γH2AX Assay Research Articles

γH2AX responses in human buccal cells exposed to ionizing radiation.

DNA double strand breaks are induced by ionizing radiation (IR), leading to the phosphorylation of the core histone protein H2AX (termed γH2AX). The understanding of the γH2AX responses in irradiated human buccal cells is still very limited. We used visual scoring and laser scanning cytometry (LSC) methods to investigate γH2AX signaling following exposure of human buccal cells (from six individuals) to ionizing radiation at 0-4 Gy. The frequency of nuclei containing 15-30 γH2AX foci was significantly elevated 30 min post-IR exposure (by visual scoring). Concomitantly, there was a significant decrease in the frequency of cells without foci following exposure to IR. IR-induced γH2AX signal as determined by laser scanning cytometry (which included γH2AX integral and MaxPixel value) increased significantly in all individual's 2N nuclei 30 min post-IR and was similar for all three nuclear shapes identified. Individuals with the lowest baseline γH2AX integral (i.e., in nonirradiated cells) showed the greatest fold stimulation of γH2AX and significant dose-responses to IR doses of 1, 2, and 4 Gy. In 5 out of 6 individuals, the frequency of visually scored γH2AX in nuclei showed a strong correlation (up to r = 0.999) with LSC scored γH2AX integrals. The γH2AX response and subsequent decline varied between individuals but remained elevated above baseline levels 24 h post IR exposure. γH2AX response in irradiated human buccal cells has potential to be used as an index of baseline DNA damage in population studies. The variable response to IR exposure between individuals should be taken into consideration when using the γH2AX assay for radiation biodosimetry.

Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX.

Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX.

Toxicity, genotoxicity and proinflammatory effects of amorphous nanosilica in the human intestinal Caco-2 cell line

Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15nm at the highest tested dose (32μg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15nm, and to a lower extent SiO2-55nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field

Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells is not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant increase in γ-H2AX foci formation when irradiated to 20 cGy in-field in comparison to out-of-field. However, no significant difference in cell survival or colony volume was observed whether the PNT1A cells were irradiated in-field or out-of-field. Out-of-field radiation dose alone can have a damaging effect on the proliferation of PNT1A cells when a clinically relevant dose of 2 Gy is delivered in in-field. Out-of-field radiation with the transfer of bystander factors induces an adaptive response in the PNT1A cells.

Abstract 4575: Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs

Abstract Drugs that target DNA topoisomerase II and interact with DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Using a structure-based design approach a series of epipodophyllotoxin-derived hybrid etoposide analogs were designed, synthesized and biologically characterized. It was hypothesized that combining an epipodophyllotoxin moiety that could interact with specific protein residues on topoisomerase II and DNA-interacting moieties in the same molecule would produce much stronger binding and yield more potent and wide spectrum cytotoxic anticancer drugs. Because the hybrids have unique dual targeting mechanisms, cancer cells should be much less able to develop resistance. The most potent hybrid had a mean GI50 in the NCI-60 cell screen much lower than etoposide. The compounds were much less cross-resistant to a K562-derived etoposide-resistant cell line. All of the hybrids tested targeted topoisomerase II as evidenced by their ability to inhibit kDNA decatenation, to produce linear DNA in a DNA cleavage assay and to form covalent topoisomerase II-DNA covalent complexes in a cellular ICE assay. The DNA double strand breaks the hybrids produced in a cellular γH2AX assay and the G2/M cell cycle blocks were also characteristic of topoisomerase II-targeted drugs. Cell cycle analysis was also used to characterize the targets. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles of both etoposide and the DNA-interacting moieties from which they were derived. This data supports the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA. Support: CIHR and a Canada Research Chair in Drug Development. Citation Format: Brian B. Hasinoff. Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2014-4575

Hsp90 inhibitor geldanamycin enhances the antitumor efficacy of enediyne lidamycin in association with reduced DNA damage repair.

