Radiosensitization of tumor cells by modulation of ATM kinase

Purpose: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization.

Method: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied.

Results: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts.

Conclusions: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.     

Kinetics and dose-response of residual 53BP1/γ-H2AX foci: Co-localization,relationship with DSB repair and clonogenic survival

Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (γ-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/γ-H2AX foci formation in relation to their co-localization, DSB repair and cell survival.

Materials and methods: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci.

Results: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of γ-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/γ-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair.

Conclusions: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.     

Radiosensitization of tumor cells by modulation of ATM kinase

PURPOSE: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization. METHOD: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied. RESULTS: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts. CONCLUSIONS: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.     

Long time persistence of residual 53BP1/γ-H2AX foci in human lymphocytes in relationship to apoptosis,chromatin condensation and biological dosimetry

Purpose:?Novel assay for radiosensitivity is based on measurements of residual DNA repair foci produced by several proteins including phosphorylated H2AX (γ-H2AX), recombinase Rad51 (Rad51) and tumour suppressor p53 binding protein 1 (53BP1), which co-localise with radiation-induced DNA double-strand breaks (DSB). Here, we studied dose-response for residual 53BP1, Rad51, and γ-H2AX foci in relationship to apoptosis and chromatin condensation in human G₀-lymphocytes.

Materials and methods:?Residual foci, apoptosis and condensation of chromatin were studied following irradiation with γ-rays at doses of 0.5–10 Gy.

Results:?No clear dose response for residual Rad51 was seen. Residual 53BP1/γ-H2AX foci remained in human lymphocytes up to four weeks after irradiation. No foci formed during radiation-induced apoptosis. We provide evidence that irreversible apoptotic condensation of chromatin is responsible for arrest of residual foci and preventing de novo focus formation. Similar linear dose dependences up to 2 Gy were observed for the 53BP1/γ-H2AX foci at all studied time points. At higher doses, saturation and decline were caused by preferential elimination of apoptotic lymphocytes with residual foci. While primary 53BP1 and γ-H2AX foci almost completely co-localised, co-localisation of residual foci did not exceed 17%, indicating that 53BP1 and γ-H2AX proteins may remain for different times at the locations of DSB repair.

Conclusions:?Prolonged persistence of residual 53BP1/γ-H2AX foci may be used for biological dosimetry within the dose range up to 2 Gy. While foci are not formed during radiation-induced apoptosis in human lymphocytes, elimination of apoptotic cells with residual foci may affect the dose response.     

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

Abstract

Purpose: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.

Materials and methods: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.

Results: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.

Conclusion: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.     

Persistence of γ-H2AX and 53BP1 foci in proliferating and non-proliferating human mammary epithelial cells after exposure to γ-rays or iron ions

Purpose:?To investigate γ-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionising radiation under different cell culture conditions.

Material and methods:?HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure.

Results:?Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced γ-H2AX foci were still present at 72?h after exposure, although 53BP1 foci returned to control levels at 48?h. In contrast in proliferating HMEC, both γ-H2AX and 53BP1 foci decreased to control levels during the 24–48?h time interval after irradiation under 2D conditions. Foci numbers decreased faster after γ-ray irradiation and returned to control levels by 12?h regardless of marker, cell proliferation status, and cell culture condition.

Conclusions:?The disappearance of radiation-induced γ-H2AX and 53BP1 foci in HMEC has different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent γ-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double-strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodelling.     

Radiation-inducible PTEN expression radiosensitises hepatocellular carcinoma cells

Purpose:?To test the radiosensitising effects of a tumour-suppressor gene, phosphatase and tensin homologue deleted from chromosome 10 (PTEN), in hepatocellular carcinoma cells (HCC).

Materials and methods:?Radiation-induced wild-type PTEN or mutant PTEN was transfected into the SMMC-7721 human hepatocellular carcinoma cells. The expressions of PTEN and serine/threonine protein kinase (Akt) were detected by Western blot analysis. 3-(4,5)-dimethylthiahiazo-(-z-y1)- 3,5-di-phenytetrazoliumromide (MTT) absorbance and clonogenic survival assays were used to determine cell viability, proliferation and radiosensitivity. By performing cell-cycle analysis, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP biotin nick end labelling (TUNEL) assays and gamma-histone H2A (γ-H2AX) formation assays, we were able to explore the mechanism of PTEN enhancement of radiosensitivity.

