Residual gammaH2AX foci as an indication of lethal DNA lesions.

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.

gammaH2AX expression in tumors exposed to cisplatin and fractionated irradiation.

PURPOSE: Is retention of gammaH2AX foci useful as a biomarker for predicting the response of xenograft tumors to cisplatin with X-ray? Is a similar approach feasible using biopsies from patients with cervical cancer? EXPERIMENTAL DESIGN: Mice bearing SiHa, WiDr, or HCT116 xenograft tumors were exposed to cisplatin and/or three daily doses of 2 Gy. Tumors were excised 24 h after treatment and single cells were analyzed for clonogenic fraction and retention of gammaH2AX foci. Tumor biopsies were examined using 47 paraffin-embedded sections from untreated tumors and 24 sections from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. RESULTS: Residual gammaH2AX measured 24 h after cisplatin injection accurately predicted surviving fraction in SiHa and WiDr xenografts. When a clinically equivalent protocol using cisplatin and fractionated irradiation was employed, the fraction of xenograft cells lacking gammaH2AX ranked survival accurately but underestimated tumor cell kill. Residual gammaH2AX foci were detected in clinical samples; on average, only 25% of tumor nuclei exhibited one or more gammaH2AX foci before treatment and 74% after the start of treatment. CONCLUSION: gammaH2AX can provide useful information on the response of human tumors to the combination of cisplatin and radiation, but prediction becomes less accurate as more time elapses between treatment and tumor biopsy. Use of residual gammaH2AX as a biomarker for response is feasible when cell survival exceeds approximately 20%, but heterogeneity in endogenous and treatment-induced gammaH2AX must be considered.

Synthesis, characterization, and evaluation of antimicrobial and cytotoxic effect of silver and titanium nanoparticles.

Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. Nosocomial infections represent an enormous emerging problem, especially in patients with ambulatory treatment, which requires that they wear medical devices for an extended period of time. In this work, an evaluation of the antimicrobial activity of both silver and titanium nanoparticles was carried out against a panel of selected pathogenic and opportunistic microorganisms, some of them commonly associated with device-associated infections. Cytotoxicity assays monitoring DNA damage and cell viability were evaluated using human-derived monocyte cell lines. We show that silver-coated nanoparticles having a size of 20-25 nm were the most effective among all the nanoparticles assayed against the tested microorganisms. In addition, these nanoparticles showed no significant cytotoxicity, suggesting their use as antimicrobial additives in the process of fabrication of ambulatory and nonambulatory medical devices. FROM THE CLINICAL EDITOR: In this study, antimicrobial activity of silver and titanium nanoparticles was evaluated against a panel of selected pathogenic and opportunistic microorganisms. Silver-coated nanoparticles of 20-25 nm size were the most effective among all the nanoparticles without significant cytotoxicity, suggesting their use as antimicrobial additives in the process of fabrication of ambulatory and nonambulatory medical devices.

A screen for suppressors of gross chromosomal rearrangements identifies a conserved role for PLP in preventing DNA lesions.

Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

Impact of the comet assay in radiobiology.

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.

Residual gammaH2AX after irradiation of human lymphocytes and monocytes in vitro and its relation to late effects after prostate brachytherapy.

BACKGROUND AND PURPOSE: Retention of gammaH2AX foci in irradiated cells can signify a deficiency in DNA double-strand break repair that may be useful as an indicator of individual radiosensitivity. MATERIALS AND METHODS: To examine this possibility, the retention of gammaH2AX after irradiation was compared using white blood cells from 20 prostate brachytherapy patients who developed late normal tissue toxicity and 20 patients with minimal toxicity. Peripheral blood lymphocytes and monocytes were coded for analysis, exposed in vitro to 4 doses of 0.7 Gy X-rays at 3 hourly intervals, and retention of gammaH2AX was measured by flow cytometry 18 hours after the final irradiation. RESULTS: Excellent reproducibility in duplicate samples and a range in residual gammaH2AX from 7% above background to 244% above background were observed. Residual gammaH2AX in lymphocytes showed a positive correlation with patient age. However, no relation was observed between the level of residual gammaH2AX in peripheral blood mononuclear cells and late normal tissue damage. CONCLUSIONS: We conclude that the method of detection of residual gammaH2AX after in vitro irradiation of lymphocytes and monocytes was simple, reproducible, and sensitive. However, it failed to predict for late normal tissue toxicity after brachytherapy. Possible reasons are discussed.

Radiosensitization by the histone deacetylase inhibitor PCI-24781.

PURPOSE: PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined. EXPERIMENTAL DESIGN: Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 micromol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival. RESULTS: PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in gammaH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of approximately 1 micromol/L, and cells in G(1) phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF(0.1) = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 micromol/L, and more radioresistant WiDr cells showed less sensitization (DMF(0.1) = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with gammaH2AX foci 24 h after irradiation but did not affect the initial rate of loss of radiation-induced gammaH2AX or the rate of rejoining of DNA double-strand breaks. CONCLUSIONS: PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.

