γH2AX Flow Cytometry Research Articles

Mithramycin A Radiosensitizes EWS:Fli1+ Ewing Sarcoma Cells by Inhibiting Double Strand Break Repair

The oncogenic EWS:Fli1 fusion protein is a key transcriptional mediator of Ewing sarcoma initiation, progression, and therapeutic resistance. Mithramycin A (MithA) is a potent and specific inhibitor of transcription mediated by the EWS:Fli1. We tested the hypothesis that pretreatment with MithA could selectively radiosensitize EWS:Fli1+ tumor cells by altering the transcriptional response to radiation injury. A panel of 4 EWS:Fli1+ and 3 EWS:Fli1- Ewing sarcoma cell lines and 1 nontumor cell line were subjected to MithA dose-response viability assays to determine the relative potency of MithA in cells possessing or lacking the EWS:Fli1 fusion. Radiosensitization by MithA was evaluated by clonogenic survival assays invitro and in a murine xenograft model. DNA damage was evaluated by comet assay and γ-H2Ax flow cytometry. Immunoblotting, flow cytometry, and reverse-transcription, polymerase chain reaction were used to evaluate DNA damage-induced signaling and repair processes and apoptosis. We found that MithA alone could potently and selectively inhibit the growth of EWS:Fli1+ tumor cells, but not cells lacking this fusion. Pretreatment with MithA for 24 hours before irradiation significantly reduced clonogenic survival invitro and delayed tumor regrowth invivo, prolonging survival of EWS:Fli1+ tumor-bearing mice. Although MithA did not increase the level of DNA double-strand breaks, mechanistic studies revealed that MithA pretreatment selectively inhibited DNA double-strand break repair through downregulation of EWS:Fli1-mediated transcription, leading to tumor cell death by apoptosis. Our data indicate that MithA is an effective radiosensitizer of EWS:Fli1+ tumors and may achieve better local control at lower doses of radiation.

Abstract 6189: Identifying efficacy of targeted HER2 antibodies in sensitization of HER2+ breast cancer to fractionated radiation

Abstract Background: Radiation therapy is one component of standard-of-care therapy for patients with breast cancer; however, treatment is often associated with patient toxicity. Fractionated radiation has emerged as an alternative to single dose radiation; although, there have been limited studies investigating it in combination with other therapies. Positron emission tomography (PET) imaging allows for detection of biological and molecular alternations. In this study, we utilized [18F]-fluoromisonidazole (FMISO)-PET imaging to monitor changes in tumor hypoxia to quantify tumor response to combination targeted plus radiation therapy. Methods: To assess for cell death, breast cancer cells (BT474, SKBR3, MDA MB 361, and MCF-7) were treated with 1 ug/mL trastuzumab (anti-HER2 targeted therapy) and radiation therapy and assessed for cell death four days later (N = 4 replicates per group). To assess for DNA double strand breaks (DSB), cells were probed for γ-H2AX with flow cytometry 30 minutes post treatment. For in vivo studies, 107 BT474 cells were subcutaneously injected into athymic nude mice (N = 3 for control and trastuzumab treated, N = 4 for radiation and combination therapy). Mice were treated with trastuzumab (4 mg/kg) on days 0 and 3 and radiation (6/3 Gy) on days 1, 2 and 3. Controls included single agent therapies and saline treated. Changes in hypoxia were assessed with FMISO PET imaging on days 0, 3 and 7. Mean and standard error of standardized uptake value (SUV) was quantified at each time point. Treatment synergy and statistical significance were assessed with Bliss test of independence and non-parametric T-test, respectively. Results: In vitro cell death assay revealed that trastuzumab or radiation treated groups exhibited 19% ± 2% or 50% ± 9% cell death respectively, while trastuzumab prior to radiation exhibited 76% ± 5.3% cell death (p = 0.01) in BT474 cells. Analysis of γ-H2AX flow cytometry showed that trastuzumab prior to radiation exhibited 40.8% ± 11% DSB, which significantly increased from trastuzumab (0.5% ± 0.4%) or radiation only groups (9% ± 7.2%) (p = 0.01). Bliss test of independence confirmed synergy of trastuzumab and fractionated radiation. In vivo data revealed that BT474 tumors treated with trastuzumab and radiation experienced a 45.8% ± 9.1% decrease in volume, while radiation only experienced a 23.2% ± 13.3% decrease from day 0 to 7 (p = 0.26). FMISO PET imaging indicated that tumors treated with trastuzumab and radiation therapy experienced a 20.5% ± 4% decrease in [18F]-FMISO tumor:muscle SUV, while radiation only tumors experience a 61.2% ± 33.3% increase in tumor:muscle SUV from day 0 to 7 (p = 0.01). Conclusions: Preliminary data reveals that treatment of cancer cells with trastuzumab synergistically increases the efficacy of fractionated radiation in vitro and in vivo. FMISO PET imaging may provide insight regarding the optimal treatment window of radiation therapy. Citation Format: Patrick Song, Yun Lu, Sharon Samuel, Anna Sorace. Identifying efficacy of targeted HER2 antibodies in sensitization of HER2+ breast cancer to fractionated radiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6189.

