Relationship between induction of phosphorylated H2AX and survival in breast cancer cells exposed to 111In-DTPA-hEGF

The Auger electron-emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF. METHODS: Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity. RESULTS: All data are presented as mean +/- SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 +/- 6, 8.1 +/- 0.1, and 1.1 +/- 0.9, respectively. The number of gamma-H2AX foci per nucleus was 35 +/- 15, 19 +/- 10, and 1.7 +/- 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 +/- 0.001) and MDA-MB-231 (0.5 +/- 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 +/- 0.1 and 0.53 +/- 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to gamma-irradiation, with an SF after 2 Gy of 0.45 +/- 0.04, compared with 0.7 +/- 0.1 and 0.8 +/- 0.1 for MCF-7 and MDA-MB-231, respectively. The number of gamma-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF. CONCLUSION: DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.     

Comparison of cytogenetic damage in cultured cells from cobalt-60 gamma-radiation and the Auger emitter zinc-65

PURPOSE: To assess the ability of the Auger-emitting nuclide, zinc-65 (65Zn), relative to gamma-irradiation, to cause chromosomal aberrations in cultured rat prostate cells. MATERIALS AND METHODS: Rat prostate adenocarcinoma cells in culture were exposed to doses of 1, 2, 3 or 5 Gy of external gamma-irradiation for 24h or incubated with 0.7, 1.5, 1.8 or 2.8 MBq of 65Zn for 24 h. The uptake by and clearance from cells of 65Zn was measured. Metaphase spreads prepared from washed cells were scored for chromatid- and chromosome-type aberrations. RESULTS: Following exposure to 65Zn or gamma-irradiation, chromatid-type damage was more commonly observed than chromosome-type aberrations. The relationship between induced chromatid damage and gamma dose (to 3 Gy) was best fitted by a second-order polynomial function, while the activity response relationship for chromatid damage caused by 65Zn appeared to be best fitted by a straight line. Measurements of the uptake of 65Zn by cells showed that average concentrations within cells were about 100 times the concentration in the culture medium. Assuming uniform distribution of 65Zn within cells, with 36% in the nucleus, the dose was estimated as 0.70 Gy per MBq added 65Zn, with Auger electrons contributing most (93%) of the dose. Assuming that 20% of cellular zinc was localized in the nucleus, based on previous measurements, the dose to the nucleus was calculated as 0.44 Gy per MBq added 65Zn. RBE values for chromatid damage induced by 65Zn compared to gamma-radiation range from about 1 to 3 based on a uniform dose throughout the cell and from about 2 to 5 based on 20% of 65Zn in the cell nucleus. CONCLUSION: The observed radiotoxicity of 65Zn is consistent with its behaviour as an Auger-emitting radionuclide that is localized to some extent in the nucleus.     

Comparison of cytogenetic damage in cultured cells from cobalt-60 gamma-radiation and the Auger emitter zinc-65

Purpose : To assess the ability of the Auger-emitting nuclide, zinc-65 (65 Zn), relative to γ-irradiation, to cause chromosomal aberrations in cultured rat prostate cells. Materials and methods : Rat prostate adenocarcinoma cells in culture were exposed to doses of 1, 2, 3 or 5 Gy of external gamma-irradiation for 24h or incubated with 0.7, 1.5, 1.8 or 2.8MBq of 65 Zn for 24h. The uptake by and clearance from cells of 65Zn was measured. Metaphase spreads prepared from washed cells were scored for chromatid- and chromosome-type aberrations. Results : Following exposure to 65 Zn or γ-irradiation, chromatidtype damage was more commonly observed than chromosometype aberrations. The relationship between induced chromatid damage and gamma dose (to 3 Gy) was best fitted by a secondorder polynomial function, while the activity-response relationship for chromatid damage caused by 65 Zn appeared to be best fitted by a straight line. Measurements of the uptake of 65 Zn by cells showed that average concentrations within cells were about 100 times the concentration in the culture medium. Assuming uniform distribution of 65 Zn within cells, with 36% in the nucleus, the dose was estimated as 0.70 Gy per MBq added 65 Zn, with Auger electrons contributing most (93%) of the dose. Assuming that 20% of cellular zinc was localized in the nucleus, based on previous measurements, the dose to the nucleus was calculated as 0.44Gy per MBq added 65 Zn. RBE values for chromatid damage induced by 65 Zn compared to gamma-radiation range from about 1 to 3 based on a uniform dose throughout the cell and from about 2 to 5 based on 20% of 65 Zn in the cell nucleus. Conclusion : The observed radiotoxicity of 65 Zn is consistent with its behaviour as an Auger-emitting radionuclide that is localized to some extent in the nucleus.     