Tissue distribution of endothelial cells in vivo following intravenous injection.

OBJECTIVE: Recent studies have suggested that endothelial cells derived from circulating endothelial progenitors can be used as carriers for cell-based therapy. However, the in vivo homing properties of mature endothelial cells are still unclear. In this paper, we studied the kinetics and specificity of endothelial homing to sites of angiogenesis. METHODS: The kinetics of the distribution of endothelial cells in mice following intravenous injection of 3H-thymidine-labeled microvascular endothelial cells were examined. To detect the homing of viable and apoptotic endothelial cells, GFP-labeled microvascular endothelial cells were injected intravenously in immunodeficient mice. RESULTS: We observed that endothelial cells injected intravenously transit rapidly through the lungs, but do not home specifically to any organ. We did not observe specific accumulation of endothelial cells in subcutaneously implanted tumors following intravenous injection. Rare GFP-labeled endothelial cells were observed in the proximity of tumor blood vessels. However, similar findings were seen when GFP-labeled apoptotic endothelial cells were injected intravenously. CONCLUSION: These findings suggest that integration of mature endothelial cells to the vasculature is a rare event and that engulfment of apoptotic bodies, independent of nuclear fusion, may be misinterpreted as cell plasticity, and care should be taken in the interpretation of such findings.

DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions.

Exposure of cells to hypertonic medium after X-irradiation results in a 3-4-fold increase in the phosphorylation of histone H2AX (gammaH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3-4-fold increase in H2AX phosphorylation in the presence of 0.5M Na(+). However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5M Na(+). Instead, 0.5M Na(+) caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the gammaH2AX foci.

Phosphorylated histone H2AX in relation to cell survival in tumor cells and xenografts exposed to single and fractionated doses of X-rays.

BACKGROUND AND PURPOSE: Human tumor cell lines grown as monolayers or xenograft tumors were exposed to single or multiple fractions of X-rays and the ability to use residual gammaH2AX to identify radiosensitive cells was assessed. MATERIALS AND METHODS: Twenty-four hour after exposure to single or daily fractions of X-rays, human tumor cells from monolayers or xenografts were analyzed for clonogenic surviving fraction. Cells were also fixed and labeled with anti-gammaH2AX antibodies for analysis by flow and image cytometry. The relative amount of residual gammaH2AX and the percentage of cells with <3 foci were compared with the clonogenic surviving fraction measured for the same population. RESULTS: The fraction of gammaH2AX remaining 24h after X-irradiation relative to peak levels 1h after exposure was correlated with radiosensitivity (SF2) for 18 human tumor cell lines. The fraction of SiHa, C33A and WiDr cells with <3 gammaH2AX foci was predictive of clonogenic surviving fraction for both monolayer cells exposed to either single doses or up to 5 fractions. Similar results were obtained using cells from xenograft tumors of irradiated mice. CONCLUSION: The percentage of tumor cells that retain gammaH2AX foci 24h after single or fractionated doses appears to be a useful measure of cellular radiosensitivity that is potentially applicable in the clinic.

Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks.

Phosphorylation of histone H2AX on serine 139 (gammaH2AX) occurs at sites flanking DNA double-strand breaks and can provide a measure of both number and location of these breaks within the nucleus. Because double-strand breaks are often lethal and are produced by several chemotherapeutic agents, we examined the possibility that expression of gammaH2AX after treatment might be useful as a surrogate indicator of clonogenic cell kill. Chinese hamster V79 cells were exposed for 30 min to drugs known to produce DNA double-stand breaks with different efficiencies: bleomycin, tirapazamine, doxorubicin, etoposide, 4-nitro-quinoline-N-oxide, and hydrogen peroxide. Cells were then allowed 1 h to develop foci before fixation or were plated to measure colony formation ability. Anti-gammaH2AX antibody staining was measured using flow cytometry. Flow histograms were analyzed for the percentage of cells that showed gammaH2AX levels greater than untreated cells, and this percentage was compared with the clonogenic surviving fraction. H2AX expression measured 1 h after treatment predicted cell killing for all of the drugs examined over two logs of cell kill. Moreover, predictive ability was largely independent of drug type in this cell line, and gammaH2AX levels five times background resulted in 50-90% cell kill. This method seems to provide a useful indicator of clonogenic response to treatment with selected chemotherapeutic drugs.

