Alter Radiation Response Research Articles

Microgravity Impairs DNA Damage Repair in Human Hematopoietic Stem/Progenitor Cells and Inhibits Their Differentiation into Dendritic Cells.

Astronauts on missions beyond low-Earth orbit are exposed to a hostile environment in which they are continually bombarded with unique high-energy species of radiation, while in conditions of microgravity (μG), which can alter radiation response and immunity. In the present studies, we examined the impact exposing human hematopoietic stem/progenitor cells (HSC) to μG had upon their capacity to repair DNA damage and their ability to generate immune cells critical for mounting an effective antitumor response. To this end, we first treated a human HSC-like cell line with an acute dose of the radiomimetic drug bleomycin, cultured them in normal gravity (1G) or simulated μG, and quantitated double-strand breaks through γ-H2AX foci. Calculating the median fluorescence intensity ratio at 1-to-4 h post-bleomycin revealed a 26% decrease in 1G, but a 20% increase in μG, suggesting that μG compromised HSC DNA damage repair and thus has the potential to enhance the genotoxic effects of space radiation. We next examined whether μG negatively affected the development of dendritic cells (DC), critical regulators of both the innate and acquired arms of the immune system. Primary human HSC were cytokine induced in 1G or μG and analyzed for generation of plasmacytoid (CD123+) and myeloid (CD11c+) DC. HSC cultured in 1G gave rise to significantly higher numbers of both myeloid and plasmacytoid DC than those cultured in μG, suggesting μG impairs production of these critical antigen-presenting cells. Our studies thus indicate that conditions of μG present during spaceflight perturb multiple pathways that could potentially enhance astronaut risk from exposure to space radiation.

Blocking Interleukin (IL)4- and IL13-Mediated Phosphorylation of STAT6 (Tyr641) Decreases M2 Polarization of Macrophages and Protects Against Macrophage-Mediated Radioresistance of Inflammatory Breast Cancer

To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24hours, then subjected to irradiation (0, 2, 4, or 6Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpinRNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

TOPK modulates tumour-specific radiosensitivity and correlates with recurrence after prostate radiotherapy

Background:Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers.Methods:Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy.Results:TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G1/S transition and G2/M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy.Conclusions:This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types.

Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

TH‐A‐BRD‐01: Radiation Biology for Radiation Therapy Physicists

Mechanisms by which radiation kills cells and ways cell damage can be repaired will be reviewed. The radiobiological parameters of dose, fractionation, delivery time, dose rate, and LET will be discussed. The linear‐quadratic model for cell survival for high and low dose rate treatments and the effect of repopulation will be presented and discussed. The rationale for various radiotherapy techniques such as conventional fractionation, hyperfractionation, hypofractionation, and low and high dose rate brachytherapy, including permanent implants, will be presented.The radiobiological principles underlying radiation protection guidelines and the different radiation dosimetry terms used in radiation biology and in radiation protection will be reviewed. Human data on radiation induced cancer, including increases in the risk of second cancers following radiation therapy, as well as data on radiation induced tissue reactions, such as cardiovascular effects, for follow up times up to 20–40 years, published by ICRP, NCRP and BEIR Committees, will be examined. The latest risk estimates per unit dose will be presented. Their adoption in recent radiation protection standards and guidelines and their impact on patient and workers safety in radiotherapy will be discussed.Biologically‐guided radiotherapy (BGRT) provides a systematic method to derive prescription doses that integrate patient‐specific information about tumor and normal tissue biology. Treatment individualization based on patient‐specific biology requires the identification of biological objective functions to facilitate the design and comparison of competing treatment modalities. Biological objectives provide a more direct approach to plan optimization instead of relying solely on dose‐based surrogates and can incorporate factors that alter radiation response, such as DNA repair, tumor hypoxia, and relative biological effectiveness. We review concepts motivating biological objectives and provide examples of how they might be used to address clinically relevant problems. Underlying assumptions and limitations of existing models and their proper application will be discussed.This multidisciplinary educational session combines the fundamentals of radiobiology for radiation therapy and radiation protection with the practical application of biophysical models for treatment planning and evaluation.Learning Objectives: To understand fractionation in teletherapy and dose rate techniques in brachytherapy. To understand how the linear‐quadratic models the effect of radiobiological parameters for radiotherapy. To understand the radiobiological basis of radiation protection standards applied to radiotherapy. To distinguish between stochastic effects and tissue reactions. To learn how to apply concepts of biological effective dose and RBE‐weighted dose and to incorporate biological factors that alter radiation response. To discuss clinical strategies to increase therapeutic ratio, i.e., maximize local control while minimizing the risk of acute and late normal tissue effects.

DNA Strand Breaks Signal the Induction of DNA Double-Strand Break Repair in Saccharomyces cerevisiae

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.

Paradigm Shifts in Radiation Biology: Their Impact on Intervention for Radiation-Induced Disease

New mechanistic cell and molecular studies on the effects of very low doses of radiation have resulted in three major paradigm shifts. First, the observation of bystander effects demonstrated that non-hit cells may respond as well as cells in which energy is deposited. Second, it was thought that gene mutations and chromosome aberrations were the most important early changes that represented the initiation phase of radiation-induced cancer. Now genomic instability that leads to the loss of genetic control appears to play a major role in the development of cancer. Finally, recent studies have demonstrated that radiation-induced changes in gene expression can be demonstrated at very low radiation doses. These changes can result in alterations in response pathways, many of which appear to be involved in protective or adaptive responses. The demonstration that unique genes are up- and down-regulated depending on the radiation type, dose and dose rate suggests that different molecular mechanisms are involved in responses to high and low radiation doses. The ability to alter radiation response by physical and chemical treatments suggests that it may be possible to intervene in the progression of radiation-induced diseases. Such intervention may decrease the cancer risk from radiation exposure. This new research also demonstrates that many nonlinear biological processes have an impact on the induction of cancer and the shape of dose-response functions. Thus, for low-LET radiation delivered at low dose rates, the linear, no-threshold hypothesis is not well supported, but it is adequately conservative in protecting against low-dose radiation risks.