Inhibition of heat shock protein 90 (Hsp90) leads to inappropriate processing of proteins involved in DNA damage repair pathways after DNA damage and may enhance tumor cell radio- and chemo-therapy sensitivity. To investigate the potentiation of antitumor efficacy of lidamycin (LDM), an enediyne agent by the Hsp90 inhibitor geldanamycin (GDM), and possible mechanisms, we have determined effects on ovarian cancer SKOV- 3, hepatoma Bel-7402 and HepG2 cells by MTT assay, apoptosis assay, and cell cycle analysis. DNA damage was investigated with H2AX C-terminal phosphorylation (γH2AX) assays. We found that GDM synergistically sensitized SKOV-3 and Bel-7402 cells to the enediyne LDM, and this was accompanied by increased apoptosis. GDM pretreatment resulted in a greater LDM-induced DNA damage and reduced DNA repair as compared with LDM alone. However, in HepG2 cells GDM did not show significant sensitizing effects both in MTT assay and in DNA damage repair. Abrogation of LDM-induced G2/M arrest by GDM was found in SKOV-3 but not in HepG2 cells. Furthermore, the expression of ATM, related to DNA damage repair responses, was also decreased by GDM in SKOV-3 and Bel-7402 cells but not in HepG2 cells. These results demonstrate that Hsp90 inhibitors may potentiate the antitumor efficacy of LDM, possibly by reducing the repair of LDM-induced DNA damage.

P03.07 * RESVERATROL ENHANCES RADIATION EFFECTS ON CLONOGENIC SURVIVAL IN HUMAN MEDULLOBLASTOMA CELL LINES

BACKGROUND AND OBJECTIVE: In recent years, the natural plant polyphenol resveratrol (RV) attracted an increasing attention in tumor therapy. Currently, clinical trials for colon cancer and multiple myeloma have been completed. RV has to been shown to exhibit tumor preventive as well as anticancer effects. These are of particular interest for the therapy of pediatric medulloblastoma (MB), as low-risk MB patients (5-year survival up to 95 %) require a therapy focused on the reduction of late adverse effects (i.e. neurological and endocrine disorders), whereas patients belonging to the high-risk group (i.e. such owning MYC amplification; 5-year survival 13 %) urgently need a more intensive therapy to improve survival. In the present study, we analyzed RV-mediated changes on the clonogenic survival and proliferation of irradiated medulloblastoma cells. Furthermore, we investigated if RV may induce double-strand breaks (DSB), and if combined RV/irradiation (IR) treatment affects the number of potential tumor stem cells (TSC). METHODS: Three MB cell lines were treated with RV doses ranged from 9.5 µM to 40 µM (based on preliminary experiments examining metabolic activity or clonogenic survival of each cell line) for three or six days. 2 Gy IR was applied immediately before RV (3 d treatment) or after three days (6 d treatment). Clonogenic assays were conducted at the end of treatment time. DSB were measured by γH2AX assay 1 h and 24 h after IR or RV. To quantify potential TSC, we detected vital CD15+ and CD133+ cells by flow cytometry three days after IR and RV treatment. Effects on proliferation were measured flow cytometrically in CSFE-labeled cells. RESULTS: The post-treatment of irradiated MB cells with RV for three days significantly decreased the clonogenic survival 1.8-fold (DAOY) to 36-fold (MEB-Med8a). The longer RV treatment period reached stronger effects decreasing the clonogenic survival by up to 900-fold in irradiated D283-Med cells. Both, IR and RV alone, induced DSB. An increase of residual, unrepaired IR-induced DSB was observed after RV treatment in two-out-of-three MB cell lines (DAOY, MEB-Med8a). Examining the number of potential TSC, we revealed an enrichment of CD15+ and CD133+ cells after RV or IR alone, which was slightly enhanced by combination of both. We found no differences in proliferation rates between TSC and non-TSC. Therefore, we assume that the enrichment of TSC is caused by a higher treatment resistance of potential TSC compared to non-TSC. CONCLUSION: RV may enhance the radiation effects on clonogenic survival of human MB cells, possibly through an increased induction of additional DSB. Further studies in animal MB models are warranted to clarify if the observed in vitro effects translate into improved survival rates in vivo.

Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cells

Photothermal ablation (PTA) is a promising avenue in the area of cancer therapeutics that destroys tumor cells through conversion of near-infrared (NIR) laser light to heat. Hollow gold nanospheres (HGNs) are one of the few materials that are capable of converting light to heat and have been previously used for photothermal ablation studies. Selective delivery of functional nanoparticles to the tumor site is considered as an effective therapeutic approach. In this paper, we demonstrated the anti-cancer potential of HGNs. HGNs were conjugated with monoclonal antibody (anti-TROP2) in order to target cervical cancer cells (HeLa) that contain abundant trophoblast cell surface antigen 2 (TROP2) on the cell surface. The efficient uptake and intracellular location of these functionalized HGNs were studied through application of inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy (TEM). Cytotoxicity induced by PTA was measured using CCK-8 assay. HeLa cells incubated with naked HGNs (0.3–3 nmol L−1) within 48 h did not show obvious cytotoxicity. Under laser irradiation at suitable power, anti-TROP2 conjugated HGNs achieved significant tumor cell growth inhibition in comparison to the effects of non-specific PEGylated HGNs (P < 0.05). γH2AX assay results revealed higher occurrences of DNA-DSBs with anti-TROP2 conjugated HGNs plus laser radiation as compared to treatment with laser alone. Flow cytometry analysis showed that the amount of cell apoptosis was increased after laser irradiation with anti-TROP2 conjugated HGNs (P < 0.05). Anti-TROP2 conjugated HGNs resulted in down-regulation of Bcl-2 expression and up-regulation of Bax expression. Our study results confirmed that anti-TROP2 conjugated HGNs can selectively destroy cervical cancer cells through inducing its apoptosis and DNA damages. We propose that HGNs have the potentials to mediate targeted cancer treatment.

Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening.

BackgroundThe genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI).MethodsWhole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out.ResultsThe results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected.ConclusionsOverall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks.

Monoglucosyl-rutin as a potential radioprotector in mammalian cells.

In the present study, the role of monoglucosyl-rutin as a potential radioprotector was investigated using mammalian cell culture models. Cell survival and DNA damage were assessed using colony formation, sister chromatid exchange and γH2AX assays. It was demonstrated that monoglucosyl-rutin was able to increase cell survival when exposed to ionizing radiation, possibly by decreasing the amount of base damage experienced by the cell. However, the present study also demonstrated that, despite monoglucosyl-rutin exhibiting radioprotective effects at low doses, high doses of monoglucosyl-rutin led to a decrease in plating efficiency and an increased doubling time. This effect may be due to double-strand breaks caused by high concentrations of monoglucosyl-rutin.

Effects of dna-dependent protein kinase inhibition by NU7026 on dna repair and cell survival in irradiated gastric cancer cell line N87.

