Cis-9,trans-11-conjugated linoleic acid affects lipid raft composition and sensitizes human colorectal adenocarcinoma HT-29 cells to X-radiation.

BACKGROUND: Investigations concerned the mechanism of HT-29 cells radiosensitization by cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), a natural component of human diet with proven antitumor activity. METHODS: The cells were incubated for 24h with 70µM c9,t11-CLA and then X-irradiated. The following methods were used: gas chromatography (incorporation of the CLA isomer), flow cytometry (cell cycle), cloning (survival), Western blotting (protein distribution in membrane fractions), and pulse-field gel electrophoresis (rejoining of DNA double-strand breaks). In parallel, DNA-PK activity, γ-H2AX foci numbers and chromatid fragmentation were estimated. Gene expression was analysed by RT-PCR and chromosomal aberrations by the mFISH method. Nuclear accumulation of the EGF receptor (EGFR) was monitored by ELISA. RESULTS AND CONCLUSIONS: C9,t11-CLA sensitized HT-29 cells to X-radiation. This effect was not due to changes in cell cycle progression or DNA-repair-related gene expression. Post-irradiation DSB rejoining was delayed, corresponding with the insufficient DNA-PK activation, although chromosomal aberration frequencies did not increase. Distributions of cholesterol and caveolin-1 in cellular membrane fractions changed. The nuclear EGFR translocation, necessary to increase the DNA-PK activity in response to oxidative stress, was blocked. We suppose that c9,t11-CLA modified the membrane structure, thus disturbing the intracellular EGFR transport and the EGFR-dependent pro-survival signalling, both functionally associated with lipid raft properties. GENERAL SIGNIFICANCE: The results point to the importance of the cell membrane interactions with the nucleus after injury inflicted by X -rays. Compounds like c9,t11-CLA, that specifically alter membrane properties, could be used to develop new anticancer strategies.

Intraclonal recovery of 'slow clones'-a manifestation of genomic instability: are mitochondria the key to an explanation?

Intraclonal recovery following X-irradiation in an in vitro study of L5178Y-S murine leukaemic cells is reviewed. This phenomenon was first described in 1994 occurring in the slowly growing clones ('slow clones') present among the survivors in irradiated cell populations. An attempt to explain these experimental data is given in terms of the present knowledge of the role of mitochondria in nontargeted radiation effects, with the focus on genomic instability and mtDNA-related epigenetic modifications of the nuclear genome. An understanding of this intraclonal recovery may be important in preventing tumour regrowth following radiotherapy, as well as in decreasing the risk of secondary radiation-induced malignancies.

Signalling loops and linear pathways: NF-kappaB activation in response to genotoxic stress.

The signalling loop concept was introduced in 1991 to explain activation of membrane and cytoplasmic kinases in response to DNA damage inflicted by ionizing radiation. Damage to the chromosomal DNA was thought to provide a primary signal and a secondary signal from a nucleus to cytoplasm was assumed. This scenario was confirmed although not as originally proposed. A complex of nuclear factor-kappaB (NF-kappaB) essential modulator and ataxia telangiectasia-mutated kinase activated by genotoxic agents is sent to cytoplasm, prompting nuclear translocation of the active transcription factor NF-kappaB. In parallel, linear signalling pathways are initiated in the cytoplasm, mostly by reactive oxygen species, resulting in NF-kappaB activation and nuclear translocation. The choice of NF-kappaB activation pathway and the extent of activation of various pathways may be influenced by the relative degree of damage inflicted by genotoxic agents in the nuclear and cytoplasmic compartments. The ultimate pattern of cellular response is determined by availability, abundance and localization of the proteins participating in the signal transduction.

Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.