Absence of DNA double-strand breaks in human peripheral blood mononuclear cells after 3 Tesla magnetic resonance imaging assessed by γH2AX flow cytometry

Magnetic resonance imaging (MRI) is regarded as a non-harming and non-invasive imaging modality with high tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities, MRI does not use ionising radiation. Recently, however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double-strand breaks in human lymphocytes. In this study we investigated the impact of 3-T cardiac MRI examinations on the induction of DNA double-strand breaks in peripheral mononuclear cells by γH2AX staining and flow cytometry analysis. The study cohort consisted of 73 healthy non-smoking volunteers with 36 volunteers undergoing CMRI and 37 controls without intervention. Differences between the two cohorts were analysed by a mixed linear model with repeated measures. Both cohorts showed a significant increase in the γH2AX signal from baseline to post-procedure of 6.7 % (SD 7.18 %) and 7.8 % (SD 6.61 %), respectively. However, the difference between the two groups was not significant. Based on our study, γH2AX flow cytometry shows no evidence that 3-T MRI examinations as used in cardiac scans impair DNA integrity in peripheral mononuclear cells. • No evidence for DNA double-strand breaks after cardiac MRI. • Prospective study underlines safe use of MRI with regard to DNA damage. • Controlled trial involving both genders investigating DNA DSBs after 3-T MRI.

Abstract 519: Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair

Abstract Mutant KRAS is found in approximately 30% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Despite the use of radiation (RT) therapy for the treatment of locally advanced disease, local recurrence remains an issue. The observation that mutations in KRAS lead to both replication stress and DNA double-strand breaks (DSBs) suggests these cancers may have a greater reliance on DNA damage response pathways and therefore may be preferentially radiosensitized by therapies targeting DNA repair. In this study, we investigated the combined inhibition of WEE1 and PARP1 using the small molecule inhibitors, AZD1775 and olaparib, respectively as a radiosensitizing strategy in KRAS mutant NSCLC. We began by comparing radiosensitization by AZD1775+olaparib, in KRAS isogenic H1703 lung cancer cells and found that KRAS mutant cells were preferentially radiosensitized (ER, enhancement ratio 1.8) compared to KRAS wild type cells (ER 1.4). Combined WEE1 and PARP1 inhibition also radiosensitized KRAS mutant Calu-6 and NCI-H23 lung cancer cells (ER 1.9 and 1.5, respectively). These findings were further confirmed in vivo: Calu-6 tumor xenografts were significantly radiosensitized by AZD1775+olaparib, as evidenced by an 11 day delay in tumor volume doubling time relative to RT treatment. Given that WEE1 and PARP1 function to prevent and manage replication stress, respectively, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent replication stress. While replication stress did contribute to AZD1775-mediated radiosensitization in Calu-6 cells, as evidenced by pan-nuclear γH2AX staining, and the ability of exogenous nucleosides to protect cells from radiosensitization by AZD1775 alone, nucleosides had little effect on radiosensitization by AZD1775+olaparib. These results suggest that replication stress is not required for radiosensitization by AZD1775+olaparib. As WEE1 and PARP1 both promote repair of radiation-induced DNA damage, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent, unrepaired DSBs. Assessment of the kinetics of DSB repair by γH2AX flow cytometry demonstrated that while total γH2AX levels in cells treated with RT alone had returned to control levels within 24 h, AZD1775+olaparib treatment significantly delayed the resolution of γH2AX following RT, with 44.8% or 46.2% of Calu-6 or KRAS mutant H1703 cells, respectively remaining γH2AX-positive 24 h post-RT. This delay corresponded with the inhibition of radiation-induced RAD51 foci by AZD1775, indicating inhibition of homologous recombination repair. Taken together these data demonstrate the efficacy of combined inhibition of WEE1 and PARP1 with radiation in KRAS mutant lung cancer. Furthermore, these results suggest that although replication stress occurs in response to AZD1775 and olaparib, persistent DSBs are the cause of radiosensitization. Citation Format: Leslie A. Parsels, David Karnak, Joshua D. Parsels, Zachery Reichert, Jonathan Maybaum, Theodore S. Lawrence, Meredith Ann Morgan. Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 519.