The relationship of DNA double-strand break induction to radiosensitivity in human tumour cell lines

Recent data suggest that the differences in radiosensitivity between cell lines can be related to differences in dsb induction (Radford 1986a). In the light of this we have set out to assess the extent to which differences in radiation survival between human tumour cell lines can be attributed to differences in dsb induction. For nine human tumour lines survival was assayed by clonogenic assay and compared with dsb induction by irradiation at ice-bath temperature as measured by neutral filter elution. The lines varied widely in their sensitivity, ranging from a sensitive neuroblastoma (surviving fraction at 2 Gy, SF2 = 0.13) to a resistant bladder carcinoma (SF2 = 0.62). Dsb induction was found to vary between the cell lines, such that resistant cells generally suffered less damage than sensitive ones. However, the relationship between damage induction and cellular sensitivity was not a simple one, and other factors which may influence sensitivity need to be invoked. These data suggest that, in human tumour cell lines, differences in radiosensitivity may at least in part be due to different levels of damage induction, but that some lines may vary in their tolerance of damage due to differences in biological characteristics such as repair capacity.     

Relation between DNA double-strand breaks and energy spectra of secondary electrons produced by different X-ray energies

Purpose: In a radiological examination, low-energy X-radiation is used (<100?keV). For other radiological procedures, the energy used is several MeV. ICRP in publication 103 has currently considered that photons irrespective of their energy have the same radiation weighting factor. Nevertheless, there are topological differences at the nanoscale of X-ray energy deposition as a function of its energy spectrum, meaning that the different interactions with living matter could vary in biological efficacy.

Materials and methods: To study these differences, we characterized our irradiation conditions in terms of initial photon energies, but especially in terms of energy spectra of secondary electrons at the cell nucleus level, using Monte Carlo simulations. We evaluated signaling of DNA damage by monitoring a large number of γH2A.X foci after exposure of G0/G1-phase synchronized human primary endothelial cells from 0.25 to 5?Gy at 40?kV, 220?kV and 4?MV X-rays. Number and spatial distribution of γH2A.X foci were explored. In parallel, we investigated cell behavior through cell death and ability of a mother cell to produce two daughter cells. We also studied the missegregation rate after cell division.

Results: We report a higher number of DNA double-strand breaks signaled by γH2A.X for 40?kVp and/or 220?kVp compared to 4?MVp for the highest tested doses of 2 and 5?Gy. We observed no difference between the biological endpoint studies with 40?kVp and 220?kVp X-ray spectra. This lack of difference could be explained by the relative similarity of the calculated energy spectra of secondary electrons at the cell monolayer.

Conclusion: The energy spectrum of secondary electrons seems to be more closely related to the level of DNA damage measured by γH2A.X than the initial spectrum of photon energy or voltage settings. Our results indicate that as the energy spectrum of secondary electrons increases, the DNA damage signaled by γH2A.X decreases and this effect is observable beyond 220?kVp.     

Radiotoxicity of Intranuclear 125I Atoms Not Bound to DNA

Summary

The radiotoxicity of ¹²⁵I covalently bound to DNA is unusually high. This has been attributed both to the Auger electrons which result from the electron capture process accompanying ¹²⁵I decay and to local transmutation effects which cause extensive damage to nearby structures. We introduced ¹²⁵I into cell nuclei in the form of iodoantipyrine, a molecule which diffuses freely through cells, and we have compared the survival of these cells to those exposed to radiation from extracellular ¹²⁵I-labelled albumin or ⁵⁵Fe-labelled transferrin. We found a value for D₀ of 34 rad for ¹²⁵I decays occurring within the cell nucleus compared to 362 rad for extracellular ¹²⁵I and 277 rad for extracellular ⁵⁵Fe. Since transmutation effects are very short range and ¹²⁵I was distributed uniformly throughout the nucleus rather than bound to DNA, most of the radiotoxicity of intranuclear ¹²⁵I-labelled iodoantipyrine must be due to Auger electrons.     

