A DNA double-strand break repair-deficient mutant of CHO cells shows reduced radiosensitization after exposure to hyperthermic temperatures in the plateau phase of growth

Iliakis, George; Seaner, Robert

doi:10.3109/02656739009140827

Cited by 35 publications

(13 citation statements)

References 45 publications

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“…However, a di erent cell line and di erent temperatures were used than in the study by Wong et al (1995), and thus the results may not be directly comparable. In general these studies agree with and support the earlier ® ndings that hyperthermia can reduce the repair of radiation induced dsb's, even though in many of those experiments the DNA break studies were done at much higher doses than the survival studies (Corry et al 1977, Mills and Meyn 1981, Dikomey 1982, Jorritsma and Konings 1983, Radford 1983, 1986, Warters et al 1987, Raaphorst 1989, Iliakis and Seaner 1990, Dikomey and Franzke 1992, Warters and Axtell 1992, Warters 1993, Nevaldine et al 1994, Wong et al 1995, DahmDaphi et al 1997. Thus the studies of comparing survival and dsb production and repair at equal doses help validate the earlier studies, in which the dsb experiments tended to have much higher doses than the survival experiments.…”

Section: Discussionsupporting

confidence: 80%

“…However, in order to assess the damage at the same dose range as used in survival, many investigators evaluated the e ects on DNA single stranded breaks (ssb), which do not correlate well with survival (Corry et al 1977, Clark et al 1981, Mills and Meyn 1981, Jorritsma and Konings 1983, Warters et al 1987, Dikomey and Franzke 1992, or others have attempted to measure survival at low doses whilst measuring DNA double stranded breaks (dsbs) at very high doses (Dikomey 1982, Radford 1983, Iliakis and Seaner 1990, Warters and Axtell 1992. Most of these studies showed that hyperthermia had a inhibitory e ect on DNA strand break repair and support the concept that its radiosensitizing e ect is through the inhibition of repair of DNA damage.…”

Section: Introductionmentioning

confidence: 99%

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Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double strand breaks

Raaphorst

1999

International Journal of Hyperthermia

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Human melanoma cells (SK-mel-3) were treated with combinations of radiation and hyperthermia treatment and survival (using the colony forming assay) and DNA double strand breaks (dsb's) (using pulsed field gel electrophoresis) were measured for immediate and delayed plating. The cells were treated in plateau phase, so that delayed plating would result in repair of potentially lethal damage (PLD). Delayed plating showed PLDR for both the survival and the dsb end-point. One hour of heating after irradiation showed a temperature dependent increase in radiosensitization for both the survival endpoint and the dsb endpoint for the temperature range from 42 to 45 degrees C. One hour of heating at 43 degrees C after irradiation resulted in the partial inhibition of PLDR and recovery of dsb's. For heating at 45 degrees C the inhibition of dsb repair was complete. There was good correlation between the survival endpoint and the dsb endpoint for the thermal radiosensitization for both the immediate plating and the PLDR protocols. These data indicate that hyperthermia inhibition of repair of PLD is probably due to the inhibition of dsb rejoining. These correlations were made at the same dose levels for survival and dsb analysis, thus avoiding the potential complications of many earlier studies which used much higher doses for dsb analysis than for survival studies.

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Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double strand breaks

Raaphorst

1999

International Journal of Hyperthermia

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“…Our results do not prove that degradation of BRCA2 is the sole or even most important effect of heat on DNA repair or on other cellular processes relevant for cell killing. In fact, multiple hyperthermia targets have been described (29)(30)(31)(32)(33)(34). They do demonstrate, however, that hyperthermia can be a powerful, noninvasive tool to locally introduce BRCA2 degradation and HR deficiency.…”