Antiproliferation of Cryptocarya concinna-derived cryptocaryone against oral cancer cells involving apoptosis, oxidative stress, and DNA damage

BackgroundCryptocarya-derived crude extracts and their compounds have been reported to have an antiproliferation effect on several types of cancers but their impact on oral cancer is less well understood.MethodsWe examined the cell proliferation effect and mechanism of C. concinna-derived cryptocaryone (CPC) on oral cancer cells in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial depolarization, and DNA damage.ResultsWe found that CPC dose-responsively reduced cell viability of two types of oral cancer cells (Ca9-22 and CAL 27) in MTS assay. The CPC-induced dose-responsive apoptosis effects on Ca9-22 cells were confirmed by flow cytometry-based sub-G1 accumulation, annexin V staining, and pancaspase analyses. For oral cancer Ca9-22 cells, CPC also induced oxidative stress responses in terms of ROS generation and mitochondrial depolarization. Moreover, γH2AX flow cytometry showed DNA damage in CPC-treated Ca9-22 cells. CPC-induced cell responses in terms of cell viability, apoptosis, oxidative stress, and DNA damage were rescued by N-acetylcysteine pretreatment, suggesting that oxidative stress plays an important role in CPC-induced death of oral cancer cells.ConclusionsCPC is a potential ROS-mediated natural product for anti-oral cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1073-5) contains supplementary material, which is available to authorized users.

Absence of DNA double strand breaks in human peripheral blood mononuclear cells after magnetic resonance imaging assessed by γH2AX flow cytometry: a prospective blinded trial

Background Magnetic Resonance Imaging (MRI) is regarded as a non harming and non invasive imaging modality with superb tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities MRI does not use ionizing radiation. Recently however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double strand breaks in human lymphocytes.

Deoxynucleoside Salvage Facilitates DNA Repair During Ribonucleotide Reductase Blockade in Human Cervical Cancers

Cells generate 2'-deoxyribonucleoside triphosphates (dNTPs) for both replication and repair of damaged DNA predominantly through de novo reduction of intracellular ribonucleotides by ribonucleotide reductase (RNR). Cells can also salvage deoxynucleosides by deoxycytidine kinase/thymidine kinase 1 in the cytosol or by deoxyguanosine kinase/thymidine kinase 2 in mitochondria. In this study we investigated whether the salvage dNTP supply pathway facilitates DNA damage repair, promoting cell survival, when pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC no.663249) impairs the de novo pathway. Human cervical cancer cells were subjected to radiation with or without 3-AP under medium deoxynucleoside concentrations of 0, 0.05, 0.5 and 5.0µM. Efficacy of DNA damage repair was assessed by γ-H2AX flow cytometry and focus counts, by single cell electrophoresis (Comet assay), and by caspase 3 cleavage assay as a marker of treatment-induced apoptosis. Cell survival was assessed by colony formation. We found that deoxyribonucleotide salvage facilitates DNA repair during RNR inhibition by 3-AP and that salvage reduces the radiochemosensitivity of human cervical cancer cells.

Detecting DNA damage of human lymphocytes exposed to 1,2-DCE with γH2AX identified antibody using flow cytometer assay

To study DNA damage of human peripheral blood lymphocytes exposed to 1,2-dichloroethane (1,2-DCE) with flow cytometry (FCM) assay. The lymphocytes were obtained from 21 workers who are occupationally exposed to 1,2-DCE (exposed group) and 27 workers who were not exposed to 1,2-DCE in the same factory (inner control) and 28 island residents who had never been occupationally exposed to adverse factors (external control). FCM assay was adopted to detect DNA damage of the lymphocytes of each group. Lymphocytes of the health people were incubated with 1,2-DCE at different doses, and FCM assay was used to detect DNA damage. DNA damage rate (%) of the exposed group of exposed workers (4.05% ± 2.55%) was significantly higher than the inner control group of workers (1.97% ± 1.40%) and external control groups of island residents (0.23% ± 0.13%), and the DNA damage of inner control was higher than the external control, all the differences were statistically significant (P < 0.01 or P < 0.05). The geometric mean fluorescence intensity of the workers in the exposed group (3.33 ± 3.01) was significantly higher than the (2.07 ± 0.58) only (P < 0.05). There was no significant difference in the DNA damage rate as well as the geometric mean fluorescence intensity among the exposed group of workers with different years of working period (P > 0.05). In vitro, the fluorescence intensity at the dose of 20, 30 µmol/L for 0.5 h exposure showed statistical significance compared with the negative control group (P < 0.01). The DNA damage rate at the dose of 20, 30 µmol/L for 1.0 h exposure was statistically significant compared with the negative control group (P < 0.05, P < 0.01); The fluorescence intensity at the dose of 10, 20, 30 µmol/L for 1.0 h exposure was statistically significant compared with the negative control group (P < 0.05, P < 0.01). 1,2-DEC can cause DNA damage. And γH2AX FCM assay can be a sensitive, objective and effective method of detecting DNA damage of peripheral blood lymphocytes.

H2AX phosphorylation as a genotoxicity endpoint

The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4 h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60 μg/ml), the alkylating agent methyl methanesulfonate (1.3–65 μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10 μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500 μg/ml) and sodium chloride (0.058–580 μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay. These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.

Short‐term culture and γH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.