Cellular origin of ionizing radiation‐induced NF‐κB activation in vivo and role of NF‐κB in ionizing radiation‐induced lymphocyte apoptosis

Purpose: To investigate the cellular origin of ionizing radiation (IR)‐induced NF‐κB activation in vivo and the role of NF‐κB in IR‐induced lymphocyte apoptosis.

Materials and methods: NF‐κB activities were analysed by gel shift/supershift assay in isolated murine T‐ and B‐cells, macrophages (M?) and tissues from normal and T‐ and B‐cell‐deficient Rag1 mice with or without exposure to IR. IR‐induced lymphocyte apoptosis was determined by analysis of 3,3′‐dihexyloxacarbocyanine iodide (DiOC₆) uptake, annexin‐V staining and the sub‐G0/1 population, or by TUNEL assay.

Results: The results showed that IR activated NF‐κB in lymphocytes, including both T‐ and B‐cells, but failed to do so in M?. Furthermore, T‐ and B‐cell‐deficient Rag1 mice exposed to IR exhibited a significant reduction in NF‐κB activation as compared with normal mice. Although NF‐κB1 (p50) gene knockout or NF‐κB decoy oligonucleotide treatment specifically inhibited IR‐induced lymphocyte NF‐κB activation, they had no significant effect on IR‐induced lymphocyte apoptosis.

Conclusions: This finding suggests that lymphocytes are the main cellular origin of IR‐induced NF‐κB activation in vivo. However, NF‐κB activation has no significant effect on IR‐induced lymphocyte apoptosis.     

Role of Ca2+ in radiation‐induced damage in murine splenocytes

Purpose: To address the links between calcium, peroxidation, cell damage and death and the response of the enzymes involved in free radical metabolism, in splenocytes of mice irradiated with gamma‐rays.

Materials and methods: Splenocytes of Swiss albino mice were irradiated with various doses (0–7?Gy) of gamma‐rays (⁶⁰Co) at a dose‐rate of 0.0575?Gy?s^?1. Membrane peroxidation and fluidity were determined by the thiobarbituric acid‐reactive substances (TBARS) method, and fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), respectively. Apoptosis was analysed by nucleosomal ladder formation and activity of NF‐κB by electrophoretic mobility shift assay (EMSA). The specific activities of the antioxidant enzymes, lactate dehydrogenase (LDH), levels of nitric oxide (NO^?) and glutathione were determined spectrophotometrically. Modulatory effects of Ca²⁺ were examined at 3?Gy using different concentrations (1, 3 and 5?mM) in the presence or absence of the ionophore A23187.

Results: Irradiation of splenocytes resulted in enhanced peroxidative damage, membrane fluidity, apoptosis and DNA binding activity of NF‐κB. The specific activities of LDH and antioxidant enzymes superoxide dismutase (SOD), DT‐diaphorase (DTD), glutathione S‐transferase (GST) and levels of glutathione (GSH) and NO^? were increased with radiation dose up to 4?Gy. Ca²⁺ augmented the radiation‐induced responses. The presence of ionophore A23187 potentiated the modulatory effects of Ca²⁺.

Conclusions: These findings show that Ca²⁺ augments radiation damage and is more effective intracellularly. Ca²⁺, peroxidation, cellular damage and apoptosis are possibly interlinked through signals, as is evident from the increased activity of NF‐κB and generation of NO^?. The enhanced antioxidant status suggests an attempt made by the irradiated cells to maintain their normal functions.     

Bowman–Birk protease inhibitor activates DNA‐dependent protein kinase and reduces formation of radiation‐induced dicentric chromosomes

Purpose: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes.

Materials and methods: An effect of BBI upon DNA repair was investigated by quantification of radiation‐induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA‐dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53‐derived peptide.

Results: The formation of radiation‐induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI‐mediated reduction of dicentric chromosome formation depended on the presence of wild‐type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA‐dependent protein kinase (DNA‐PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA‐PK) activity. In SCID mouse fibroblasts deficient in DNA‐PK activity, BBI failed to reduce the amount of radiation‐induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI.

Conclusions: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA‐PK activity.     

Sister chromatid exchanges in ring chromosomes following X‐irradiation of human lymphocytes

Purpose: To examine whether X‐rays induce sister chromatid exchanges (SCE).