Study on cell survival, induction of apoptosis and micronucleus formation in SCL-II cells after exposure to the auger electron emitter (99m)Tc

OBJECTIVE: To study the biological effectiveness of Auger electrons emitted by (99m)Tc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation. MATERIAL AND METHODS: Cells were either gamma(60Co)-irradiated (0.67 Gy/min) or exposed to (99m)Tc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of (99m)Tc under the given geometry. RESULTS: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SF 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The (99m)Tc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure. CONCLUSION: No overall increased biological effectiveness due to the emitted Auger electrons of (99m)Tc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after (99m)Tc-exposure at very low doses has to be further investigated.     

阿魏酸钠对ox-LDL诱导血管内皮细胞凋亡的影响

目的观察阿魏酸钠(SF)对氧化修饰的低密度脂蛋白(ox-LDL)诱导血管内皮细胞凋亡的保护作用。方法将体外培养的人脐静脉内皮细胞(HUVEC)分为5组,分别为对照组,ox-LDL(50mg/L)组,SF低浓度(5μmol/L)组,SF中浓度(10μmol/L)组及SF高浓度(20μmol/L)组。SF低、中、高浓度组在加入含相应浓度的SF培养1h后,加入终浓度为50mg/L的ox-LDL。采用MTT比色法确定SF的作用浓度。分别采用Hochest 33258细胞核荧光染色法(定性)和流式细胞仪(定量)检测SF对ox-LDL致内皮细胞凋亡的影响。结果一定浓度的SF能够抑制ox-LDL引起的HUVEC存活率降低,对HUVEC损伤具有拮抗作用,40μmol/L以下是SF拮抗细胞损伤的最佳浓度范围。Hochest33258染色后ox-LDL组细胞核显示较强蓝色荧光,可见核固缩、凋亡小体等典型的凋亡形态学特征;加入20μmol/L SF后,细胞核基本恢复其正常形态。流式细胞术结果显示,加入低、中、高浓度的SF后,其细胞凋亡率分别为25.21%±4.07%、12.45%±2.65%、10.85%±1.23%,与ox-LDL组(31.14%±4.29%)比较均明显降低(P<0.05),且存在量效关系。结论 SF具有拮抗ox-LDL诱导的HUVEC损伤,减少血管内皮细胞凋亡的作用。     

Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line

PURPOSE: Lepidopteran insect cells are known to exhibit very high radioresistance. Although very effective DNA excision-repair has been proposed as a contributing factor, a detailed understanding of insect cell radiation responses has not yet been obtained. Therefore, the study was carried out to understand the in vitro radiation responses of Sf9 lepidopteran cells. MATERIALS AND METHODS: Exponentially growing asynchronous Sf9 cells (derived from ovaries of Spodoptera frugiperda) were exposed to gamma-radiation doses of 2-200 Gy. Cell survival, growth inhibition, cell cycle progression delay, alterations in cell morphology as well as induction of DNA damage, micronuclei and apoptosis were studied at various post-irradiation time intervals. RESULTS: Biphasic survival response curves were obtained with D0 rising from 20 Gy (at doses < or = 60 Gy) to 85 Gy (between 60 and 200 Gy), corroborating earlier reports on lepidopteran cells. An additional downward deviation at 2 Gy indicated a hypersensitive response. Dose-dependent growth inhibition with a transient G2 delay starting 12 h and extending up to 48-96 h was observed at doses of 10-200 Gy, while a brief G1/S transition delay was observed only at higher doses (> or = 100 Gy). Significant DNA damage was detected only at 20 Gy and higher doses, in contrast with human cells that showed similar damage at 2 Gy. Interestingly, micronuclei were not induced at any of the doses tested, although spontaneous micronucleation was evident in <1% of cells. Lack of micronucleus induction even at doses that induced significant DNA damage and a transient G2 block (20-50 Gy) strongly indicated a role of holocentric lepidopteran chromosomes. Apoptosis was detected only in a small proportion of cells (3%) exposed to 200 Gy, and cell/nucleus size and granularity increased by 72-96 h post-irradiation in a dose-dependent manner. Sf9 nucleoids extracted at 2 M NaCl showed higher compactness than the nucleoids prepared from human cells. CONCLUSIONS: It is clearly shown that lepidopteran cells are highly resistant to the induction of DNA damage and micronuclei, and display very low induction of apoptosis at doses up to 200 Gy. While the lack of micronucleus induction seems to be primarily due to the holocentric nature of their chromosomes, certain unique signalling pathways might be responsible for the low induction of apoptosis. Factors causing protection of Sf9 cellular DNA from radiation-induced damage are presently being investigated.     

