L. De Ridder

Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition.

The chromosomal radiosensitivity of breast cancer patients with a known or putative genetic predisposition was investigated and compared to a group of healthy women. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co γ-rays after 71 h incubation, and chromatid breaks were scored in 50 metaphases. For the micronucleus assay lymphocytes were exposed in vitro to 3.5 Gy 60Co γ-rays at a high dose rate or low dose rate. 70 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a known or putative genetic predisposition was on the average more radiosensitive than a population of healthy women, and this with the G2 as well as with the high dose rate and low dose rate micronucleus assay. With the G2 assay 43% of the patients were found to be radiosensitive. A higher proportion of the patients were radiosensitive with the micronucleus assay (45% with high dose rate and 61% with low dose rate). No correlation was found between the G2 and the G0-micronucleus chromosomal radiosensitivity. Out of the different subgroups considered, the group of the young breast cancer patients without family history showed the highest percentage of radiosensitive cases in the G2 (50%) as well as in the micronucleus assay (75–78%).

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique.

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide−). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.

Flow cytometry as a quantitative and sensitive method to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes

Human peripheral blood lymphocytes, irradiated in vitro, die by an apoptotic process. The number of apoptotic cells after in vitro gamma-irradiation (0, 0.1, 0.2, 1, 2 and 5 Gy) was measured by flow cytometry using Annexin V and DiOC6 (a cationic dye) after 24 and 48 h incubation. The mean dose-response curves for apoptosis of six healthy volunteers obtained with both methods were steep below 1 Gy and flatter at higher doses. A slightly higher number of apoptotic cells was observed with DiOC6, compared to Annexin V. This can be assigned to a minor DiOC6-int/PI- population. Forty-eight hour cultures contained higher numbers of apoptotic cells compared with 24 h cultures. For both culture times, DiOC6 and Annexin V detected a statistically significant difference between a control sample and a 0.1 Gy irradiated one, illustrating the high sensitivity of the methods.

In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes

One of the major drawbacks of the in vitro micronucleus (MN) assay for human lymphocytes is its reduced sensitivity for the detection of damage induced by low radiation doses, due to the high variability among the spontaneous MN frequencies. In this paper we investigated the enhancement of the sensitivity of the MN assay by analysing spontaneous and radiation-induced MN for the presence of centromeres. For this, in situ hybridization (FISH) with the human pancentromeric DNA probe, p82H, was performed. Our results revealed that a high percentage (73%) of the spontaneous MN contain a centromere. These centromere-positive MN indicate the presence of a whole chromosome/chromatid. After in vitro irradiation with low doses (0.1-2 Gy) 60Co gamma-rays mainly centromere-negative MN were induced while only a very small number of additional centromere-positive MN were formed. This demonstrates that radiation-induced MN mainly contain acentric fragments pointing to the clastogenic action of ionizing radiation. Furthermore, our data show that the sensitivity of the MN assay for low dose detection is increased by scoring only centromere-negative MN.

Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers

Purpose: The chromosomal radiosensitivity of a selected group of familial breast cancer patients carrying a mutation in BRCA1 (n=11) or BRCA2 (n=9) and a group of healthy mutation carriers (n=12) was investigated and compared to a reference group of breast cancer patients without a BRCA1/2 mutation (n=78) and a group of healthy women carrying no mutation (n=58). Materials and methods: The chromosomal radiosensitivity was assessed with the G2 and the G0‐micronucleus (MN)‐assay on fresh blood samples and on Epstein‐Barr virus (EBV)‐transformed lymphoblastoid cell lines. For the MN‐assay, lymphocytes were exposed in vitro to 3.5 Gy and 2 Gy 60Co γ‐rays at a high dose rate (HDR) or low dose rate (LDR). 70‐h post‐irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. For the G2‐assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co γ‐rays after 71h incubation. Cultures were arrested 90 min after irradiation and chromatid breaks were scored in 50 metaphases. Results: The group of breast cancer patients with a BRCA1 or 2 mutation was on average more radiosensitive than the control group, but not different from breast cancer patients without a BRCA mutation. The radiation response of healthy BRCA1/2 carriers was not significantly different from the control group and also not different from relatives without a BRCA mutation. Comparing the radiation response in EBV cell lines derived from breast cancer patients with or without a BRCA1 mutation revealed no significant difference. Conclusions: Our results reveal that chromosomal radiosensitivity observed in breast cancer patients heterozygous for BRCA1 or 2 mutations, could not be demonstrated in healthy BRCA1/2 mutation carriers. This suggests that mutations in BRCA1 or 2 genes are not playing a main role in chromosomal radiosensitivity, this although BRCA1 and 2 are both involved in DNA repair/signalling processes.

Changes in peripheral blood lymphocyte subsets in patients undergoing radiotherapy.

