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Residual gammaH2AX foci as an indication of lethal DNA lesions.

Banáth JP, Klokov D, MacPhail SH, Banuelos CA, Olive PL - BMC Cancer (2010)

Bottom Line: This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication.To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity.Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Biophysics Department, BC Cancer Agency Research Centre, 675 W, 10th Ave, Vancouver, BC V5Z 1L3, Canada.

ABSTRACT

Background: Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible gammaH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication.

Methods: To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci.

Results: For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained gammaH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained gammaH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.

Conclusion: Retention of DNA damage-induced gammaH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain gammaH2AX foci.

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The fraction of cells with residual RAD51 foci is correlated with the fraction of cells that die. Panel a: SiHa cells were exposed to X-rays and allowed to recover for 24 hours. Cells were fixed and co-immunostained for γH2AX and RAD51, and cells with foci were scored. Results are the means and SD for 3 experiments. Panel b: The fraction of SiHa cells lacking RAD51 foci is compared with the clonogenic fraction measured 24 hours after exposure to X-rays. Panel c: Several cell lines were exposed to 2 Gy, allowed to recover for 24 hours, and then examined for the fraction of cells that lacked RAD51 foci. Panel d: RAD51 (green) and γH2AX (red) antibody staining of SiHa cells 24 hours after exposure to 2 Gy. Nuclei are stained blue with DAPI. Panel e: 24 hours after exposure to 8 Gy. Note the micronuclei stained with antibodies to RAD51 and/or γH2AX foci and the co-localization of some foci in some cells but not all cells.
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Figure 5: The fraction of cells with residual RAD51 foci is correlated with the fraction of cells that die. Panel a: SiHa cells were exposed to X-rays and allowed to recover for 24 hours. Cells were fixed and co-immunostained for γH2AX and RAD51, and cells with foci were scored. Results are the means and SD for 3 experiments. Panel b: The fraction of SiHa cells lacking RAD51 foci is compared with the clonogenic fraction measured 24 hours after exposure to X-rays. Panel c: Several cell lines were exposed to 2 Gy, allowed to recover for 24 hours, and then examined for the fraction of cells that lacked RAD51 foci. Panel d: RAD51 (green) and γH2AX (red) antibody staining of SiHa cells 24 hours after exposure to 2 Gy. Nuclei are stained blue with DAPI. Panel e: 24 hours after exposure to 8 Gy. Note the micronuclei stained with antibodies to RAD51 and/or γH2AX foci and the co-localization of some foci in some cells but not all cells.

Mentions: As it was not possible to confirm directly that cells that retained γH2AX foci 24 hours after treatment were the cells that died, we attempted to accomplish this in another way by using live-cell analysis of SiHa cells expressing RAD51-GFP. First, it was important to confirm results of a previous study that showed that the fraction of cells that died after irradiation was correlated with the fraction of cells with RAD51 foci 24 hours after irradiation [6]. Exponentially growing SiHa cells were exposed to ionizing radiation and allowed to recover for 24 hours before plating for survival or fixing in 2% paraformaldehyde for analysis of RAD51 and γH2AX foci. The fraction of SiHa cells that expressed γH2AX and RAD51 foci 24 hours after irradiation increased with dose, and the majority of foci-positive cells exhibited both foci (Fig. 5a), although not necessarily co-localized. About 5-10% of the cells that lacked RAD51 foci exhibited γH2AX foci, but <5% of cells showed RAD51 foci in the absence of γH2AX foci. As mitotic cells did not exhibit RAD51 foci, this could account in part for the presence of residual γH2AX foci in the absence of RAD51 foci. Differences in rate of foci development and removal may also account for minor discrepancies. The correlation between the fraction of SiHa cells that lacked RAD51 foci and surviving fraction after irradiation was excellent (Fig. 5b). To determine whether this correlation would hold for other cell types, several cell lines were exposed to 2 Gy and examined for the presence of RAD51 foci 24 hours after irradiation. In agreement with results of Sak et al. [6], the fraction of cells that exhibited RAD51 foci 24 hours after exposure to 2 Gy was higher for the more radiosensitive cell lines (Fig. 5c). RAD51 foci appeared in many micronuclei 24 hours after exposure, either alone or with γH2AX foci, and daughter cell pairs showed similar RAD51 foci patterns as has been reported for γH2AX foci (Fig 5d, e). Although RAD51 foci may only mark a subset of the double-strand breaks, the number of residual RAD51 foci per cell was not invariably lower than the number of residual γH2AX foci (Fig. 5e). Therefore, residual RAD51 foci appear to behave much like residual γH2AX foci, at least at 24 hours post-treatment, and should therefore be useful for predicting response to treatment.


Residual gammaH2AX foci as an indication of lethal DNA lesions.