定量超声基因导入与效应控制

目的研究低频超声裸基因导入新方法与基因导入效应的控制,为疾病的基因治疗提供安全可靠、无细胞毒性的导入新方法。方法用不同参数的35.1kHz超声进行裸基因导入细胞实验,用激光共聚焦显微镜、荧光显微镜、流式细胞仪和分光光度法分析细胞膜形态、细胞膜通透性与损伤阈值、细胞存活率和基因表达。结果实验所见90%细胞存活点的超声能量积分是细胞膜通透安全控制参数,80%细胞存活点的能量积分是细胞的损伤阈值。绿色荧光蛋白(GFP)在安全超声参数下成功导入了S180细胞,细胞转染率为35.83%±2.53%(n=6),荧光表达强度明显高于病毒转染法和控制组(P<0.001)。结论安全参数下低频超声可有效地将裸基因导入S180肿瘤细胞,其基因表达强度随超声能量积累而增强,并有细胞凋亡倾向。     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts

PURPOSE: To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. MATERIALS AND METHODS: Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. RESULTS: The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7+/-0.2 to 5.0+/-0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. CONCLUSIONS: The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

Cellular effects of high-intensity focused continuous wave ultrasound alone and in combination with X-rays

PURPOSE: To investigate the cellular effects of 1.1 MHz high-intensity focused continuous wave (CW) ultrasound alone and in combination with X-rays. MATERIALS AND METHODS: V79 cells were exposed to ultrasound of different intensities for 30s (non-hyperthermic conditions). In the combined treatments, cells were exposed to ultrasound (63 W/cm2) either before or after 2 Gy X-irradiation. Cellular effects studied were clonogenic survival, DNA strand breaks (pulsed-field gel electrophoresis, DNA precipitation assay) and membrane integrity (morphological analysis). RESULTS: With increasing ultrasound intensity, cell survival decreased in a dose-dependent manner as the induction of DNA strand breaks, the fraction of cells with lost membrane integrity and cell lysis increased. In the treatments with combined exposures, the regimen with X-rays before ultrasound had a nearly additive effect on cell kill, whereas the reverse regimen with ultrasound exposure before X-irradiation resulted in a synergistic effect (p<0.012). CONCLUSIONS: High-intensity focused CW ultrasound induces an intensity-dependent reduction in clonogenic survival in V79 cells, which seems to depend on both DNA and membrane damage. Combined exposures of ultrasound and X-rays resulted in a synergistic reduction in cell survival when cells were exposed to ultrasound before X-rays but not for the reverse regimen. Thus, a larger fraction of the repairable sublethal cell damage induced by an initial ultrasound exposure was rendered non-repairable by a subsequent X-ray exposure than if the reverse treatment order was used.     

Calcium-dependent injury of human microvascular endothelial cells induced by a variety of iodinated radiographic contrast media

RATIONALE AND OBJECTIVES: The aim of the present study was to determine the possible mechanisms underlying the endothelial cell damage induced by iodinated radiographic contrast materials (RCM). METHODS: The cultured human skin microvascular endothelial cells (HMVECs) were exposed to various contrast media, and the cell viability was measured by mitochondrial enzyme activity. Nuclear damage was assessed by Hoechst 33342 staining and a fluorescent single-cell gel electrophoresis. The effects of contrast materials on the cellular ATP content and intracellular free Ca2+ concentration were subsequently examined. RESULTS: Although the iodinated RCM tested all caused the cell injury in HMVECs, ionic RCM including amidotrizoate and ioxaglate were more potent in producing the cell damage than nonionic RCM. It is unlikely that the contrast material-induced cell damage is associated with hyperosmolality, since hyperosmolar solution of mannitol or NaCl had no marked influence on the endothelial cell viability. Nuclear damage was noted in cells exposed to amidotrizoate. Amidotrizoate lowered cellular ATP content while elevating the intracellular free Ca2+ concentration. It was notable that the RCM-induced endothelial cell damage was reversed by the chelation of intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the removal of extracellular Ca2+. CONCLUSIONS: Both ionic and nonionic contrast materials caused nuclear damage of endothelial cells. The decrease in tissue ATP content and elevation of intracellular Ca2+ are likely to contribute to the contrast materials-induced endothelial cell damage.     

