The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

Dynlacht, Joseph R.; Bittner, Michaela; Bethel, Jody A.; Beck, Brian D.

doi:10.1002/jcp.10334

Cited by 17 publications

(13 citation statements)

References 63 publications

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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.

302

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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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.

302

288

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“…Since we have previously shown that Ku-dependent NHEJ is not involved in heat radiosensitization (39), by deduction one might assume that either D-NHEJ (alternative NHEJ) or HR, or both pathways, must also be implicated in heat radiosensitization, since Mre11 is known to function in both pathways. Using an siRNA approach, cells in which Mre11 was knocked down to 30–40% of control levels were significantly radiosensitized (42).…”

Section: Discussionmentioning

confidence: 99%

“…Such a finding (loss of activity of a critical repair enzyme after heat shock) would not be without precedent. Although Ku-dependent NHEJ is not involved in heat radiosensitization (39), the kinase and DNA-binding activity of DNA-PK and both Ku subunits, respectively, are known to decrease rapidly during hyperthermia treatment (45, 46). …”

Section: Discussionmentioning

confidence: 99%

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Identification of Mre11 as a Target for Heat Radiosensitization

et al. 2011

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Thermal radiosensitization is believed to be mediated by an inhibition of double-strand break (DSB) repair, but the exact mechanism of radiosensitization remains to be elucidated. Previously, we demonstrated that proteins of the Mre11/Rad50/Nbs1 complex (MRN) translocate from the nucleus to the cytoplasm in cells have that been heated or heated and then irradiated; this finding led us to propose that heat radiosensitization was due at least in part to translocation of MRN. In the current study, we used leptomycin B to inhibit MRN translocation in heated, irradiated cells, but we found that heat radiosensitization was not altered. Thus enhanced radiosensitivity was not attributed to translocation of MRN proteins. To determine which of the MRN subunits contributed to heat radiosensitization, we compared the extent of heat radiosensitization in wild-type cells with that of cells hypomorphic for Mre11 or Nbs1 or cells in which the level of Rad50 was suppressed. We found that neither Nbs1 nor Rad50 is involved in heat radiosensitization, because a similar amount of heat radiosensitization was observed in cells deficient in those proteins compared to cells expressing normal levels. However, heat radiosensitization was not observed in A-TLD1 cells deficient in Mre11. Measurement of exonuclease activity of purified Mre11 heated at 42.5°C or 45.5°C indicated that the protein is very heat-labile. Immunoprecipitation of Mre11 from heated HeLa cells also revealed that hsp70 associates with Mre11 and that this association is maintained long after heating. Taken together, these findings implicate Mre11 as a target for heat radiosensitization and suggest that heat radiosensitization and inhibition of DSB repair may be mediated by heat-induced conformational changes in Mre11.

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“…Ku80 becomes aggregated 54,55 and loses its activity rapidly after cells are heated 47,48,56,57 , but even before these findings (and indeed, the identification of the role of the Ku70/80 heterodimer in DSB repair), evidence for a role for Ku80 and for the NHEJ pathway itself in heat radiosensitization had been proposed 58 . However, Woudstra et al 59 and Dynlacht et al 55 have concluded that Ku and DNA ligase IV (and thus NHEJ) are not critical for heat radiosensitization and are suggestive that other pathways for DSB repair, notably HR, may be a target for radiosensitization. A review of pertinent studies concerning the role of NHEJ and HR in heat radiosensitization may be found elsewhere in this issue 60 .…”

Section: Introductionmentioning

confidence: 96%

Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells

Dynlacht

Pandita

et al. 2004

International Journal of Hyperthermia

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The mechanism by which hyperthermia sensitizes mammalian cells to ionizing radiation remains to be elucidated, but an overwhelming amount of circumstantial evidence suggests that heat radiosensitization might be mediated by inhibition of double-strand break repair, particularly after exposure of irradiated cells to heat treatments in excess of about 43 degrees C. In mammalian cells, double-strand break repair usually occurs via two pathways, non-homologous end-joining and homologous recombination. Several reports suggest a role for non-homologous end-joining in heat radiosensitization, while others implicate homologous recombination as a target. However, cell lines that are compromised in either the non-homologous end-joining or homologous recombination pathway are still capable of being radiosensitized, suggesting that heat affects both pathways. Indeed, several of the proteins involved in one or both of these pathways have been observed to undergo alterations or translocation after unirradiated or irradiated cells are exposed to heat shock. The work summarized in this review implicates proteins of the Mre11/Rad50/Nbs1 complex as targets for heat radiosensitization.

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The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

Cited by 17 publications

References 63 publications

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

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

Identification of Mre11 as a Target for Heat Radiosensitization

Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells

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