γH2AX-positive Cells Research Articles

Measurement of gamma-H2AX in circulating tumor cells (CTC) from patients receiving ABT-888 (veliparib) in combination with carboplatin on a phase I dose-escalation study (OSU-10080/NCI8609).

58 Background: Non-invasive methods to accurately measure DNA damage induced by PARP inhibitors in the tumors are crucial in identifying the optimal biological dose and schedule of these novel agents. With the use of an antibody that targets the histone H2AX, which becomes phosphorylated on serine residue 139 (γ-H2AX), it is possible to measure DNA DSB in the cell. But repeated biopsy in patients with metastatic breast cancer is not feasible. We propose here to quantify γ-H2AX in CTCs as a measurement of DNA damage in the tumor in patients receiving veliparib (ABT-888), an oral PARP inhibitor and carboplatin on a phase I study. Methods: CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points. Immunocytochemical staining for nucleus (DAPI), pan-cytokeratin CD45, and γ-H2AX was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. The γ-H2AX foci present in the nucleus of the CTCs will be counted by immunocytochemistry (ICC) analysis. Results: We negatively enriched blood sample from enrolled patients with metastatic breast cancer (n=6). CTCs were observed in all samples (55CTCs/mL of blood - 2000CTCs/mL of blood). Thus far 3 out of 6 patient samples have been analyzed. The number of CTCs per mL of blood after enrichment at baseline has a mean of 293, a median of 140 and a range of (684-140) At time point 1 the mean is 1071.33, the median is 1151 and the range is (1151-63). ICC analysis on the CTCs demonstrated the presence of γ-H2AX positive cells. Conclusions: Our novel negative depletion method for isolating CTCs allows for separation of a wider range of CTCs, since it does not assume presence of epithelial markers before separation. This leads to improved yield and purity allowing better ICC detection of various markers directly on CTCs. Development and validation of the above methodology to determine γ-H2AX foci in CTCs will help in identifying a novel, non-invasive biomarker that can be used to assess the effect of DNA damaging agents during therapy.

Effect of Pre-treatment Imaging Dose on the Radiosensitivity of Tumor Cells

Effect of Pre-treatment Imaging Dose on the Radiosensitivity of Tumor Cells

Monitoring Drug-Induced γH2AX as a Pharmacodynamic Biomarker in Individual Circulating Tumor Cells

Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker gammaH2AX in individual CTCs of patients treated with chemotherapeutic agents. An Alexa Fluor 488-conjugated monoclonal gammaH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage-dependent nuclear gammaH2AX signals in individual CTCs. Time-course changes in both CTC number and gammaH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment. The percentage of gammaH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent gammaH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count. These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research.

Development of Laser Plasma X-ray Microbeam Irradiation System and Radiation Biological Application

Laser plasma x-ray source has the features such as ultra short pulse, high brilliance, monochromaticity, and focusing ability. These features are excellent compared with conventional x-ray source. In order to apply the laser plasma x-ray source to the biomedical study and to more closely research the radiobilogical responce of the cancer cell such as radiation induced bystander effect, we have developed x-ray microbeam system using laser plasma x-ray source. The absorbed dose of laser plasma x-ray was estimated with Gafchromic EBT film and DNA double strand breaks on the cells were detected by immunofluorescence staining. When the cells were irradiated with laser plasma x-ray, the circular regions existing γ-H2AX positive cells were clearly identified. The usefulness of the laser plasma x-ray on the radiobiological study was proved in this research.

DNA damage response to the Mdm2 inhibitor Nutlin-3

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM 0 colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin-1). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a ∼50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double-strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double-stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander γ-H2AX induction. In this paper, we used normal (ρ +) and mtDNA-depleted (ρ 0) human–hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with γ-H2AX, we found that the fraction of γ-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated ρ + cells at 10 min post-irradiation (ρ + ICCM 10 min ) caused larger increases of bystander γ-H2AX induction comparing to ρ 0 ICCM 10 min , which only caused a slight increase of bystander γ-H2AX induction. The ρ + ICCM 10 min could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with ρ 0 ICCM 10 min . We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched γ-H2AX induction by ρ + ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59 − gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of ρ + ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser(139) (gamma-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of gamma-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser(1981) was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation.

XRCC3 Depletion Induces Spontaneous DNA Breaks and p53-Dependent Cell Death

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G2/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of γH2AX expression shows that S and G2/M phase cells express the highest fraction of γH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of γH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of γH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.