Residual gammaH2AX foci as an indication of lethal DNA lesions

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.

Figure 1

Development of γH2AX after MNNG treatment. Flow cytometric analysis was used to detect γH2AX formation in V79 cells exposed to MNNG for 30 min and then allowed 1 h to develop γH2AX foci. Single cells were fixed and analyzed for γH2AX antibody binding in relation to DNA content using flow cytometry. Cells that expressed control levels of γH2AX are contained within the boxes and are given as percentages.

Figure 2

Toxicity, DNA damage, and γH2AX formation in V79 cells exposed to MNNG. Panel a: Clonogenic survival after a 30 min exposure of V79 cells to MNNG. Panel b: Expression of γH2AX in V79 cells relative to untreated cells as a function of dose of MNNG. Panel c: Time-dependence of γH2AX formation in V79 cells after exposure to low doses of MNNG. Panel d: Alkali-labile lesions measured using the alkaline comet assay showing drug dose dependence for different times of recovery after a 30 min exposure. Panel e: DNA double-strand breaks measured using the neutral comet assay as a function of drug dose and time after a 30 min exposure. Panel f: Rejoining of alkali-labile lesions as a function of time after exposure of V79 cells to MNNG.

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.

Figure 4

Comparison between clonogenic fraction and fraction of cells lacking residual γH2AX foci for SiHa cells exposed to 8 drugs. With the exception of tirapazamine treatment that was conducted in suspension culture under anoxia, attached cells were exposed at 37°C in complete medium for either 30 min or 60 min (cisplatin, temozolomide). After rinsing, cells were allowed to recover for 24 hours before microscopic analysis of γH2AX foci and plating to measure clonogenic fraction. Combined results from 2-4 experiments are shown.

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.

Figure 6

RAD51-GFP as a live cell marker. Panel a: RAD51-GFP foci in live SiHa cells stably transfected with RAD51-GFP are shown 24 hours after exposure to 8 Gy. The phase contrast image is indicated by red outlines. Panel b: Co-localization between RAD51-GFP and anti-RAD51 antibody staining (red) in an untreated SiHa cell. Panel c: Co-localization between RAD51-GFP (green) and γH2AX antibody staining (red) in SiHa cells 24 hours after exposure to 8 Gy. Panel d: Analysis of the fraction of SiHa-RAD51-GFP cells that develop foci as a function of time after exposure to 4 Gy or 8 Gy. Results from several experiments are pooled. The dotted line and open circles show the development of RAD51 antibody-labelled foci after exposure to 4 Gy. The single open triangle shows the fraction of RAD51 antibody-labeled cells 24 hours after 8 Gy. Panel e: The fraction of cells that exhibit RAD51-GFP foci or γH2AX foci 24 hours after exposure to radiation, measured microscopically. Panel f: SiHa-RAD51-GFP cells expressing high levels of RAD51-GFP after 24 hours after irradiation were sorted on the basis of GFP, fixed and stained for γH2AX. The average intensity of the populations was measured using flow cytometry. The mean and standard error for 3 sorted populations is shown. Panel g: Clonogenicity of SiHa-RAD51-GFP cells after 0 or 3 Gy exposure. Cells in 8-well dishes were irradiated and 24 hours later, wells containing one or two doublets were scored for the presence or absence of γH2AX foci (daughter cell pairs show the same foci patterns). Dishes were returned to the incubator for 2 weeks to form colonies. The fraction of doublets with foci that survived treatment or the fraction lacking foci that survived treatment was calculated.