Repair of radiation-induced dna double-strand breaks is a key mechanism in cancer cell radio-resistance. The synthesized compound NU7026 specifically inhibits dna-dependent protein kinase (dna-pk) within the non-homologous end-joining repair mechanism. Earlier studies demonstrated increased radiosensitivity in dna-pk deficient cells compared with wild-type cells. In chronic leukemia cells, NU7026 appears to enhance the cytotoxic effect of chlorambucil. The radio-modifying effects of NU7026 on cell survival, cell cycle, apoptosis, and dna double-strand break repair have yet to be studied in gastric cancer cells. The gastric cancer cell line N87 was treated with 0 Gy or 4 Gy in the presence of NU7026 at a dose range of 0-20 μmol/L. Clonogenic assays were used to assess cell survival after treatment. Cell-cycle distribution was analyzed using propidium iodide with fluorescence-activated cell sorting. Apoptosis was detected using annexin-V and propidium iodide with fluorescence-activated cell sorting. The γH2AX assay was used to measure dna double-strand breaks. Statistically significant increases in G2/M arrest were observed in N87 cells treated with radiation and NU7026 compared with those treated with radiation alone (p = 0.0004). Combined treatment also led to an increase in apoptosis (p = 0.01). At 24 hours, the γH2AX analysis revealed more dna double-strand breaks in N87 cells treated with radiation and NU7026 than in those treated with radiation alone (p = 0.04). Clonogenic assays demonstrated declining cell survival as both the radiation and the NU7026 dose increased. The dose enhancement factor at 0.1 survival fraction was 1.28 when N87 cells were treated with 4 Gy radiation and 5 μmol/L NU7026. In gastric cancer cells, NU7026 appears to enhance the cytotoxic effect of irradiation as assessed by clonogenic assays. This increased cytotoxicity might be the result of an increase in dna double-strand breaks resulting in G2/M cell arrest and possibly higher levels of apoptosis.

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO2) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO2 NPs. The purpose of this study is to evaluate the genotoxicity of TiO2 NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO2 NPs (75±15nm) through intragastric administration at 0, 10, 50 and 200mg/kg body weight every day for 30 days. The γ-H2AX assay showed TiO2 NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO2 NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO2 NPs at the dose of 0, 5, 10, 20, 50 and 100μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24h and 48h exposure. Significant DNA damage was only observed at the concentration of 100μg/mL after 24h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100μg/mL after 2h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO2 NPs, and TiO2 NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO2 NPs induced genotoxic effects.

The human polynucleotide kinase/phosphatase (hPNKP) inhibitor A12B4C3 radiosensitizes human myeloid leukemia cells to Auger electron-emitting anti-CD123 111In-NLS-7G3 radioimmunoconjugates

Leukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131⁻ phenotype uniquely displayed by LSCs. The surviving fraction (SF) of CD123⁺/CD131⁻ AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33-266 nmols/L; 0.74MBq/μg) or to γ-radiation (0.25-5Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) or with γ-radiation (0.25-2Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation. Binding of (111)In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131⁻ epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25-0.5Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6Gy to AML-5 cells. We conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.

Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

Abstract P4-12-14: Metal chelating polymer-conjugated trastuzumab labeled to high specific activity with indium-111 is cytotoxic to trastuzumab-sensitive and resistant breast cancer cells through the emission of auger electrons