Results:?Restoration of wild-type PTEN induced growth suppression and sensitised the cells to ionising radiation specifically by its lipid phosphatase activity through the PTEN-phosphatidylinositol 3-kinase (PI3K)-Akt signalling pathway. Restoring PTEN function correlated with G2/M arrest, apoptosis and the retardation of repair of radiation-induced double strand breaks (DSB).

Conclusions:?Our study suggests that strategies designed to restore the expression of PTEN may represent promising new therapies for sensitising HCC cells to ionising radiation.     

Kinetics and dose-response of residual 53BP1/gamma-H2AX foci: co-localization, relationship with DSB repair and clonogenic survival

PURPOSE: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (gamma-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/gamma-H2AX foci formation in relation to their co-localization, DSB repair and cell survival. MATERIALS AND METHODS: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci. RESULTS: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of gamma-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/gamma-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair. CONCLUSIONS: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.     

Human papillomavirus 16 E6 increases the radiosensitivity of p53-mutated cervical cancer cells,associated with up-regulation of aurora A

Purpose:?To examine the effect of the human papillomavirus (HPV) type 16-E6 (HPV ‘early’ gene) oncoprotein on in vitro radiosensitivity of HPV-negative/p53 mutant C33a cervical cancer cells.

Methods and materials:?The human cervical cancer cell line C33a was stably transfected with either the HPV16 E6 cDNA cloned into the vector pcDNA?3.0 (C33aE6) or the empty-vector control (C33aV). Radiosensitivity, DNA damage, and cell cycle measurements were made using standard clonogenic assays, immunofluorescent assessment of nuclear histone H2AX phosphorylated on serine-139 (γ-H2AX) foci, and flow cytometry. Western immunoblotting and fluorescence confocal microscopy were used to analyse the changes in cellular proteins. Real-time polymerase chain reaction (PCR) was used to compare levels of aurora A mRNA.

Results:?Compared to C33aV cells, C33aE6 cells showed enhanced radiation cell killing. This was associated with a large amount of polyploidy which was followed by late cell death in C33aE6 cells. Aurora A was highly expressed in C33aE6 cells at pre- and post-irradiation times compared to C33aV cells. Silencing aurora A resulted in a reduced amount of residual γ-H2AX foci in C33aE6 cells, and diminished the difference in radiosensitivity between the C33aE6 and C33aV cells.

Conclusion:?Our in vitro results indicate that genetic instability could be augmented in the HPV-infected cancer cells by up-regulation of aurora A, especially against a background of dysfunctional p53. Further studies are needed to examine whether aurora A could be a viable therapeutic target in HPV-related tumours.     

Comparison of methods to quantify histone H2AX phosphorylation and its usefulness for prediction of radiosensitivity

Abstract

Purpose: The number of radio-induced double-strand breaks is correlated with the number of histone gamma-H2AX (γ-H2AX) foci. For this reason, foci quantification is a useful tool to measure radiation-induced DNA damage and the number of foci has been suggested as a predictive biomarker of radiosensitivity. The aim of the present study was to evaluate the reproducibility of different microscopic methodologies and flow cytometry analysis to score γ-H2AX induction, and its suitability to distinguish a radiosensitive (RS) cell line from a radioresistant (RR) one.

Materials and methods: γ-H2AX analyses were performed by semi-automated and automated microscopic methods and by flow cytometry before and after irradiation in two human lymphoblastoid cell lines and in lymphocytes from three healthy donors.

Results: Reproducible results were obtained by all the methodologies tested, although not all showed the same sensitivity. The RS cell line always showed higher foci counts and higher levels of immunofluorescence intensity after irradiation than the RR cell line.

Conclusions: Our results suggest that microscopic methodologies with z-stage capacity give the most accurate results after 1?Gy irradiation. However, for high doses of ionizing radiation, flow cytometry gives reliable results. Further studies will be necessary to determine the usefulness of γ-H2AX analysis to predict adverse side reactions in radiotherapy patients.     

ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy

Abstract

Purpose: In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied.

Materials and methods: Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA).

Results: Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway.

Conclusions: Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.     

Comparison of organic and inorganic germanium compounds in cellular radiosensitivity and preparation of germanium nanoparticles as a radiosensitizer

Purpose: The aim of this work is to compare the radiosensitizing effect between organic and inorganic germanium compounds and to investigate whether nanometer-sized germanium particles can act as radiosensitizers.

Materials and methods: Bis (2-carboxyethylgermanium) sesquioxide (Ge-132), germanium oxide (GeO₂) and germanium nanoparticles were used in this study. Cell viability was determined by clonogenic survival assay. Cellular DNA damage was evaluated by alkaline comet assay, confocal microscopy and the cellular level of phospho-histone H2AX (γ-H2AX).

Results: Nanometer-sized germanium particles were fabricated. They have a similar radiosensitizing effect as that of GeO₂. Conversely, Ge-132 did not enhance the radiosensitivity of cells. Comet assay was employed to evaluate the level of DNA damage and confirmed that inorganic germanium compounds enhanced cellular radiosensitivity. Notably, the comet assay indicated that the nanoparticle itself caused a higher level of DNA damage. The possibility that germanium nanoparticles per se caused DNA damage was ruled out when the cellular level of γ-H2AX was examined.

Conclusions: We demonstrated that inorganic but not organic germanium compounds exerted radiosensitizing effect in cells. Nanometer-sized germanium particles were fabricated and were able to enhance the radiosensitivity of cells. Confounding effect may occur when comet assay is used to estimate the level of DNA damage in the presence of germanium nanoparticles.     

DNA repair foci and late apoptosis/necrosis in peripheral blood lymphocytes of breast cancer patients undergoing radiotherapy

Abstract

Purpose: Double-strand breaks (DSB) repair and apoptosis are assumed to be key factors in the determination of individual variability in response to radiation treatment. In this study we investigated tumor protein p53 (TP53) binding protein 1 (53BP1) and phosphorylated histone 2A family member X (γH2AX) foci, γH2AX pan-staining and late apoptosis/necrosis (LAN) in lymphocytes from breast cancer (BC) patients undergoing radiotherapy.

Materials and methods: BC patients were subjected to local radiotherapy with fractionated doses using linear accelerator. Adverse reactions of patients were classified according to the Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) criteria. Blood samples were collected before treatment, at various time-points during and after radiotherapy. Residual 53BP1 and γH2AX foci, γH2AX pan-staining were analyzed in peripheral blood lymphocytes (PBL) using the Metafer system and confocal laser scanning microscopy. LAN cells were counted by the trypan blue (TB) exclusion assay. Statistical analysis was performed using Mann–Whitney test, Spearman rank correlation test and analysis of covariance (ANCOVA).

Results: No statistically significant changes were observed in the levels of γH2AX foci during radiotherapy. In contrast, radiation-induced residual 53BP1 were detected already after the first fraction. Increased individual variability in the 53BP1 focus formation was observed during treatment. The background level of DNA repair foci and its individual variability in response to radiotherapy decreased after the end of radiotherapy indicating successful removal of DNA-damaging effects. A correlation between stage of cancer and 53BP1 focus formation was established which suggests the prognostic value of this test. We show that the fraction of LAN cells negatively correlates with the level of 53BP1 and positively correlates with individual radiosensitivity. Only weak correlation was observed between γH2AX pan-staining and LAN cells. Due to large interindividual variability, both in vivo assays, LAN and focus formation, have shown relatively low predictive power at the individual level.

Conclusions: It is likely that radiosensitive patients have less efficient mechanisms of elimination of apoptotic cells with DNA damage resulting in accumulation of LAN cells and facilitating adverse reactions. Our data suggested that the grade of adverse reaction may positively correlate with LAN cells in PBL before and during radiotherapy.     

Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells

Purpose:?To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing.

Materials & methods:?We treated the G1-phase cultures of wild-type and DNA repair deficient mutants of CHO cells with various concentrations of CSC and examined the cell survival by colony formation assay and the induction of DNA double strand breaks by constant field gel electrophoresis as well as the phophorylated histone H2-A variant X (γ-H2AX) assay.

Results:?Gel analysis and γ-H2AX focus assay showed significantly fewer, but still detectable levels of DSB per cell after CSC treatment compared to ionizing radiation (IR) exposures, even when equitoxic radiation exposures were delivered at a low dose rate over the same 8-hour exposure used for CSC treatments. None of the three non-homologous end joining (NHEJ) deficient mutants were remarkably hypersensitive to CSC compared to wild-type cells. In contrast, UV-1 cells that are hypersensitive to several base damage and cross-linking agents showed a higher sensitivity to CSC compared to the other CHO cell lines.

Conclusions:?DNA DSB produced directly by CSC are not principally responsible for its cytotoxicity. Further, the present study does not rule out the possibility that some of these lesions may secondarily result in DSB, such as may occur during impeded DNA replication and whose repair may require systems other than NHEJ.     

Famitinib enhances nasopharyngeal cancer cell radiosensitivity by attenuating radiation-induced phosphorylation of platelet-derived growth factor receptor and c-kit and inhibiting microvessel formation

Purpose: Famitinib is a novel tyrosine kinase inhibitor. We investigated the effects of famitinib on the radiosensitivity of human nasopharyngeal carcinoma (NPC) cell radiosensitivity in vitro and in vivo, and explored its possible mechanisms.

Materials and methods: Human nasopharyngeal carcinoma cell line (CNE-2) were treated with famitinib and radiation, and analyzed by3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic survival assay, and Western blot. A xenograft model using CNE-2 cells was established to analyze the effects of famitinib and radiation on tumor volume and microvessel density (MVD).

Results: Famitinib dose-dependently inhibited CNE-2 cells growth and significantly reduced clonogenic survival (p < 0.05), with a sensitivity enhancement ratio (SER) of 1.45. The tumor inhibition rate of the combined treatment group was 91%, which was significantly higher than the radiation group (35%, p < 0.05) and famitinib group (46%, p < 0.05). Famitinib attenuated radiation-induced phosphorylation of the platelet-derived growth factor receptor (PDGFR) and stem cell factor (c-kit) at 0, 30, 60 min after radiation treatment. Furthermore, radiation combined with famitinib decreased tumor MVD (p < 0.05).

Conclusions: Famitinib significantly increased CNE-2 cell radiosensitivity in vitro and in vivo by attenuating radiation-induced PDGFR and c-kit phosphorylation and by inhibiting microvessel formation.     

Computational analysis of the number,area and density of γ-H2AX foci in breast cancer cells exposed to 111In-DTPA-hEGF or γ-rays using Image-J software

Purpose: To develop a simple method for the quantification of γ-H2AX focus number, density and size.

Methods: MDA-MB-468 human breast cancer cells were treated overnight with ¹¹¹In-diethylenetriaminepentaacetic acid human epidermal growth factor (¹¹¹In-DTPA-hEGF, 0–142 kBq/pmol) or exposed to γ-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (γ-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of γ-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify γ-H2AX foci.

Results: The number of γ-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R² = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R² values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of γ-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R² values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the γ-ray absorbed dose up to 3.0 Gy.

Conclusions: The density, area and IntDen of individual foci, as well as the number of γ-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.     

Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

Purpose:?To investigate radiation-induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response.

Materials and methods:?We used medium transfer and targeted irradiation to examine radiation-induced bystander effects in primary human fibroblast (AG01522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10–100 cGy.

Results:?The results showed no evidence of a low-LET radiation-induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor did we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV α-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation.

Conclusions:?From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in?vitro models to radiation risks in humans.     

Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats

Abstract

Purpose: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation.

Materials and methods: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24–72 hours after irradiation in a dose range of 0–5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry.

Results: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation.

Conclusion: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.     

DNA-dependent protein kinase: effect on DSB repair,G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2?Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1?h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24?h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4?Gy, but decreased both at higher doses of 20?Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.