PURPOSE To investigate the changes in peripheral blood lymphocyte subpopulations in patients undergoing radiotherapy. MATERIALS AND METHODS In 8 patients undergoing external beam radiotherapy to the pelvis, the different lymphocyte subpopulations were followed during treatment. The lymphocyte populations were determined using two-colour flow cytometry. The study comprises the T-helper, T-suppressor/cytotoxic cells, the B-lymphocytes and natural killer (NK) cells. RESULTS The B-cells were characterized by a steep decrease at the beginning of the radiotherapy. They reached their lowest level at an equivalent total body dose of approximately 1.5 Gy and remained constant during the rest of the therapy (10% of the initial level). In T-cells (both T-helper and T-suppressor subsets) the steep decrease was less pronounced. T-lymphocytes reached a base level at 2.5 Gy equivalent total body dose (20% of the initial level). No significant differences between the T-helper and the T-suppressor/cytotoxic cells were observed. NK cells were characterized by a weak decline during the first weeks of therapy, being less pronounced than in the other populations. Near the end of therapy, the NK cells reached the level of the T-lymphocytes. CONCLUSION In vivo, NK cells were the most radioresistant and B-cells the most radiosensitive lymphocytes. No significant differences between T-helper and T-suppressor/cytotoxic cells were observed. These data are in agreement with the differences in apoptosis induction in peripheral blood lymphocyte subpopulations after in vitro gamma-irradiation of whole blood lymphocytes.

Micronucleus Induction in Peripheral Blood Lymphocytes of Patients under Radiotherapy Treatment for Cervical Cancer or Hodgkin's Disease

The genetic damage present in peripheral blood lymphocytes of patients treated with fractionated partial-body radiation therapy for cervical cancer or Hodgkins disease was followed during treatment by means of the cytokinesis-block micronucleus assay. For each patient a dose-response relationship with respect to the number of micronuclei after in vitro irradiation of blood samples pretreatment was also determined. Comparing the individual in vivo-in vitro data, the micronucleus yields after the equivalent whole-body dose during radiotherapy were found to differ substantially from the in vitro dose-response. Contrary to the linear-quadratic dose dependence after in vitro irradiation the initial increase in the micronucleus yield during radiotherapy levelled off at elevated doses. The observed differences cannot be attributed only to the effects of interphase death and the partial irradiation of the lymphocyte pool. The correlation between the micronucleus yield and the equivalent whole-body dose for values up to 2 Gy, observed for the pooled data of the first part of the radiotherapy treatment, demonstrates the suitability of the cytokinesis-block micronucleus assay as a biological dosemeter after accidents involving partial-body irradiation.

Micronuclei Induced by Fast Neutrons Versus 60Co γ-rays in Human Peripheral Blood Lymphocytes

Here we compared the effectiveness of neutrons (〈E〉 = 5·5 MeV) versus 60Co γ-rays in producing micronuclei (MN) in human lymphocytes. To obtain dose-response data, blood samples of six donors were irradiated with doses ranging from 0·1 to 5 Gy for γ-rays and 0·1–3 Gy for neutrons. A linear dependence of MN yield with dose was found for fast neutrons while for γ-rays a nonlinear dependence existed. For both radiation qualities no significant interindividual differences were found. Derived relative biological effectiveness values decreased with increasing dose. The MN frequency distributions were overdispersed with respect to the Poisson distribution, with neutrons showing higher dispersion values than with γ-rays. To compare the repair kinetics of both radiation qualities split-dose experiments were performed. A dose of 4 Gy γ-rays (3 Gy neutrons) was delivered either as a single exposure or in two equal fractions separated by time intervals ranging from 30 min to 10h (30 min to 7h for neutrons). The data ...

Study of Dose-rate and Split-dose Effects on the in Vitro Micronucleus Yield in Human Lymphocytes Exposed to X-rays

This paper reports the effects of changes in dose-rate and dose-fractionation on the micronucleus (MN) yield in human lymphocytes exposed to 250 kV X-rays. For the investigation of dose-rate effects whole blood samples of four healthy donors were irradiated with doses ranging from 1 to 4 Gy given at various dose-rates between 0.2 and 40 Gy/h. For the higher doses (3 and 4 Gy) a decline in the MN yield became apparent when the dose-rate was reduced below 1.6 Gy/h. This effect was enhanced systematically by a further lowering of the dose-rate. For lower doses (1 and 2 Gy) the reduction in the MN yield was less pronounced: only a small effect was observed for two donors when a dose of 2 Gy was administered at a dose-rate of 0.2 Gy/h. In the split-dose experiment a dose of 4 Gy was delivered either as a single exposure or in two fractions of 2 Gy, separated by time intervals ranging from 30 min to 10 h. A continuous decrease of the MN yield with increasing interfraction time is observed: after an initial fast decline a further slight reduction in the MN yield occurs. The observed dose-rate and split-dose effects on the MN yield can be attributed to repair of sublethal damage.

Invasiveness of human glioma cell lines in vitro: relation to tumorigenicity in athymic mice

SummaryFive established cell lines derived from human anaplastic astrocytomas or glioblastoma multiforme were tested for invasiveness into precultured chick heart fragments in vitro. Four of the cell lines (U118 MG, D54 MG, U373 MG and A172) were strongly invasive into the heart tissue. A fifth cell line, U251 MG sp, which was only tumorigenic at doses of greater than 1×108 cells in athymic mice, was non-invasive in vitro. One line, A172, was invasive but not tumorigenic in athymic mice, although a related invasive subline, D54 MG, at later passage levels was tumorigenic even at low cell doses. Invasion of the glioma cells was characterized by progressive and irreversible replacement of the precultured chick heart tissue. Both by light and transmission electron microscopy, a similar pattern of invasion was observed as earlier found with experimental rat glioma cells in the same system. Some human cell lines established from human gliomas retain invasive properties after a prolonged culture period in vitro.