Delayed cell death and bystander effects in the progeny of Chinook salmon embryo cells exposed to radiation and a range of aquatic pollutants

PURPOSE: To determine whether delayed and bystander effects can be seen in both a non malignant teleost fish cell line, (CHSE) and a malignant teleost fish cell line (EPC) when exposed to low doses of ionising radiation and genotoxic pollutants. METHODS: Teleost fish cells were briefly exposed to radiation and chemical toxins at low doses. Clonogenic survival was measured in the exposed population and the distant progeny of exposed cells to assess early and delayed cell death. Clonogenic survival was also measured in cultures, which received medium from briefly exposed cells to determine bystander effects. RESULTS: The dose response pattern for both early and delayed cell death was found to differ for different stressors. Different mechanisms of cell death appear to be involved in the early cytotoxic effect and the delayed effect. No delayed cell death occurred in a transformed fish cell line (EPC). Bystander effects occurred in CHSE cells and were similar in intensity to previously reported mammalian cell bystander effects. CONCLUSIONS: The results may have implications for radiation and environmental protection of biota. They demonstrate that damage caused by low doses of radiation and common aquatic pollutants is not only similar but occurs in both acute and delayed forms.     

Cellular effects of high-intensity focused continuous wave ultrasound alone and in combination with X-rays

Purpose : To investigate the cellular effects of 1.1 MHz high-intensity focused continuous wave (CW) ultrasound alone and in combination with X-rays. Materials and methods : V79 cells were exposed to ultrasound of different intensities for 30 s (non-hyperthermic conditions). In the combined treatments, cells were exposed to ultrasound (63 W/cm 2) either before or after 2 Gy X-irradiation. Cellular effects studied were clonogenic survival, DNA strand breaks (pulsed-field gel electrophoresis, DNA precipitation assay) and membrane integrity (morphological analysis). Results : With increasing ultrasound intensity, cell survival decreased in a dose-dependent manner as the induction of DNA strand breaks, the fraction of cells with lost membrane integrity and cell lysis increased. In the treatments with combined exposures, the regimen with X-rays before ultrasound had a nearly additive effect on cell kill, whereas the reverse regimen with ultrasound exposure before X-irradiation resulted in a synergistic effect (p < 0.012). Conclusions : High-intensity focused CW ultrasound induces an intensity-dependent reduction in clonogenic survival in V79 cells, which seems to depend on both DNA and membrane damage. Combined exposures of ultrasound and X-rays resulted in a synergistic reduction in cell survival when cells were exposed to ultrasound before X-rays but not for the reverse regimen. Thus, a larger fraction of the repairable sublethal cell damage induced by an initial ultrasound exposure was rendered non-repairable by a subsequent X-ray exposure than if the reverse treatment order was used.     

Dose per unit cumulated activity (S-values) for e− and beta emitting radionuclides in cancer cell models calculated by Monte Carlo simulation

Cell dosimetry is relevant regarding new efforts in specific molecular radiotherapy using Auger, CE and beta emitters. Absorbed dose in cells can be obtained by means of the dose per unit cumulated activity (S-values), together with the activity distribution. In this work, Monte Carlo simulation codes PENELOPE and MCNPX were used to obtain cellular S-values for point and extended sources of electrons and beta emitting radionuclides in the nucleus of breast (MDA-MB231, MCF7) and prostate (PC3) cancer cell models.     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal,NBS and AT fibroblasts

Purpose : To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. Materials and methods : Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. Results : The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7 ±0.2 to 5.0 ±0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. Conclusions : The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line

Purpose: Lepidopteran insect cells are known to exhibit very high radioresistance. Although very effective DNA excision–repair has been proposed as a contributing factor, a detailed understanding of insect cell radiation responses has not yet been obtained. Therefore, the study was carried out to understand the in vitro radiation responses of Sf9 lepidopteran cells.

Materials and methods: Exponentially growing asynchronous Sf9 cells (derived from ovaries of Spodoptera frugiperda) were exposed to gamma‐radiation doses of 2–200?Gy. Cell survival, growth inhibition, cell cycle progression delay, alterations in cell morphology as well as induction of DNA damage, micronuclei and apoptosis were studied at various post‐irradiation time intervals.