Abstract Purpose: Auger electrons are very low energy electrons emitted by Indium-111(111In) that cause lethal DNA damage when emitted in close to the cell nucleus. Our laboratory reported previously that trastuzumab modified with nuclear localizing sequence (NLS) peptides and labeled with 111In(111In-NLS-DTPA-trastuzumab) was internalized and transported to the nucleus of HER2-overexpressing breast cancer (BC) cells, reducing their clonogenic survival (CS) to ≤10%. However, a limitation was that at the specific activity (SA) achieved (0.24 MBq/μg) for 111In-DTPA-trastuzumab without or with NLS, only 1 in 50 molecules was radiolabeled. This reduced the potency for cells with low or intermediate HER2 density. Our purpose was to dramatically increase the SA of 111In-labeled trastuzumab by conjugation to a metal chelating polymer (MCP) that displays multiple DTPA chelators for 111In and compare its potency to 111In-NLS-trastuzumab on BC cells with a wide range of HER2 density including cells resistant to trastuzumab. Methods: An MCP with a polyglutamide backbone, 29 DTPA groups, and a hydrazide reactive end-group was site-specifically linked to periodate-oxidized carbohydrates on the Fc domain of trastuzumab. HER2 immunoreactivity, internalization and nuclear importation were evaluated by cell binding and fractionation studies using HER2-overexpressing SK-Br-3 human BC cells. The SA achievable was assessed by radiolabeling 1, 5, 50 and 100 μg of the immunoconjugates with 9.25 MBq of 111In. The effect of high SA (5.5 MBq/μg) 111In-MCP-trastuzumab to on the CS of human BC cells with a wide range of HER2 density (5×105-1.3×106 HER2 receptors/cell) that were sensitive or resistant to trastuzumab was compared to low SA (0.37 MBq/μg) 111In-labeled trastuzumab conjugates. Studies are also in progress to assess DNA double-strand breaks caused by the Auger electrons emitted by 111In using the γH2AX assay. Results: MCP-trastuzumab bound specifically to HER2 with high affinity (KD = 10.7±1.5nM) and was internalized and imported into the nucleus of HER2-positive BC cells. The smallest mass that could be labeled (labeling efficiency&amp;gt;80%) with 111In for MCP-trastuzumab was 1 μg (SA = 8.9 MBq/μg) compared to 50 μg for DTPA-trastuzumab (SA = 0.08 MBq/μg). High SA 111In-MCP-trastuzumab reduced the CS of SK-Br-3 cells with high HER2 density to 1.8±1.3% and the CS of TrR1 trastuzumab-resistant BC cells with intermediate HER2 density to 17.1±1.6%. These represent a 4-5 and 5-6 fold greater potency compared to low SA 111In-trastuzumab conjugates on cells with high or intermediate HER2 expression, respectively. Conclusion: 111In-MCP-trastuzumab was labeled to 37-fold higher SA than 111In-DTPA-trastuzumab. High SA increased its cytotoxicity potency for HER2-positive BC cells that were sensitive or resistant to trastuzumab. Auger electron radioimmunotherapy is a promising strategy for HER2-positive BC which may extend to trastuzumab-resistant tumours. Supported by a grant from the Canadian Breast Cancer Research Alliance with funds from the Canadian Cancer Society. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-14.

Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time, Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis

BackgroundTo establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines. ResultsCompared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05). ConclusionBleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

Use of the γ-H2AX assay to investigate DNA repair dynamics following multiple radiation exposures.

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Dovitinib induces mitotic defects and activates the G2DNAdamage checkpoint

Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.

Risk Assessment of Esophageal Adenocarcinoma Using γ-H2AX Assay

Mutagen-induced DNA damage as measured in peripheral blood lymphocytes (PBL) has been associated with increased risks of cancers. The formation of γ-H2AX is an early cellular response to DNA double-strand breaks (DSB). We hypothesize that higher level of radiation-induced γ-H2AX in PBLs may be associated with an increased risk of esophageal adenocarcinoma. Laser scanning cytometer-based immunocytochemical method was used to measure baseline and irradiation-induced γ-H2AX levels in PBLs from 211 patients with esophageal adenocarcinoma and 211 healthy controls. The ratio of induced γ-H2AX level to baseline level was used to evaluate individual susceptibility to DSBs. Relative risks for esophageal adenocarcinoma associated with γ-H2AX were assessed by multivariable logistic regression analysis. Radiation-induced γ-H2AX level and the γ-H2AX ratio were significantly higher in cases than in controls. Dichotomized at the median in controls, a significantly increased risk for esophageal adenocarcinoma was observed in association with high γ-H2AX ratio [OR = 2.94; 95% confidence interval (CI), 1.83-4.72]. Quartile analyses showed significant dose-response associations between higher γ-H2AX ratio and increased risk of esophageal adenocarcinoma (Ptrend, 1.64E-06). In addition, joint effect between γ-H2AX ratio and smoking was observed: smokers who had high γ-H2AX ratio exhibited the highest risk of esophageal adenocarcinoma (OR = 5.53; 95% CI, 2.71-11.25) compared with never smokers with low γ-H2AX ratio. Radiation-induced DNA damage assessed by γ-H2AX ratio is associated with an increased risk of esophageal adenocarcinoma. γ-H2AX assay is a new and robust method to measure DSB damage in PBLs, which can be used to assess mutagen sensitivity and esophageal adenocarcinoma risk.

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics.