Adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture.

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose-response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture. When the single-cell microbeam delivered from 1 to 12 alpha particles through the nuclei of 10% of C3H 10T(1/2) cells, more cells were inactivated than were actually traversed by alpha particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of gamma rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the alpha particles.     

Protein kinase C epsilon is involved in ionizing radiation induced bystander response in human cells

Our earlier study demonstrated the induction of PKC isoforms (βII, PKC-α/β, PKC-θ) by ionizing radiation induced bystander response in human cells. In this study, we extended our investigation to yet another important member of PKC family, PKC epsilon (PKC?). PKC? functions both as an anti-apoptotic and pro-apoptotic protein and it is the only PKC isozyme implicated in oncogenesis. Given the importance of PKC? in oncogenesis, we wished to determine whether or not PKC? is involved in bystander response. Gene expression array analysis demonstrated a 2–3-fold increase in PKC? expression in the bystander human primary fibroblast cells that were co-cultured in double-sided Mylar dishes for 3 h with human primary fibroblast cells irradiated with 5 Gy of α-particles. The elevated PKC? expression in bystander cells was verified by quantitative real time PCR. Suppression of PKC? expression by small molecule inhibitor Bisindolylmaleimide IX (Ro 31-8220) considerably reduced the frequency of micronuclei (MN) induced both by 5 Gy of γ-rays (low LET) and α-particles (high LET) in bystander cells. Similar cytoprotective effects were observed in bystander cells after siRNA mediated silencing of PKC? suggestive of its critical role in mediating some of the bystander effects (BE). Our novel study suggests the possibility that PKC signaling pathway may be a critical molecular target for suppression of ionizing radiation induced biological effects in bystander cells.     

Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom

In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3-20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM-irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.     

Radio-adaptive response: characterization of a cytogenetic repair induced by low-level ionizing radiation in cultured Chinese hamster cells

Pretreatment with low doses of beta-rays from incorporated tritiated thymidine ([3H]dThd) or of Co-60 gamma-rays (1 or 5 cGy) rendered actively growing Chinese hamster V79 cells more resistant to the induction of micronuclei or sister-chromatid exchanges (SCEs) by a subsequent high dose of gamma-rays (1 Gy). This adaptive response to ionizing radiation (radio-adaptive response) can be induced by an optimal range of low doses of 3H beta-rays, but not by much lower or higher adapting doses. Full expression of the adaptive response induced by the exposure to low doses of 60Co gamma-rays occurred 4 h after the adapting dose. The cells pre-exposed to low doses of gamma-rays showed cross-resistance to challenge doses of gamma-rays themselves and also of mitomycin C (MMC) and near ultraviolet light (UV-B, 313 nm), but not to those of ethyl methanesulfonate (EMS) or cis-platinum (II) diammine dichloride (cisplatin) for SCE induction. These results suggest that the radio-adaptive response mechanistically couples to the repair network which copes with chromatin lesions induced by MMC and UV-B.     

Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects

The shape of the dose-response curve for cancer induction by low doses of ionizing radiation is of critical importance to the assessment of cancer risk at such doses. Epidemiologic analyses are limited by sensitivity to doses typically greater than 50-100 mGy for low LET radiation. Laboratory studies allow for the examination of lower doses using cancer-relevant endpoints. One such endpoint is neoplastic transformation in vitro. It is known that this endpoint is responsive to both adaptive response and bystander effects. The relative balance of these processes is likely to play an important role in determining the shape of the dose-response curve at low doses. A factor that may influence this balance is cell density at time of irradiation. The findings reported in this paper indicate that the transformation suppressive effect of low doses previously seen following irradiation of sub-confluent cultures, and attributed to an adaptive response, is reduced for irradiated confluent cultures. However, even under these conditions designed to optimize the role of bystander effects the data do not fit a linear no-threshold model and are still consistent with the notion of a threshold dose for neoplastic transformation in vitro by low LET radiation.     

Biophysical models of radiation bystander effects: 1. Spatial effects in three-dimensional tissues

Non-targeted (bystander) effects of ionizing radiation are caused by intercellular signaling; they include production of DNA damage and alterations in cell fate (i.e. apoptosis, differentiation, senescence or proliferation). Biophysical models capable of quantifying these effects may improve cancer risk estimation at radiation doses below the epidemiological detection threshold. Understanding the spatial patterns of bystander responses is important, because it provides estimates of how many bystander cells are affected per irradiated cell. In a first approach to modeling of bystander spatial effects in a three-dimensional artificial tissue, we assume the following: (1) The bystander phenomenon results from signaling molecules (S) that rapidly propagate from irradiated cells and decrease in concentration (exponentially in the case of planar symmetry) as distance increases. (2) These signals can convert cells to a long-lived epigenetically activated state, e.g. a state of oxidative stress; cells in this state are more prone to DNA damage and behavior alterations than normal and therefore exhibit an increased response (R) for many end points (e.g. apoptosis, differentiation, micronucleation). These assumptions are implemented by a mathematical formalism and computational algorithms. The model adequately describes data on bystander responses in the 3D system using a small number of adjustable parameters.     

T-cell induced pathogenesis in HIV: bystander effects and latent infection.

The progress of HIV is accompanied by the infection and decline of the population of CD4+ cells. This reduction in cells results from both cytolytic influences of the virus and virus-specific cytotoxic T-cell (CTL) responses. We seek to characterize the extent of CD4+ reduction caused by HIV-specific CTLs at equilibrium. Here we show that intermediate levels of cytotoxic killing of infected cells can be inferior to both strong and weak or absent immune responses. We further show that the deleterious effects of the CTL response are made worse by a slow immune response. Bystander effects in which uninfected cells are thought to be eliminated by non-specific CTL activation lead to small or negligible reductions in uninfected CD4+ cells. Latently infected cells containing pro-viral DNA and which become activated at a constant rate ensure that the immune response is more effective for a larger range of CTL activities and reduces T-cell associated pathology.     

Chemical and radiation induced late dominant lethal effects in mice.

Although theoretically expected, experimental data to date have not shown dominant lethal expression to occur throughout the developmental period. Specifically, late post-implantation effects have not been demonstrated. We routinely use an experimental technique in which parental females mated to mutagenically treated males are allowed to give birth and wean their litter, and their uterine horns are then inspected for uterine scars indicative of live and dead embryos. In a number of experiments in which males were mutagenically treated with either chemicals or X-irradiation, a discrepancy was observed between the number of live embryos as determined by the scar technique and the number of live observed at birth, suggesting the possibility of embryonic losses at a late stage in development. Initial analyses showed that mutagenic treatment increased the percentage of these late losses. These differences were statistically significant in 2 of 3 analyses. Factors affecting statistical significance and an understanding of dominant lethal mutations are discussed.     

Position-specific growth of mouse limb bud cells in vitro.

The relationship between cellular position and growth control has been studied in cultures of dissociated fragments of mouse limb bud cells. Using cells derived from various positions along the anterior-posterior axis of the limb bud we have developed culture conditions that optimize growth of positionally isolated cells. Under these conditions limb bud cells display an inherent, position-specific growth response; proliferation of cells derived from anterior and central regions of the limb is enhanced over that of posterior derived cells. Thus, within the total population of limb bud cells the in vitro growth of posterior cells is unique and correlates with the positional activity associated with the zone of polarizing activity. Anterior and posterior cells were cocultured to determine whether interactions between these two groups of positionally distinct cells lead to the stimulation of growth that has been observed in vivo. We observe a slight but consistent position-dependent stimulation of growth that is indicative of a mitogenic signal passing between these positionally disparate cells. Similarities between position-related growth dynamics in vivo and in vitro suggest that positional interactions that are important for limb formation can occur between dissociated cells cultured under standard conditions.     

Comparison between ectoderm-conditioned medium and fibronectin in their effects on chondrogenesis by limb bud mesenchymal cells

Limb bud ectoderm inhibits chondrogenesis by limb bud mesenchymal cells cultured at high density or on collagen gels. This ectodermal antichondrogenic influence has been postulated to function in vivo in regulating the spatial patterning of cartilage and soft connective tissue in the limb. We have developed a method for preparing ectoderm-conditioned medium containing antichondrogenic activity. Using a simple bioassay, we have investigated some characteristics of the ectodermal products and their effects on limb bud mesenchymal cells. Inhibition of chondrogenesis by ectoderm-conditioned medium was tested on limb bud mesenchymal cells cultured on collagen gels. The antichondrogenic influence involves enhanced cell spreading and is alleviated by agents, such as cytochalasin D, that induce cell rounding. Fibronectin resembles ectoderm-conditioned medium in its ability to inhibit chondrogenesis and promote cell spreading in collagen gel cultures of limb bud mesenchymal cells. However, Western blot analysis shows that the antichondrogenic activity of ectoderm-conditioned medium is not due to fibronectin in the medium. Peptides related to the fibronectin cell-binding domain block the antichondrogenic effect of fibronectin, but not that of ectoderm-conditioned medium. On the other hand, an antibody to an integrin, as well as heparan sulfate, alleviates the antichondrogenic effects of both fibronectin and ectoderm-conditioned medium. The antichondrogenic effect of ectoderm-conditioned medium may be mediated by an integrin and by a cell surface heparan sulfate proteoglycan, but it does not depend directly upon fibronectin-mediated cell spreading.     

Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes

The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA(R) isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA(R) produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA(R) extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.     

The nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by gamma-rays or ethyl methanesulphonate

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from gamma-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the gamma-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of gamma-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while gamma-ray-induced mutations may arise mostly through larger genetic changes.     

Adaptive response for chromosomal inversions in pKZ1 mouse prostate induced by low doses of X radiation delivered after a high dose

Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01-1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage.     

Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

Changes in cell morphology and cell-to-cell adhesion induced by extracellular Ca2+ in cultured taste bud cells

Cell morphology and cell-to-cell adhesion of taste bud cells were significantly altered by extracellular Ca2+ during in vitro culture. Under high Ca2+ condition (above 0.5 mM), the cells were tightly associated with each other and formed packed aggregates. Under low Ca2+ condition (below 0.1 mM), the cells were dispersed and had an elongated shape. These two forms were reversible and specifically dependent on Ca2+. The results indicate that extracellular Ca2+ regulates cell shape and cell-to-cell adhesion of taste bud cells.     

热休克反应对高温诱导的低血压的保护作用及其机制

目的:探讨热休克反应对高温诱导的低血压的保护作用及机制.方法:给予热休克预处理,观察热休克大鼠致死热暴露后生存时间、血压动态变化及热暴露73 min时心肌一氧化氮含量.结果:热暴露后,热休克组血压显著高于对照组,而心肌一氧化氮含量却低于对照组.结论:HSR通过抑制热暴露大鼠心肌NO的过量生成,对高温诱导的低血压起到了明显的保护作用.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

Interaction between radiation and drug damage in mammalian cells. IV. Radiation response of adriamycin-resistant V79 cells

Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties. A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve. Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers. A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury. LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties. These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics. LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium. Reverted cells also lost their large capacity for sublethal damage. It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.