Studies on radiation-induced apoptosis in G0 human lymphocytes.

PURPOSE: To determine the relationships between the frequencies of radiation-induced chromosomal alterations and the extent of apoptosis in G0 human lymphocytes. MATERIAL AND METHODS: G0 human peripheral blood lymphocytes (HPBL) were X or gamma-irradiated, in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). Directly after irradiation, a part of the lymphocytes were stimulated to grow while the rest were stimulated 48 h after irradiation. These lymphocyte cultures were analysed for induction of chromosomal aberrations. A subset of lymphocytes was kept in G0 and analysed for cell viability, apoptosis and p53 expression. RESULTS: The fraction of cells bearing dicentrics was reduced in lymphocytes stimulated to grow 48 h post irradiation as compared to lymphocytes stimulated immediately after irradiation. The decrease in the frequency of dicentrics correlated with the increase in the number of apoptotic cells. The operative apoptotic pathway in irradiated Go lymphocytes was dependent on the expression of p53. CONCLUSIONS: The radiation-induced apoptotic response of G0 lymphocytes is p53 dependent and increases with the time they are held in G0. When mitogen was added 48 h after irradiation, cells with dicentrics were either preferentially eliminated or did not enter mitosis. Thus the radiation-induced damage can be underevaluated depending on the time between radiation exposure and the induction of proliferation. These results may have relevance for biodosimetry studies or for evaluations of the efficacy of radiotherapy which are based on the frequencies of chromosomal aberrations.

Induction of homologous recombination in the hprt gene of V79 Chinese hamster cells in response to low- and high-LET irradiation.

Dense ionization tracks from high linear energy transfer (LET) radiations form multiple damaged sites (MDS), which involve several types of DNA lesions in close vicinity. The primary DNA damage triggers sensor proteins that activate repair processes, cell cycle control or eventually apoptosis in subsequent cellular responses. The question how homologous recombination (HR) and non-homologous end joining (NHEJ) interact in the repair of radiation-induced DNA damage of MDS type has been addressed in different model systems but several questions remain to be answered. We have therefore challenged cells with treatments of ionizing radiation of different qualities to investigate whether primary DNA damages of different complexity are reflected in the processes of repair by HR as well as cell survival. We used the V79 derived SPD8 cell line to determine the induction of HR in the hprt exon 7 and clonogenic assay for survival in response to radiation. SPD8 cells were irradiated with gamma-rays (137Cs 0.5 keV/microm), boron ions (40 and 80 keV/microm) and nitrogen ions (140 keV/microm), with doses up to 5 Gy. Analysis of clonogenic survival showed that B-ions (80 keV/microm) and N-ions were more toxic than gamma-rays, 4.1 and 5.0 times respectively, while B-ions at 40 keV/microm were 2.0 times as toxic as gamma-rays. Homologous recombination in the cells exposed to B-ions (80 keV/microm) increased 2.9 times, a significant response as compared to cells exposed to gamma-rays, while for B-ions (40 keV/microm) and N-ions a nonsignificant increase in HR of 1.2 and 1.4, respectively, was observed. We hypothesize that the high-LET generated formation of MDS is responsible for the enhanced cytotoxicity as well as for the mobilization of the HR machinery.

Changes in chromatin conformation during radiation-induced apoptosis in human lymphocytes.

Human peripheral lymphocytes in G(0) phase were irradiated with 1-5 Gy of gamma rays. The biochemical and morphological changes characteristic of apoptosis were examined for 72 h after irradiation. In parallel, changes in chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) and by measurements of nuclear halo size. An immediate and dose-dependent relaxation of chromatin, which became saturated at doses above 2-3 Gy, was revealed by the AVTD method. The state of relaxed chromatin lasted up to 12-24 h after irradiation, a response considerably longer than the time attributable to repair of radiation-induced DNA breaks. Measurements of nuclear halo size also indicated the initial relaxation of chromatin in the irradiated cells and its subsequent condensation. This condensation of chromatin as revealed with AVTD correlated well with nuclear condensation, as measured with dual fluorescence staining, and with DNA fragmentation, as measured by conventional and pulsed-field gel electrophoresis (PFGE). Late apoptotic cells did not contribute significantly to the AVTD signal, showing that the chromatin of these cells was completely condensed and fragmented.

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes.

PURPOSE: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. MATERIALS AND METHODS: Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3 Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). RESULTS: A significant decrease in reproductive cell death was observed after 3 Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDRI analysed day 6, although not of statistical significance. Radiation-induced apoptosis was efficiently counteracted by growth factors up to 24-48 h after 3 Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR 1 cells plated day 6), a significant increase in reproductive cell death was found (3 Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR 1 plated day 1 and HDR6). CONCLUSIONS: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.

pH-dependent DNA cleavage in permeabilized human fibroblasts.

In several cell types, apoptosis is associated with intracellular acidification and activation of a pH-dependent endonuclease. We have examined the effect of acidic pH on the DNA of permeabilized human fibroblasts, and observed cleavage of DNA into high-molecular-mass fragments. This pH-dependent DNA breakage was modulated by temperature, the presence of histones and diethyl pyrocarbonate. Superoxide dismutase and chelators with high affinity for Cu prevented DNA fragmentation, whereas catalase, DMSO and Desferal (desferrioxamine mesylate) offered no protection. Fragmentation of DNA into high-molecular-mass fragments, which is occasionally observed as an early phase of apoptosis, is thought to result from the activation of endonuclease(s). Our results suggest that such fragmentation also occurs through induction of copper-mediated site-specific DNA damage that is enhanced by intracellular acidification.

Detection of single-strand breaks and formamidopyrimidine-DNA glycosylase-sensitive sites in DNA of cultured human fibroblasts.

Under oxidative stress 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), a damaged base with mutagenic potential, and single-strand breaks (SSB) are formed in DNA. Both lesions are frequently used as a parameter for oxidative damage of DNA. Here we report on results from the evaluation of a modified nick translation assay, where 8-oxodG and SSB formation in cellular DNA of cultured human fibroblasts were simultaneously detected. The assay is based on a method previously described by others, with several modifications in reaction conditions and type of substrate. We used formamidopyrimidine-DNA glycosylase (FPG) in our assay in order to measure the formation of FPG-sensitive lesions (which include 8-oxodG) in DNA of human fibroblasts in response to ionising radiation. The quantification of the DNA damage was based on calibration experiments with plasmid DNA pUC19. Dose-response curves of SSB and FPG-sensitive lesion formation in human fibroblasts VH-10 were established. A very low background level of 8-oxodG was detected in unirradiated fibroblasts (approx. 500 residues per cell).

Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies.

A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs). Four recombinant phages with inserts of about 2-5 kbp were isolated. One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30. This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells. The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs. Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260. All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51. The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.