Materials and methods: Peripheral lymphocytes irradiated in vitro or in vivo were cultured and treated with okadaic acid to generate premature chromosome condensation (PCC). When identical spreads were analysed using conventional Giemsa staining and pan‐centromeric fluorescence in situ hybridization painting, ring chromosomes were observed.

Results: In PCC preparations, cells in the late G₂ phase and late M phase were observed. In late M phase cells, 17–20% of ring chromosomes lacked one chromatid (single‐chromatid ring), irrespective of dose. Both the distribution patterns of centromeres in rings and intercentromere distances in dicentric rings indicate that a considerable number of single‐chromatid rings might be formed by SCE occurring in a chromosome‐type ring, thereby joining strands of two rings, followed by a transformation into one ring. These single‐chromatid rings were less stable in vivo than chromosome‐type rings.

Conclusion: Single‐chromatid rings visualized clearly using PCC techniques indicate SCE in the respective rings. Contrary to the conventional SCE‐detecting technique, this approach does not require the use of bromodeoxyuridine, which itself leads to SCE. Some of the observed SCE might be secondary products resulting from the repair of radiation‐induced DNA damage, while others may be spontaneous.     

Chromatin‐ and temperature‐dependent modulation of radiation‐induced double‐strand breaks

Purpose: To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double‐strand breaks (DSB) in structures derived from human diploid fibroblasts.

Materials and methods: Agarose plugs with different chromatin structures (intact cells±wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X‐rays at 2 or 37°C and lysed using two different lysis protocols (new ice‐cold lysis or standard lysis at 37°C). Induction of DSB was determined by constant‐field gel electrophoresis.

Results: The dose‐modifying factor (DMF_temp) for irradiation at 37 compared with 2°C was 0.92 in intact cells (i.e. more DSB induced at 2°C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMF_temp was influenced by the presence of 0.1?M DMSO or 30?mM glutathione, but not by post‐irradiation temperature.

Conclusion: The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post‐irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.     

Androgen and the blocking of radiation‐induced sensitization to Fas‐mediated apoptosis through c‐jun induction in prostate cancer cells

Purpose: To clarify the key mechanism by which androgen makes prostate cancer cells highly resistant to Fas‐mediated apoptosis.

Materials and methods: The role of c‐jun induction by 10?nM dihydrotestosterone (DHT) in 5?Gy radiation‐induced up‐regulation of Fas and sensitization to the apoptosis was studied by using the human prostate cancer cell line LNCaP.

Results: On exposure to 5?Gy radiation, LNCaP cells demonstrated high sensitization to Fas‐mediated apoptosis through increased Fas expression, stabilized p53 expression and binding to p53 response elements within the promoter and first intronic region of the Fas gene. Following treatment with DHT, in vivo binding of p53 to its response elements was strongly inhibited. In addition, DHT significantly up‐regulated c‐jun expression through extracellular stress‐regulated kinase (ERK) activation, and transfection of an antisense oligonucleotide for c‐jun or ERK inhibition by PD98059 cancelled DHT‐mediated suppression of radiation‐induced transactivation of Fas gene and sensitization to Fas‐mediated apoptosis.

Conclusions: Radiation‐induced Fas sensitization in prostate cancer cell was mediated through p53‐dependent transactivation of the Fas gene, which can be blocked by androgen stimulation mainly through induction of c‐jun.     

Cytological basis for enhancement of radiation‐induced mortality by Friend leukaemia virus infection

Purpose: To analyse the cytological basis for enhancement of radiation‐induced mortality by Friend leukaemia virus infection.

Materials and methods: Cellularity in haematopoietic tissues of C3H mice infected with FLV and/or whole‐body irradiation was examined.

Results: When mice were treated with a sublethal dose (3?Gy) of irradiation at 1 week after virus infection, most manifested a severe loss of cellularity in the spleen, bone marrow and peripheral blood 2 weeks after irradiation. More than 90% of the mice died within 1 month post‐irradiation. However, this deleterious effect of virus infection on the survival of irradiated mice was observed only when they were irradiated at around 1 week after virus inoculation. Strain differences in the sensitivity to this effect were observed among virus‐sensitive strains of mice.

Conclusions: The results indicate that Friend leukaemia virus infection can cause enhancement of radiation sensitivity of haematopoietic cells in host animals in a restricted manner in terms of genetic background and the interval between infection and irradiation.     