Results: Biphasic survival response curves were obtained with D₀ rising from 20?Gy (at doses ≤60?Gy) to 85?Gy (between 60 and 200?Gy), corroborating earlier reports on lepidopteran cells. An additional downward deviation at 2?Gy indicated a hypersensitive response. Dose‐dependent growth inhibition with a transient G2 delay starting 12?h and extending up to 48–96?h was observed at doses of 10–200?Gy, while a brief G1/S transition delay was observed only at higher doses (≥100?Gy). Significant DNA damage was detected only at 20?Gy and higher doses, in contrast with human cells that showed similar damage at 2?Gy. Interestingly, micronuclei were not induced at any of the doses tested, although spontaneous micronucleation was evident in <1% of cells. Lack of micronucleus induction even at doses that induced significant DNA damage and a transient G2 block (20–50?Gy) strongly indicated a role of holocentric lepidopteran chromosomes. Apoptosis was detected only in a small proportion of cells (3%) exposed to 200?Gy, and cell/nucleus size and granularity increased by 72–96?h post‐irradiation in a dose‐dependent manner. Sf9 nucleoids extracted at 2?M NaCl showed higher compactness than the nucleoids prepared from human cells.

Conclusions: It is clearly shown that lepidopteran cells are highly resistant to the induction of DNA damage and micronuclei, and display very low induction of apoptosis at doses up to 200?Gy. While the lack of micronucleus induction seems to be primarily due to the holocentric nature of their chromosomes, certain unique signalling pathways might be responsible for the low induction of apoptosis. Factors causing protection of Sf9 cellular DNA from radiation‐induced damage are presently being investigated.     

CELLDOSE: a Monte Carlo code to assess electron dose distribution--S values for 131I in spheres of various sizes.

Monte Carlo simulation can be particularly suitable for modeling the microscopic distribution of energy received by normal tissues or cancer cells and for evaluating the relative merits of different radiopharmaceuticals. We used a new code, CELLDOSE, to assess electron dose for isolated spheres with radii varying from 2,500 mum down to 0.05 mum, in which (131)I is homogeneously distributed. METHODS: All electron emissions of (131)I were considered, including the whole beta(- 131)I spectrum, 108 internal conversion electrons, and 21 Auger electrons. The Monte Carlo track-structure code used follows all electrons down to an energy threshold E(cutoff) = 7.4 eV. RESULTS: Calculated S values were in good agreement with published analytic methods, lying in between reported results for all experimental points. Our S values were also close to other published data using a Monte Carlo code. Contrary to the latter published results, our results show that dose distribution inside spheres is not homogeneous, with the dose at the outmost layer being approximately half that at the center. The fraction of electron energy retained within the spheres decreased with decreasing radius (r): 87.1% for r = 2,500 mum, 8.73% for r = 50 mum, and 1.18% for r = 5 mum. Thus, a radioiodine concentration that delivers a dose of 100 Gy to a micrometastasis of 2,500 mum radius would deliver 10 Gy in a cluster of 50 mum and only 1.4 Gy in an isolated cell. The specific contribution from Auger electrons varied from 0.25% for the largest sphere up to 76.8% for the smallest sphere. CONCLUSION: The dose to a tumor cell will depend on its position in a metastasis. For the treatment of very small metastases, (131)I may not be the isotope of choice. When trying to kill isolated cells or a small cluster of cells with (131)I, it is important to get the iodine as close as possible to the nucleus to get the enhancement factor from Auger electrons. The Monte Carlo code CELLDOSE can be used to assess the electron map deposit for any isotope.     

Delayed cell death and bystander effects in the progeny of Chinook Salmon Embryo cells exposed to radiation and a range of aquatic pollutants

Purpose: To determine whether delayed and bystander effects can be seen in both a non malignant teleost fish cell line, (CHSE) and a malignant teleost fish cell line (EPC) when exposed to low doses of ionising radiation and genotoxic pollutants.

Methods: Teleost fish cells were briefly exposed to radiation and chemical toxins at low doses. Clonogenic survival was measured in the exposed population and the distant progeny of exposed cells to assess early and delayed cell death. Clonogenic survival was also measured in cultures, which received medium from briefly exposed cells to determine bystander effects.

Results: The dose response pattern for both early and delayed cell death was found to differ for different stressors. Different mechanisms of cell death appear to be involved in the early cytotoxic effect and the delayed effect. No delayed cell death occurred in a transformed fish cell line (EPC). Bystander effects occurred in CHSE cells and were similar in intensity to previously reported mammalian cell bystander effects. Conclusions: The results may have implications for radiation and environmental protection of biota. They demonstrate that damage caused by low doses of radiation and common aquatic pollutants is not only similar but occurs in both acute and delayed forms.