Banáth JP, Klokov D, MacPhail SH, Banuelos CA, Olive PL - BMC Cancer (2010)

The fraction of cells with residual RAD51 foci is correlated with the fraction of cells that die. Panel a: SiHa cells were exposed to X-rays and allowed to recover for 24 hours. Cells were fixed and co-immunostained for γH2AX and RAD51, and cells with foci were scored. Results are the means and SD for 3 experiments. Panel b: The fraction of SiHa cells lacking RAD51 foci is compared with the clonogenic fraction measured 24 hours after exposure to X-rays. Panel c: Several cell lines were exposed to 2 Gy, allowed to recover for 24 hours, and then examined for the fraction of cells that lacked RAD51 foci. Panel d: RAD51 (green) and γH2AX (red) antibody staining of SiHa cells 24 hours after exposure to 2 Gy. Nuclei are stained blue with DAPI. Panel e: 24 hours after exposure to 8 Gy. Note the micronuclei stained with antibodies to RAD51 and/or γH2AX foci and the co-localization of some foci in some cells but not all cells.
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Related In: Results  -  Collection

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Figure 5: The fraction of cells with residual RAD51 foci is correlated with the fraction of cells that die. Panel a: SiHa cells were exposed to X-rays and allowed to recover for 24 hours. Cells were fixed and co-immunostained for γH2AX and RAD51, and cells with foci were scored. Results are the means and SD for 3 experiments. Panel b: The fraction of SiHa cells lacking RAD51 foci is compared with the clonogenic fraction measured 24 hours after exposure to X-rays. Panel c: Several cell lines were exposed to 2 Gy, allowed to recover for 24 hours, and then examined for the fraction of cells that lacked RAD51 foci. Panel d: RAD51 (green) and γH2AX (red) antibody staining of SiHa cells 24 hours after exposure to 2 Gy. Nuclei are stained blue with DAPI. Panel e: 24 hours after exposure to 8 Gy. Note the micronuclei stained with antibodies to RAD51 and/or γH2AX foci and the co-localization of some foci in some cells but not all cells.
Mentions: As it was not possible to confirm directly that cells that retained γH2AX foci 24 hours after treatment were the cells that died, we attempted to accomplish this in another way by using live-cell analysis of SiHa cells expressing RAD51-GFP. First, it was important to confirm results of a previous study that showed that the fraction of cells that died after irradiation was correlated with the fraction of cells with RAD51 foci 24 hours after irradiation [6]. Exponentially growing SiHa cells were exposed to ionizing radiation and allowed to recover for 24 hours before plating for survival or fixing in 2% paraformaldehyde for analysis of RAD51 and γH2AX foci. The fraction of SiHa cells that expressed γH2AX and RAD51 foci 24 hours after irradiation increased with dose, and the majority of foci-positive cells exhibited both foci (Fig. 5a), although not necessarily co-localized. About 5-10% of the cells that lacked RAD51 foci exhibited γH2AX foci, but <5% of cells showed RAD51 foci in the absence of γH2AX foci. As mitotic cells did not exhibit RAD51 foci, this could account in part for the presence of residual γH2AX foci in the absence of RAD51 foci. Differences in rate of foci development and removal may also account for minor discrepancies. The correlation between the fraction of SiHa cells that lacked RAD51 foci and surviving fraction after irradiation was excellent (Fig. 5b). To determine whether this correlation would hold for other cell types, several cell lines were exposed to 2 Gy and examined for the presence of RAD51 foci 24 hours after irradiation. In agreement with results of Sak et al. [6], the fraction of cells that exhibited RAD51 foci 24 hours after exposure to 2 Gy was higher for the more radiosensitive cell lines (Fig. 5c). RAD51 foci appeared in many micronuclei 24 hours after exposure, either alone or with γH2AX foci, and daughter cell pairs showed similar RAD51 foci patterns as has been reported for γH2AX foci (Fig 5d, e). Although RAD51 foci may only mark a subset of the double-strand breaks, the number of residual RAD51 foci per cell was not invariably lower than the number of residual γH2AX foci (Fig. 5e). Therefore, residual RAD51 foci appear to behave much like residual γH2AX foci, at least at 24 hours post-treatment, and should therefore be useful for predicting response to treatment.

Bottom Line: This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication.To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity.Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Biophysics Department, BC Cancer Agency Research Centre, 675 W, 10th Ave, Vancouver, BC V5Z 1L3, Canada.

ABSTRACT

Background: Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible gammaH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication.

Methods: To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci.

Results: For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained gammaH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained gammaH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.

Conclusion: Retention of DNA damage-induced gammaH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain gammaH2AX foci.

Show MeSH
Related in: MedlinePlus