Section: Discussionmentioning

confidence: 99%

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Krawczyk

Eppink²,

Essers³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

300

288

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Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5°C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized ondemand induction of HR deficiency, an approach that we term induced synthetic lethality.anti-cancer treatment | RAD51 | double-strand break M any anti-cancer therapies are based on cytotoxicity of DNA double strand breaks (DSBs) induced by ionizing radiation or, indirectly, by chemical agents. However, efficient DSB repair mechanisms protect cells from the genotoxic effects of DSBs, thereby reducing the effectiveness of the therapies. Two major pathways are involved in DSB repair in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR uses intact homologous DNA sequences, usually the sister chromatid in postreplicative chromatin, to faithfully restore DNA breaks (1), whereas NHEJ operates throughout the entire cell cycle and does not require a DNA template (2). Agents inhibiting DNA repair processes potentiate the cytotoxicity of DSBs in cancer therapy (3). Elevated temperature is one such agent that, via unclear mechanisms, interferes with multiple pathways of DNA repair (4-6) and is clinically applied (7). ResultsTo investigate if HR, among other processes and DSB repair pathways, is influenced by elevated temperature, we used an isogenic set of mouse embryonic stem (ES) cells that are either HR proficient (wild-type) or HR deficient (Rad54 −/− ) due to the disruption of the Rad54 gene, which is important for HR activity (1). We compared radiosensitization of these cells by incubating them at 37°C or 41°C before irradiation. For this and subsequent experiments we chose temperatures below 43°C, because they are relevant in clinical practice (8). Interestingly, we observed that wild-type but not Rad54 −/− cells were radiosensitized by preincubation at 41°C compared with cells incubated at 37°C (Fig. 1A). Similarly, HeLa cells, in which the important HR factors XRCC3 or BRCA2 were down-regulated using siRNA, were refractory to further temperature-mediated radiosensitization (Fig. 1B and Fig. S1). These results suggest that elevated temperature inactivates HR. To directly measure the effect of temperature on HR, we quantitated HR-mediated gene targeting in ES cells (9) and found that the efficiency of gene targeting was significantly reduced by preincubation at 41°C compared with 37°C (Fig. 1C). Similarly, preincubation at 41°C reduced the frequency of spontaneous and mitomycin C-induced sister chromatid exchanges in SW-1573 cells (Fig. S2A), w...

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“…Interestingly, some cell lines deficient in Ku protein or DNA-PKcs do not show thermal radiosensitization (i.e. their intrinsically high sensitivity to radiation is not further increased by heat) (21,22). This implicates DNA-PK in the thermal radiosensitization phenomenon.…”

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confidence: 99%

DNA-dependent Protein Kinase Protects against Heat-induced Apoptosis

Nueda¹,

Hudson

Mivechi

et al. 1999

Journal of Biological Chemistry

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Purified heat shock transcription factor 1 (HSF1) binds to both the regulatory and catalytic components of the DNA-dependent protein kinase (DNA-PK). This observation suggests that DNA-PK may have a physiological role in the heat shock response. To investigate this possibility, we performed a comparison of cell lines that were deficient in either the Ku protein or the DNA-PK catalytic subunit versus the same cell lines that had been rescued by the introduction of a functional gene. DNA-PK-negative cell lines were up to 10-fold more sensitive to heat-induced apoptosis than matched DNA-PK-positive cell lines. There may be a regulatory interaction between DNA-PK and HSF1 in vivo, because constitutive overexpression of HSF1 sensitized the DNA-PK-positive cells to heat but had no effect in DNA-PK-negative cells. The initial burst of hsp70 mRNA expression was similar in DNA-PK-negative and -positive cell lines, but the DNA-PK-negative cells showed an attenuated rate of mRNA synthesis at later times and, in some cases, lower heat shock protein expression. These findings provide evidence for an antiapoptotic function of DNA-PK that is experimentally separable from its mechanical role in DNA double strand break repair.The DNA-dependent protein kinase (DNA-PK) 1 is composed of a 470-kDa catalytic subunit (DNA-PKcs) and a 70/80-kDa heterodimeric regulatory component known as Ku protein. Ku protein binds avidly to DNA ends (1) and recruits DNA-PKcs to form an active complex (2-4). Mutations in DNA-PKcs or Ku protein have been described in mammals, flies, and yeast, and in each case, the mutant organism is deficient in double strand break repair, sensitive to ionizing radiation, or both (reviewed in Refs. 5 and 6). Binding of DNA-PK components to DNA is believed to be the first step in a repair pathway that involves a number of other gene products (reviewed in Ref. 7).It is not clear if the ability of DNA-PK to protect against the cytotoxic effect of ionizing radiation is attributable solely to the mechanical role of the enzyme in DNA repair or whether DNA-PK also participates in antiapoptotic signaling. There have been several reports that DNA-PK physically interacts with signaling molecules, including poly(A)DP-ribose polymerase, IB, Vav, c-Abl, and certain transcription factors (8 -12), but the physiological significance of these interactions has not yet been fully explored.In the present study, we investigate the in vivo role of a previously described interaction between DNA-PK and the heat shock transcription factor, HSF1 (12, 13). These prior studies have shown that purified HSF1 binds to both the Ku protein and DNA-PKcs (13). The binding requires a phylogenetically conserved region of HSF1 that includes amino acids 203-280 and results in a stimulation of DNA-PK phosphorylation activity of up to 20-fold in an in vitro reaction (13). These observations suggest that DNA-PK may be involved in regulating some aspect of the heat shock response in vivo.Other evidence also suggests an involvement of DNA-PK in the response to ...

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A DNA double-strand break repair-deficient mutant of CHO cells shows reduced radiosensitization after exposure to hyperthermic temperatures in the plateau phase of growth

Cited by 35 publications

References 45 publications

Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double strand breaks

Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double strand breaks

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

DNA-dependent Protein Kinase Protects against Heat-induced Apoptosis

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