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 (gammaH2AX). 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, gammaH2AX representing up to 30% of the initial signal still present. SW756 cells showed almost four times higher background levels of gammaH2AX and no residual gammaH2AX compared with the most radiosensitive HPV-negative C33A cells that showed the lowest background and retained 30% of the maximum level of gammaH2AX. Radiation sensitivity, measured as clonogenic-surviving fraction after 2 Gy, was correlated with the fraction of gammaH2AX remaining 24 hours after irradiation. A substantial correlation with gammaH2AX 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 gammaH2AX loss half-times than p53-deficient cell lines. We conclude that cell line-dependent differences in loss of gammaH2AX after irradiation are related in part to intrinsic radiosensitivity. The possibility that the presence of gammaH2AX foci may not always signify the presence of a physical break, notably in some tumor cell lines, is also supported by these results.

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; gammaH2AX). Using flow cytometry to analyze gammaH2AX antibody-stained cells 1 h after a 30-min drug treatment, the fraction of cells that showed the control levels of gammaH2AX 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, gammaH2AX 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 gammaH2AX signal for tirapazamine was smaller than expected for the observed amount of cell killing. Frozen sections of tumors confirmed the greater intensity of gammaH2AX 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 gammaH2AX antibody staining as a method to aid in prediction of tumor and normal tissue response to treatment.

Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase.

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.

Measurement of tumor hypoxia using single-cell methods.

A growing appreciation for the importance of hypoxia in tumor progression and response to treatment has driven efforts to develop methods that could be used routinely in the clinic to identify tumors containing hypoxic cells. The ideal method would be noninvasive and could be used both before treatment to determine the presence of hypoxia and during therapy to assess tumor reoxygenation. Although this goal is being approached, there are still questions about how best to measure tumor oxygenation and whether noninvasive imaging methods can provide the necessary sensitivity. Analysis of hypoxia at the level of the individual cell can provide the following information that cannot be obtained in other ways: the degree of hypoxia, the lifetime of hypoxic cells, and the dynamic nature of hypoxia. This review will describe methods that have been used to detect hypoxia in individual cells, the relation between these measurements and patient response to treatment, and indicate where these methods can provide important additional insights into the consequences of tumor hypoxia.

Phosphorylation of histone H2AX as a measure of radiosensitivity.

PURPOSE: Phosphorylation of histone H2AX (gammaH2AX) occurs rapidly in response to the presence of DNA double-strand breaks and is thought to recruit repair enzymes to these sites. We examined the possibility that expression of phosphorylated H2AX could provide information on tumor and/or normal tissue sensitivity to radiation. METHODS: Flow cytometry of gammaH2AX antibody-stained single cells was used to measure gammaH2AX intensity in cultured cell lines, tumor cells, and normal tissues. RESULTS: The rate of disappearance of gammaH2AX during the first few hours after irradiation was generally faster in more radioresistant tumor and normal cell lines, but slower in radiosensitive cells and normal tissues from C3H mice. An exception was testis, which showed a high background and rapid loss rate. Levels of gammaH2AX were at least three times higher in well-oxygenated cells than in anoxic cells, and the oxygen concentration that produced a half-maximal response was 0.55%. Hypoxic cells could be detected in SiHa xenografts as a subpopulation with lower expression of gammaH2AX. CONCLUSIONS: Analysis of gammaH2AX has the potential to provide useful information on tumor and normal cell response to ionizing radiation after exposure to clinically relevant doses of radiation.

The range of oxygenation in SiHa tumor xenografts.

Tumor cells at very low oxygen tensions are known to be about three times more resistant to killing by ionizing radiation. Since cells at intermediate oxygen tensions (defined here as greater than 0.1% and less than 2% O(2)) show partial radioresistance, they should be a consideration in tumor treatment. In an effort to estimate the extent and range of oxygenation in SiHa human cervical carcinoma xenografts, patterns of cell killing and DNA damage by radiation and two bioreductive drugs, PD-144872 and RSU-1069, were compared to those seen in SiHa cells grown as spheroids. These drugs produce DNA interstrand crosslinks that are largely responsible for cell killing, and the degree of crosslinking increases as the oxygenation is reduced. About 60% of the cells in SiHa xenografts exhibited drug-induced crosslinks, but only about 35% showed extensive crosslinking indicative of hypoxia below 0.1% oxygen. Patterns of toxicity and DNA damage in xenografts were comparable to those of spheroids equilibrated with about 2% oxygen, indicating that most cells in the xenografts exhibit some radioresistance due to lack of oxygen. Similarly, pimonidazole binding indicated that about 60% of the cells in SiHa xenografts were either intermediate in oxygenation or hypoxic, but only about half of those were consistent with extreme oxygen depletion. The apparent size of the population of "intermediately hypoxic" cells has implications for the use of ionizing radiation, hypoxic cell cytotoxins, and other antitumor agents whose cytotoxicity is dependent on cellular oxygen content.

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells.

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) 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 gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-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 G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.

Hypertonic saline enhances expression of phosphorylated histone H2AX after irradiation.

Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called "foci" that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na(+) increased gammaH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of gammaH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced gammaH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that gammaH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.