Judit P. Banáth

Radiation Sensitivity, H2AX Phosphorylation, and Kinetics of Repair of DNA Strand Breaks in Irradiated Cervical Cancer Cell Lines

Six human cervical cancer cell lines [five human papillomavirus (HPV) positive, one HPV negative] for induction and rejoining of DNA strand breaks and for kinetics of formation and loss of serine 139 phosphorylated histone H2AX (γH2AX). X-rays induced the same level of DNA breakage for all cell lines. By 8 hours after 20 Gy, <2% of the initial single-strand breaks remained and no double-strand breaks could be detected. In contrast, 24 hours after irradiation, γH2AX representing up to 30% of the initial signal still present. SW756 cells showed almost four times higher background levels of γH2AX and no residual γH2AX compared with the most radiosensitive HPV-negative C33A cells that showed the lowest background and retained 30% of the maximum level of γH2AX. Radiation sensitivity, measured as clonogenic-surviving fraction after 2 Gy, was correlated with the fraction of γH2AX remaining 24 hours after irradiation. A substantial correlation with γH2AX loss half-time measured over the first 4 hours was seen only when cervical cell lines were included in a larger series of p53-deficient cell lines. Interestingly, p53 wild-type cell lines consistently showed faster γH2AX loss half-times than p53-deficient cell lines. We conclude that cell line-dependent differences in loss of γH2AX after irradiation are related in part to intrinsic radiosensitivity. The possibility that the presence of γH2AX foci may not always signify the presence of a physical break, notably in some tumor cell lines, is also supported by these results.

Radiation-Induced Apoptosis Measured in TK6 Human B Lymphoblast Cells Using the Comet Assay

The comet assay, a sensitive method of measuring DNA strand breaks in individual cells, is also capable of identifying apoptotic cells which contain highly fragmented DNA. This method requires embedding cells in agarose, lysing cells to remove proteins, and providing a brief exposure to an electric field to allow broken pieces of DNA to migrate. TK6 human B lymphoblast cells undergo fragmentation which is dependent on both time after irradiation and radiation dose. While some TK6 cells undergo apoptosis within 2 h after irradiation, the fragmentation rate increases approximately 10 h after exposure to radiation doses of 2.5 to 15 Gy. Results confirm that apoptosis is a very rapid event since few cells with intermediate amounts of DNA damage were detected. The comet assay detected apoptotic TK6 cells much earlier than a flow cytometry method.

Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells

Abstract MacPhail, S. H., Banáth, J. P., Yu, Y. T., Chu, E. and Olive, P. L. Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells. Radiat. Res. 159, 759–767 (2003). Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (γH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in γH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced γH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of γH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of γH2AX. With the exception of xrs5 cells, γH2AX level was approximately 3 times lower in unirradiated G1-phase cells than S- and G2-phase cells, and the slope of the G1-phase dose–response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G1-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5–10-fold higher levels of γH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of γH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G2-phase cells.

Kinetics of H2AX phosphorylation after exposure to cisplatin

Cisplatin is a widely used cancer chemotherapeutic drug that causes DNA crosslinking and stimulates H2AX phosphorylation. Our goal was to assess the potential of γH2AX to help predict tumor response to cisplatin treatment.

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix

Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF‐1α and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix.

Analysis of DNA damage in individual cells

Publisher Summary DNA is the most common intracellular target for the action of agents used to treat malignant disease, and it is an essential target for mutagenic agents. The comet or single-cell gel electrophoresis method can detect, either directly or indirectly, a variety of DNA lesions, including single-strand breaks, doublestrand breaks, interstrand cross-links, and damage to the DNA bases. Apoptotic cells can be easily identified because highly fragmented DNA in apoptotic cells migrates efficiently. As few cells are required for this method, fluorescence-activated cell sorting becomes a practical way to provide purified, populations for analysis of DNA damage. Comets can be stained with antibodies against incorporated bromodeoxyuridine (BrdUrd) or against specific DNA adducts. Much of the popularity of the comet assay is due to the ability of this method to measure DNA damage in virtually any single cell. A more unique aspect of this method is the ability to detect heterogeneity in response within different cells of a single population. Under alkaline conditions, the comet assay detects DNA single-strand breaks, double-strand breaks, and alkali labile lesions.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

Phosphorylated Histone H2AX in Spheroids, Tumors, and Tissues of Mice Exposed to Etoposide and 3-Amino-1,2,4-Benzotriazine-1,3-Dioxide

We reported recently that exposure of hamster V79 fibroblasts to 6 drugs that varied in their ability to produce DNA double-strand breaks stimulated formation of phosphorylated histone H2AX (serine 139 phosphorylated histone H2AX; γH2AX). Using flow cytometry to analyze γH2AX antibody-stained cells 1 h after a 30-min drug treatment, the fraction of cells that showed the control levels of γH2AX correlated well with the fraction of cells that survived to form colonies. This observation is now extended to V79 and SiHa human cervical carcinoma cells grown as multicell spheroids and SiHa xenografts and SCCVII tumors in mice. Animals were injected with etoposide, a topoisomerase-II inhibitor that targets proliferating cells or 3-amino-1,2,4-benzotriazine-1,3-dioxide (tirapazamine), a bioreductive cytotoxin that targets hypoxic cells. For spheroids, γH2AX intensity predicted clonogenic cell survival for cells recovered 90 min after drug injection, regardless of position of the cells within the spheroid. Similar results were obtained for etoposide in tumors; however, the γH2AX signal for tirapazamine was smaller than expected for the observed amount of cell killing. Frozen sections of tumors confirmed the greater intensity of γH2AX staining in cells close to blood vessels of tumors soon after treatment with etoposide and the opposite pattern for tumors exposed to tirapazamine. Analysis of cells or frozen sections from mouse spleen and kidney suggests that information can also be obtained on initial damage in normal tissues. These results support the possibility of using γH2AX antibody staining as a method to aid in prediction of tumor and normal tissue response to treatment.

Homologous Recombination Is the Principal Pathway for the Repair of DNA Damage Induced by Tirapazamine in Mammalian Cells

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective cytotoxin that has shown significant activity in advanced clinical trials in combination with radiotherapy and cisplatin. The current study aimed to advance our understanding of tirapazamine-induced lesions and the pathways involved in their repair. We show that homologous recombination plays a critical role in repair of tirapazamine-induced damage because cells defective in homologous recombination proteins XRCC2, XRCC3, Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with the involvement of homologous recombination repair, we observed extensive sister chromatid exchanges after treatment with tirapazamine. We also show that the nonhomologous end-joining pathway, which predominantly deals with frank double-strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In addition, we show that tirapazamine preferentially kills mutants both with defects in XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable DNA-topoisomerase I cleavable complexes. We further show that gamma H2AX, an indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle. These observations lead us to an overall model of tirapazamine damage in which DNA single-strand breaks, base damage, and DNA-protein cross-links (including topoisomerase I and II cleavable complexes) produce stalling and collapse of replication forks, the resolution of which results in DSB intermediates, requiring homologous recombination and XPF/ERCC1 for their repair.