The purpose of this review is to discuss the relation between the repair of DNA double strand breaks (DSB)--the main lethal lesion inflicted by ionising radiation-and the function of receptors of epidermal growth factor (EGFR) and similar ligands (other members of the ERBB family). The reviewed experimental data support the assumption that in mammalian cells, one consequence of EGFR/ERBB activation by X-rays is its internalisation and nuclear translocation together with DNA-dependent protein kinase (DNA-PK) subunits present in lipid rafts or cytoplasm. The effect of EGFR/ERBB stimulation on DSB rejoining would be due to an increase in the nuclear content of DNA-PK subunits and hence, in activity increase of the DNA-PK dependent non-homologous end-joining (D-NHEJ) system. Such mechanism explains the radiosensitising action of "membrane-active drugs", hypertonic media, and other agents that affect nuclear translocation of proteins. Also, one radiosensitising effect of the recently introduced into clinical practice EGFR/ERBB inhibitors would consist on counteracting the nuclear translocation of DNA-PK subunits. In result, D-NHEJ may be less active in inhibitor-treated cells and this will contribute to an enhanced lethal effect of irradiation. The reviewed observations point to a heretofore not understood mechanism of the cell's self-defence against X-rays which can be exploited in combined radio- and chemotherapy.

Sirtuin inhibition increases the rate of non-homologous end-joining of DNA double strand breaks.

Sirtuins (type III histone deacetylases) are an important member of a group of enzymes that modify chromatin conformation. We investigated the role of sirtuin inhibitor, GPI 19015, in double strand break (DSB) repair in CHO-K1 wt and xrs-6 mutant cells. The latter is defective in DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end-joining (D-NHEJ). DSB were estimated by the neutral comet assay and histone gammaH2AX foci formation. We observed a weaker effect of GPI 19015 treatment on the repair kinetics in CHO wt cells than in xrs6. In the latter cells the increase in DNA repair rate was most pronounced in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in the number of histone gammaH2AX foci was faster in xrs6 than in CHO-K1 cells. The altered repair rate did not affect survival of X-irradiated cells. Since in G1 xrs6 cells DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ, to a greater extent than the other DSB repair system, D-NHEJ.

Sirtuins (histone deacetylases III) in the cellular response to DNA damage--facts and hypotheses.

Histone deacetylases (HDAC) are an important member of a group of enzymes that modify chromatin conformation. Homologues of the yeast gene SIR2 in mammalian cells code type III histone deacetylases (HDAC III, sirtuins), dependent on NAD(+) and inhibited by nicotinamide. In yeast cells, Sir2 participates in repression of transcriptional activity and in DNA double strand break repair. It is assumed that certain sirtuins may play a similar role in mammalian cells, by modifying chromatin structure and thus, altering the accessibility of the damaged sites for repair enzymes. A relation between poly(ADP-ribosylation) and sirtuin function in cells with damaged DNA has been also postulated. Interconnections between NAD(+) metabolism, poly(ADP-ribosylation), DNA repair and gene expression should allow to modulate the cellular response to agents that damage DNA. Preliminary results, reviewed in this paper indicate that such possibility exists. We propose a hypothetical mechanism of sirtuin participation in DSB repair. It is based on the assumption that activation of PARP at the sites of DNA strand breaks leads to a local increase in nicotinamide concentration. Nicotinamide then inhibits sirtuins exactly at the site of DNA strand break. At present, however, there are no data directly confirming the effect of sirtuin inhibition on DSB repair processes in mammalian cells. Nevertheless, a connection between the acetylation status of histones and repair of DNA breaks has recently been found, indicating that all HDAC classes may modulate DNA repair processes. In addition, sirtuins exert an anti-apoptotic action in various cell types. Hence, it is possible to sensitise cells to apoptosis-inducing agents by sirtuin inhibitors.

Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster.

Cells with mutated autophosphorylation sites in the ABCDE cluster of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are defective in the repair of ionising radiation-induced DSB, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. Nevertheless, the mutated DNA-PK is able to undergo ATP-dependent autophosphorylation and inactivation. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in DSB repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line. The purpose of this work was to examine the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells. Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences coding for the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PK autophosphorylation sites (ABCDE casette).

DNA interstrand crosslinks are induced in cells prelabelled with 5-bromo-2'-deoxyuridine and exposed to UVC radiation.

It has been observed previously that 5-bromo-2'-deoxyuridine (BrdU) potentiates the effect of UVC radiation on the level of sister chromatid exchanges. It is not known which type of DNA damage is responsible for this enhancing effect and we have proposed this to be the DNA interstrand crosslink (ICL) which, theoretically, may arise in cells that are labelled with BrdU for one round of replication and exposed to UVC radiation. The aim of the present investigation was to verify if ICLs are indeed formed during this irradiation scenario. CHO-K1 cells were prelabelled with BrdU and exposed to UVC. ICLs were detected by a modified version of the comet assay that relies on the reduction of induced DNA migration in the agarose gel. Carboplatin was used as a positive control. We found that BrdU+UVC treatment indeed results in a reduction of the damage induced by gamma-radiation. Furthermore, we observed that CL-V4B cells exposed to BrdU+UVC, but not to UVC alone, showed a very high level of chromosomal damage. These cells have a deficient Rad51C paralog that renders them extremely sensitive towards ICLs. Interestingly, the cytogenetic results did not correlate with cell survival, where it was found that the CL-V4B cells tolerate BrdU+UVC better than the wild type cells. The possible reasons are discussed. Taken together our results indicate that ICLs are formed in DNA that was prelabelled with BrdU and exposed to UVC radiation.

The radiation sensitivity of human chromosomes 2, 8 and 14 in peripheral blood lymphocytes of seven donors.

PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.

From radioresistance to radiosensitivity: In vitro evolution of L5178Y lymphoma.

PURPOSE: To discuss the possible reasons for the loss of tumourigenicity and the acquisition of new phenotypic features (among them, sensitivity to X and UVC radiations) as a result of in vitro cultivation of L5178Y lymphoma cells. RESULTS: Ten years ago the phenotypic differences between LY-R (original L5178Y maintained in vivo and examined in vitro) and LY-S lines were reviewed in detail by the author. The loss of tumourigenicity of LY-R cells upon in vitro cultivation accompanying the acquirement of the LY-S phenotype had been described earlier by Beer et al. (1983). In spite of their common origin, the sublines were shown to differ in their relative sensitivity to a number of DNA damaging agents and in numerous other features. Here, selected differences between LY-R and LY-S lines are briefly reviewed. It is proposed that Wallace's concept (2010a) that mitochondria are the interface between environmental conditions and the genome may explain the LY-R-LY-S conversion under prolonged in vitro cultivation. CONCLUSION: The differences between the LY lines were probably of epigenetic rather than genetic character. The properties of LY-R cells changed as a result of exposure to an oxic in vitro milieu. The changes could be preconditioned by heteroplasmy and the selection of cells endowed with mitochondria best fitted to a high oxygen-low carbon dioxide environment.

Ionizing radiation-induced oxidative stress, epigenetic changes and genomic instability: the pivotal role of mitochondria.

PURPOSE: To review the data concerning the role of endogenously generated reactive oxygen species (ROS) in the non-targeted ionizing radiation (IR) effects and in determination of the cell population's fate, both early after exposure and after many generations. CONCLUSIONS: The short-term as well as chronic oxidative stress responses mainly are produced due to ROS generation by the electron transport chain (ETC) of the mitochondria and by the cytoplasmic NADPH oxidases. Whether the induction of the oxidative stress and its consequences occur or are hampered in a single cell largely depends on the interaction between the nucleus and the cellular population of several hundred or thousands of mitochondria that are genetically heterogeneous. High intra-mitochondrial ROS level is damaging the mitochondrial (mt) DNA and its mutations affect the epigenetic control mechanisms of the nuclear (n) DNA, by decreasing the activity of methyltransferases and thus, causing global DNA hypomethylation. These changes are transmitted to the progeny of the irradiated cells. The chronic oxidative stress is the main cause of the late post-radiation effects, including cancer, and this makes it an important adverse effect of exposure to IR and a target for radiological protection.

Radiosensitizing properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols: two modes of action.

We examined radiosensitizing properties of two novel platinum complexes (ethylenediamine(L-malato)platinum(II)), Pt1 and bis(1-ethylimidazole(L-malato)platinum(II)), Pt4. Initial double strand break (DSB) level and DSB rejoining were measured, using pulse field gel electrophoresis (PFGE) in human G1 phase lymphocytes subjected to Pt complex treatment alone and in combination with 10Gy of X-rays. Effects of Pt complex pre-treatment followed by X-irradiation were examined on survival (clonogenic ability) and growth (48 h growth tests) in Chinese hamster ovary (CHO-K1), xrs6 and L5178Y (LY) cells (LY-R and LY-S sublines). Cell cycle distributions of CHO cells after drug treatment were determined with the use of flow cytometry. Pt1 slowed down rejoining of X-ray induced DSB. It exerted a more than additive lethal effect on CHO-K1 cells but not on L5178Y cells subjected to combined Pt complex treatment and X-irradiation. In xrs6 cells the effect of combined Pt1+X treatment was additive. We conclude that, as earlier proposed for other Pt complexes, the radiosensitizing effect of Pt1 is connected with converting repairable DNA damage into irrepairable one (mode (i) of action). The requirements for this mode of sensitization are functional DNA repair systems (nucleotide excision repair (NER) and non-homologous end-joining (NHEJ)). Pt4 does not slow down DSB rejoining. It shows a considerable ability to arrest cells in G2 phase. We assume that Pt4 pre-treatment arrests cells in G2 phase and thus sensitizes to X-rays these cells that have a radiosensitive G2 phase (mode (ii) of action).

Silver nanoparticles -- allies or adversaries?

Nanoparticles (NP) are structures with at least one dimension of less than 100 nanometers (nm) and unique properties. Silver nanoparticles (AgNP), due to their bactericidal action, have found practical applications in medicine, cosmetics, textiles, electronics, and other fields. Nevertheless, their less advantageous properties which make AgNP potentially harmful to public health or the environment should also be taken into consideration. These nanoparticles are cyto- and genotoxic and accumulate in the environment, where their antibacterial properties may be disadvantageous for agriculture and waste management. The presented study reviews data concerning the biological effects of AgNP in mammalian cells in vitro: cellular uptake and excretion, localization in cellular compartments, cytotoxicity and genotoxicity. The mechanism of nanoparticle action consists on induction of the oxidative stress resulting in a further ROS generation, DNA damage and activation of signaling leading to various, cell type-specific pathways to inflammation, apoptotic or necrotic death. In order to assure a safe application of AgNP, further detailed studies are needed on the mechanisms of the action of AgNP on mammalian cells at the molecular level.

Radiation hormesis: Autophagy and other cellular mechanisms.

PURPOSE: To review the cellular mechanisms of hormetic effects induced by low dose and low dose rate ionising radiation in model systems, and to call attention to the possible role of autophagy in some hormetic effects. RESULTS AND CONCLUSIONS: Very low radiation doses stimulate cell proliferation by changing the equilibrium between the phosphorylated and dephosphorylated forms of growth factor receptors. Radioadaptation is induced by various weak stress stimuli and depends on signalling events that ultimately decrease the molecular damage expression at the cellular level upon subsequent exposure to a moderate radiation dose. Ageing and cancer result from oxidative damage under oxidative stress conditions; nevertheless, ROS are also prominent inducers of autophagy, a cellular process that has been shown to be related both to ageing retardation and cancer prevention. A balance between the signalling functions and damaging effects of ROS seems to be the most important factor that decides the fate of the mammalian cell when under oxidative stress conditions, after exposure to ionising radiation. Not enough is yet known on the pre-requirements for maintaining such a balance. Given the present stage of investigation into radiation hormesis, the application of the conclusions from experiments on model systems to the radiation protection regulations would not be justified.

Autophagy, reactive oxygen species and the fate of mammalian cells.

The paper reviews the rapidly expanding pool of information on cellular and molecular mechanisms of autophagy, including autophagy types, macroautophagy induction, formation of autophagosomes and cross-talk between autophagy and apoptosis. Special attention is given to generation of reactive oxygen species (ROS) in various cellular compartments of cells under stress conditions inducing autophagy. The roles of hydrogen peroxide and superoxide in autophagy are analysed based on the recent experimental work. The relation between ROS and life span prolongation is briefly discussed, with the final conclusion that the paradox of dual role of ROS in life and death may be solved to a considerable extent due to research on autophagy.