Decreased c‐Myc expression and its involvement in X‐ray‐induced apoptotic cell death of human T‐cell leukaemia cell line MOLT‐4

Purpose: To investigate the possible involvement of c‐Myc and ceramide‐c‐Jun N‐terminal kinase (JNK) pathway in X‐ray‐induced apoptotic cell death of MOLT‐4 cells.

Materials and methods: The expressions of c‐Myc protein and c‐myc mRNA after X‐irradiation were analysed by Western blotting and RT‐PCR between radiosensitive MOLT‐4 and radioresistant variant Rh‐1a cells with less JNK activation than the parental cells. Apoptotic cell death was determined by a dye exclusion test, the appearance of chromatin condensation and DNA fragmentation. The effect of a JNK activator anisomycin or c‐Myc inhibitor peptides (Int‐H1‐S6A, F8A) on the amount of c‐Myc protein and on the induction of apoptosis was investigated, respectively.

Results: In X‐irradiated MOLT‐4 cells, amounts of both c‐myc mRNA and c‐Myc protein rapidly decreased, which was followed by apoptotic cell death, while little change or limited reduction of c‐Myc protein was observed in X‐irradiated Rh‐1a cells with accompanying higher cell viability. Exposure of MOLT‐4 and Rh‐1a cells to c‐Myc inhibitor peptides similarly induced apoptotic cell death with decreases of c‐Myc protein. Anisomycin rapidly induced JNK activation and a subsequent decrease of c‐Myc protein, causing cell death in MOLT‐4 cells. On the other hand, Rh‐1a cells were more resistant to anisomycin than parental MOLT‐4 cells, showing less JNK activation and a delayed decrease of c‐Myc protein.

Conclusion: A decrease of c‐Myc protein was considered important in X‐ray‐induced apoptotic cell death of MOLT‐4 cells; activation of the JNK pathway caused reduction in the amounts of c‐myc mRNA and c‐Myc protein, and finally induced apoptotic cell death.     

Radiation‐induced fragmentation of cardiolipin in a model membrane

Purpose: To obtain evidence for the possibility of free‐radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes.

Materials and methods: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to γ‐rays from ⁶⁰Co or ¹³⁷Cs sources at doses between 1 and 24?kGy. Fragmentation products were identified using thin‐layer chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS).

Results: Using MALDI‐TOF MS and thin‐layer chromatography, it was shown that γ‐irradiation of liposomes consisting of TMCL was accompanied by free‐radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen.

Conclusion: It is shown for the first time that on γ‐irradiation, cardiolipin can undergo free‐radical fragmentation in its polar component.     

Issues in the control of low‐level radiation exposure*

The carcinogenic risks of exposure to low level ionizing radiation used by the International Commission on Radiation Protection (ICRP) have been challenged as being, at the same time, both too high and too low. This paper explains that the epidemiological evidence will always be limited at low doses, so that understanding the cellular mechanisms of carcinogenesis is increasingly important to assess the biological risks. An analysis is then given of the reasons why the challenges to ICRP, especially about the linear non‐threshold response model, have arisen. As a result of considering the issues, the Main Commission of ICRP is now consulting on a revised, simpler, approach based on an individual oriented philosophy. This represents a potential shift by the Commission from the past emphasis on societal‐oriented criteria. These proposals have been promulgated through the International Radiation Protection Association (IRPA) and an open literature publication was published in the Journal of Radiological Protection ¹ in June 1999. On the basis of comments received and the observations presented at the IRPA 10 Conference, the Commission will begin to develop the outline of the next Recommendations. It is now more than ten years since ICRP distributed, for comment, a draft of what was to become the publication of the 1990 Recommendations. The Commission plans to develop its new Recommendations on a time scale of the next four or five years. In this paper, many of the issues that will need to be addressed in the development of the recommendations will be identified. These issues will cover biological effects, dosimetric quantities and the establishment of those levels of dose at which different protection requirements will be put into place. Concepts of exclusion and exemption will need to be clarified as well as the meaning of how to achieve what the proposal identifies as ‘As Low as Reasonably Practicable’ (ALARP). Finally, the Commission has decided to develop an environmental radiation protection philosophy that will need to be developed as part of the new Recommendations.     

Chromosome aberrations and cell inactivation induced in mammalian cells by ultrasoft X‐rays: correlation with the core ionizations in DNA

Purpose: To study the frequency of chromosome aberrations induced by soft X‐rays. To see if the core ionization of DNA atoms is involved in this end‐point as much as it appears to be in cell killing.

