masalimov ZK, Chernikov AV Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA. Nucleic Acids Res 2002;30:1354-63.[Abstract/Free Full Text]
  38. Warters RL, Brizgys LM Apurinic site induction in the DNA of cells heated at hyperthermic temperatures. J Cell Physiol 1987;133:144-50.[CrossRef][Medline]
  39. Lindahl T, Nyberg B Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 1974;13:3405-10.[CrossRef][Medline]
  40. Hall DM, Buettner GR, Matthes RD, Gisolfi CV Hyperthermia stimulates nitric oxide formation: electron paramagnetic resonance detection of .NO-heme in blood. J Appl Physiol 1994;77:548-53.[Abstract/Free Full Text]
  41. Dewey WC, Esch JL Transient thermal tolerance for cell killing and polymerase activities. Radiat Res 1982;92:611-4.[CrossRef][Medline]
  42. Mivechi NF, Dewey WC DNA polymerase alpha and beta activities during the cell cycle and their role in heat radiosensitization in Chinese hamster ovary cells. Radiat Res 1985;103:337-50.[Medline]



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