Chromatin acetylation, ß-amyloid precursor protein and its binding partner FE65 in DNA double strand break repair.

Among post-translational modifications of chromatin proteins taking place in DNA double strand break (DSB) repair, acetylation plays a prominent role. This review lists several facts and hypotheses concerning this process. Lack of acetyltransferase TIP60 (HIV-Tat interacting protein of 60 kDa) activity results in cells with defective DSB repair. The enzyme is present in the nucleus in a multimeric protein complex. TIP60 dependent activation of ATM (ataxia telangiectasia mutated kinase) is an early event in the response to DNA breakage. Other important acetylations are those of histones H4 and γH2AX. Correct reconstruction of the damaged site is critical for survival and prevention of genetic and epigenetic changes in the cell that may affect the function of its daughter cells. Recently, two proteins with previously unsuspected functions in DSB repair have been identified as active in this process: Alzheimer ß-amyloid precursor protein (APP) and its binding partner FE65, ß-amyloid precursor binding protein. Their participation in DSB repair in both neuronal and non-neuronal cells is related to acetylation carried out by the acetyltransferase complex. The same function is ascribed to heterochromatin protein 1 (HP1). So far, the relations (if any) between TIP60 activation by HP1 and by the FE65 complex remain unidentified.

Effect of temperature during irradiation on the level of micronuclei in human peripheral blood lymphocytes exposed to X-rays and neutrons.

OBJECTIVES: It has been reported that the level of cytogenetic damage in human peripheral blood lymphocytes (PBL) is higher following irradiation at 37 degrees C than at 0-4 degrees C. The mechanisms of this cytogenetic temperature effect are not fully known. The aim of our study was to check whether the effect was related to the indirect or direct action of radiation. MATERIALS AND METHODS: PBL were kept at 37 degrees C and 0 degree C for 20 min and exposed to 2 Gy of X-rays. In some experiments PBL were isolated and 0.5 M dimethyl sulfoxide (DMSO) was added for 5 min before exposure. PBL were also irradiated at 37 degrees C and 0 degree C with 1 Gy of 6 MeV neutrons. Micronuclei were scored as the endpoint. Following exposure to X-rays the level of initial DNA damage was also measured by the alkaline and neutral comet assay. RESULTS: The frequency of micronuclei in cells exposed at 37 degrees C to X-rays or neutrons was higher than that after exposure at 0 degree C. No effect of temperature was seen when PBL were exposed to X-rays in the presence of DMSO. No effect of temperature was observed on the level of DNA damage measured with the alkaline or neutral comet assay. CONCLUSIONS: The results of experiments with DMSO indicate that the temperature effect is due to the indirect action of radiation, i.e., via reactive oxygen species. However, this is not supported by the results with neutrons and the comet assay. Possible reasons for the discrepancies are discussed.

Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells.

Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the beta-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of beta-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.

Rad51C-deficient CL-V4B cells exhibit normal levels of mitomycin C-induced SCEs but reduced levels of UVC-induced SCEs.

The mechanisms of sister chromatid exchanges (SCEs) are not known. One hypothesis is that SCE is a manifestation of Rad51-dependent homologous recombination repair. In order to test this hypothesis, we have compared the frequencies of SCEs induced by mitomycin C (MMC) and 254nm ultraviolet radiation (UVC) in wt V79B and the Rad51C-deficient CL-V4B cells. SCEs were analysed in the first (M1) and second (M2) post-treatment mitoses. In M1 MMC induced the same frequencies of SCEs in CL-V4B and V79B cells, while the UVC-induced SCE frequencies were lower in CL-V4B than V79B cells. In CL-V4B cells, MMC-induced SCEs were higher in M2 than in M1, suggesting that interstrand cross-links (ICL) are either not removed completely or are transformed into another form of DNA damage that persists until the next cell cycle. We suggest that SCEs may represent a mechanism to bypass MMC-induced ICL without their removal.