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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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Toxicity in relation to residual γH2AX in SiHa cells exposed to MNNG. Panel a: Clonogenic surviving fraction after a 30 min exposure of SiHa cells to MNNG followed by a 24 hours recovery period. Panel b: Fraction of cells without γH2AX foci 24 hours after exposure to MNNG. Results are expressed relative to the endogenous expression of γH2AX; typically 60-70% of SiHa cells lack foci. Panel c: Comparison between fraction of cells lacking residual γH2AX foci and clonogenic fraction. Panels d-f: Distribution of γH2AX foci per cell. Panels g-i: Representative antibody-stained images of cells exposed 0, 0.5 and 1 μg/ml MNNG showing foci numbers and distribution.
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Figure 3: Toxicity in relation to residual γH2AX in SiHa cells exposed to MNNG. Panel a: Clonogenic surviving fraction after a 30 min exposure of SiHa cells to MNNG followed by a 24 hours recovery period. Panel b: Fraction of cells without γH2AX foci 24 hours after exposure to MNNG. Results are expressed relative to the endogenous expression of γH2AX; typically 60-70% of SiHa cells lack foci. Panel c: Comparison between fraction of cells lacking residual γH2AX foci and clonogenic fraction. Panels d-f: Distribution of γH2AX foci per cell. Panels g-i: Representative antibody-stained images of cells exposed 0, 0.5 and 1 μg/ml MNNG showing foci numbers and distribution.

Mentions: The relation between clonogenic surviving fraction (Fig 3a) and fraction of cells with residual γH2AX foci (Fig. 3b) was then compared using SiHa human cervical carcinoma cells exposed for 30 min to MNNG. To improve resolution for detecting the relevant resistant cells within the population, the fraction of nuclei lacking foci was scored microscopically 24 hours after exposure to MNNG. The fraction of cells lacking foci at 24 hours was then compared with the fraction of cells from the same treated population that retained clonogenicity as measured two weeks later. A good correlation was observed between clonogenicity and fraction of cells lacking foci 24 hours after treatment (Fig. 3c) and there was a progressive increase in the number of foci per cell over this dose range (Fig. 3d-f). Representative images of MNNG-treated cells show the heterogeneity in foci number per nucleus (Fig. 3g-i). Therefore unlike the neutral comet assay, retention of γH2AX foci appears to have the requisite sensitivity to predict cell survival over the first log or two of cell killing.


Residual gammaH2AX foci as an indication of lethal DNA lesions.

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

Toxicity in relation to residual γH2AX in SiHa cells exposed to MNNG. Panel a: Clonogenic surviving fraction after a 30 min exposure of SiHa cells to MNNG followed by a 24 hours recovery period. Panel b: Fraction of cells without γH2AX foci 24 hours after exposure to MNNG. Results are expressed relative to the endogenous expression of γH2AX; typically 60-70% of SiHa cells lack foci. Panel c: Comparison between fraction of cells lacking residual γH2AX foci and clonogenic fraction. Panels d-f: Distribution of γH2AX foci per cell. Panels g-i: Representative antibody-stained images of cells exposed 0, 0.5 and 1 μg/ml MNNG showing foci numbers and distribution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819996&req=5

Figure 3: Toxicity in relation to residual γH2AX in SiHa cells exposed to MNNG. Panel a: Clonogenic surviving fraction after a 30 min exposure of SiHa cells to MNNG followed by a 24 hours recovery period. Panel b: Fraction of cells without γH2AX foci 24 hours after exposure to MNNG. Results are expressed relative to the endogenous expression of γH2AX; typically 60-70% of SiHa cells lack foci. Panel c: Comparison between fraction of cells lacking residual γH2AX foci and clonogenic fraction. Panels d-f: Distribution of γH2AX foci per cell. Panels g-i: Representative antibody-stained images of cells exposed 0, 0.5 and 1 μg/ml MNNG showing foci numbers and distribution.
Mentions: The relation between clonogenic surviving fraction (Fig 3a) and fraction of cells with residual γH2AX foci (Fig. 3b) was then compared using SiHa human cervical carcinoma cells exposed for 30 min to MNNG. To improve resolution for detecting the relevant resistant cells within the population, the fraction of nuclei lacking foci was scored microscopically 24 hours after exposure to MNNG. The fraction of cells lacking foci at 24 hours was then compared with the fraction of cells from the same treated population that retained clonogenicity as measured two weeks later. A good correlation was observed between clonogenicity and fraction of cells lacking foci 24 hours after treatment (Fig. 3c) and there was a progressive increase in the number of foci per cell over this dose range (Fig. 3d-f). Representative images of MNNG-treated cells show the heterogeneity in foci number per nucleus (Fig. 3g-i). Therefore unlike the neutral comet assay, retention of γH2AX foci appears to have the requisite sensitivity to predict cell survival over the first log or two of cell killing.

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