Materials and methods: V79 hamster cells were irradiated by synchrotron radiation photons iso‐attenuated in the cell (250, 350, 810?eV). The morphological chromosome aberrations detected in the first post‐irradiation cell division (dicentrics and centric rings) were studied by Giemsa staining.

Results: The chromosome aberrations at 350?eV were, respectively, 2.6±0.8 and 2.1±0.8 times more numerous than at 250 and 810?eV for the same average dose absorbed by the nucleus. These relative effectivenesses are comparable with the ones already measured for cell killing. Moreover, they roughly vary such as the relative numbers of core ionizations (including in the phosphorus L‐shell) produced in DNA and its bound water (water being involved only at 810?eV through the oxygen atoms). In particular, they reproduce the characteristic twofold enhancement at 350?eV, above the carbon K threshold.

Conclusions: Correlations suggest that the core ionization process is likely a common and essential mechanism initiating both chromosome aberration and cell killing end‐points at these photon energies.     

Lack of inverse dose‐rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T‐47D cells arrested in G2 by ionizing radiation

Purpose: To investigate the radiosensitivity of human breast cancer cells, T‐47D, irradiated with low dose‐rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation.

Materials and methods: Cells were irradiated with ⁶⁰Co γ‐rays with dose‐rates of 0.37 and 0.94?Gy?h^?1. Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear‐bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3‐245 and Hoechst 33258 was used for additional staining of DNA. Two‐parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTAR^PLUS flow cytometer.

Results: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94?Gy?h^?1 resulted in exponential dose–survival curves with nearly equal α values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2.

Conclusions: T47‐D cells accumulate in G2 during low dose irradiation, but no inverse dose‐rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose‐rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation‐induced cell death in G2.     

Low and high LET radiation‐induced apoptosis in M059J and M059K cells

Purpose: To investigate and compare the ability of DNA‐dependent protein kinase (DNA‐PK)‐deficient and ‐proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation.

Materials and methods: A human glioma cell line M059J lacking the catalytic subunit of DNA‐PK (DNA‐PKcs) and its DNA‐PKcs‐proficient counterpart, M059K, were exposed to 1 and 4?Gy of accelerated nitrogen ions (¹⁴N, 140?eV?nm^?1, 8–12?Gy?min^?1) or ⁶⁰Co γ‐rays (0.2?eV?nm^?1, 0.7?Gy?min^?1). The induction of apoptosis was studied up to 144?h post‐irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA‐PKcs and poly(ADP‐ribose) polymerase (PARP) as well as DNA‐PK and caspase‐3 activity were investigated.

Results: Low and high LET radiations (4?Gy) induced a time‐dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144?h post‐irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase‐3 activation.

Conclusions: M059J and M059K cells both display radiation‐induced apoptosis, which occur independently of caspase‐3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.     

Radiation-induced breakpoint misrejoining in human chromosomes: random or non-random?

PURPOSE: To investigate whether radiation-induced misrejoining of chromosome breakpoints is randomly or non-randomly distributed throughout the human genome. MATERIALS AND METHODS: Data were combined from as many published cytogenetic studies as possible. The percentage of radiation-induced breaks per megabase (Mb) of DNA between all human chromosomes was calculated, and the observed and expected numbers of breakpoints based on DNA content between and within chromosomes were compared. RESULTS: A DNA-proportional distribution of breakpoints in 14 autosomes and a statistically significant deviation from proportionality in the other eight autosomes and the sex chromosomes was found. Regression analysis showed no significant change in breakpoint frequency per Mb of DNA relative to autosome size. Analysis between chromosome arms showed a non-random distribution of induced breakpoints within certain autosomes, particularly the acrocentrics. In cases of non-random distributions, a prevalence of events was found at heterochromatic regions and/or telomeres, and a clustering of breakpoints was found near the centromeres of many chromosomes. CONCLUSIONS: There is an approximately linear proportionality between autosomal DNA content and observed breakpoint number, suggesting that subsets of autosomes can be used to estimate accurately the overall genomic frequency of misrejoined breakpoints contingent upon a carefully selected subset. However, this conclusion may not apply to the sex chromosomes. The results also support the influence of chromatin organization and/or preferential DNA repair/misrejoining on the distribution of induced breakpoints. However, these effects are not sufficient at a global level to dismiss the value of cytogenetic analysis using a genome subset for biodosimetry.