Stress-Inducible DNA Repair in Saccharomyces Cerevisiae

Saccharomyces cerevisiae shows altered radiation response under various stress conditions, such as nutrition depletion, nitrogen starvation, osmotic shock, heat shock, and mild chemical treatments. In general, the cells show higher levels of UV or gamma radiation resistance under the stress. However, not all the stress conditions affect the repair system in the same manner. For example, depletion of nitrogen supply in the growth medium has been shown to enhance the repair of gamma ray-induced DNA damage without significantly affecting the UV response of the cells. On the other hand, a mild treatment with alkali or hydrogen peroxide improves the response to UV light but not to gamma radiation. It has further been shown that the effect of these stresses are not additive, e.g., the alkali and hydrogen peroxide treatments given simultaneously show the same effect as either of them alone. Low levels of gamma and UV radiation exposures are also treated as stress in the present context. Studies show that irradiation of low-dose gamma rays results in enhanced excision repair of UV-induced pyrimidine dimers. However, in all the wild-type strains tested, none showed any effect on gamma rays response. The exposure to low doses of UV light did not show any effect on either the gamma rays or the UV response. It is suggested that the stress-induced enhancement of DNA repair can be of two types: 1) A general response to stress, which prepares the organism to survive in adverse circumstances (some of the proteins produced during this response also take part in the DNA repair), and 2) a particular response involving DNA damage, such as that caused by gamma irradiation. In this case, the DNA damage may act as a signal for enhancement of the DNA repair.

Cells from individuals with SOD-1 associated familial amyotrophic lateral sclerosis do not have an increased susceptibility to radiation-induced free radical production or DNA damage.

Oxidative stress may play a role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS). Superoxide dismutases (SODs) are enzymes that can influence free radical processes in irradiated cells and there is some evidence that manipulation of SODs can affect survival of cells after radiation treatments. SOD-1 associated FALS mutants may have an altered radiation response due to an enhanced generation of hydroxyl radicals or a compromised ability to neutralize free radicals. We have investigated the ability of the lymphoblastoid cell lines from FALS patients with SOD-1 gene mutations, patients with sporadic ALS and controls to handle oxidative stress induced by ionising radiation by measuring levels of intracellular reactive oxygen species and production of DNA double-strand breaks. Levels of reactive oxygen species, expressed as the slope of the relative fluorescence of a radical-reactive fluorochrome, in the cells from familial ALS patients with SOD-1 gene mutations (2.14+/-1.06 Gy(-1)) and patients with sporadic ALS (1.38+/-0.21 Gy(-1)) were not significantly different from the controls (1.54+/-0.39 Gy(-1)). No significant difference was observed in the production of DNA double-strand breaks between three groups. The ability of lymphoblastoid cells from FALS patients with SOD-1 gene mutations to scavenge radiation-induced free radicals is not compromised nor is their ability to protect DNA damage induced by ionising radiation.

Mechanism of misonidazole-linked cytotoxicity and altered radiation response: role of cellular thiols.

The effectiveness of misonidazole as a hypoxic radiosensitizer of mammalian cells is increased by prolonged exposure of hypoxic cells to the drug. Increased radiosensitization occurs even after the cells are washed free of excess Hall and Biaglow, 1977; Whitmore et al., 1978; Varnes et al., 1981). Hall et al. (1977) suggested and Biaglow et al. 1978) found that drug intermediates, produced during hypoxic incubation of cells with misonidazole, might react with endogenous non-protein thiols (NPSH). These thiols function to protect the cell against deleterious intermediates that could otherwise attack and modify critical macromolecules such as DNA, RNA and protein. This hypothesis is in agreement with the cytotoxic mechanisms proposed earlier for many different nitro compounds (Biaglow, 1981). With respect to the effects of preincubation with misonidazole on the radiation response, alterations in both NPSH and protein thiols (Varnes et al., 1981) would be expected to alter the shoulder of the radiation response curve if hydrogen-donating species such as NPSH are involved in chemical repair of radiation damage (Chapman et al., 1973) or if thiol-containing enzymes are necessary for biochemical repair of damaged macromolecules (Han et al., 1976).

The Effects of Nitrobenzene Derivatives on Oxygen Utilization and Radiation Response of an in Vitro Tumor Model

The effects of several nitrobenzene derivatives on the oxygen utilization and radiation response of multicell spheroids have been determined. In this system, the best radiosensitizer was nitrotoluene, previously reported to be a poor sensitizer of anoxic cells in monolayer cultures. The sensitization of the spheroid is interpreted as due to nitrotoluene's immediate inhibitory effect on cellular oxygen utilization resulting in a reoxygenation of the centrally located cells. The anoxic radiosensitizre dinitrobenzonitrile markedly stimulated oxygen utilization producing an immediate protection, presumably because the hypoxic fraction of the spheroid was increased. Some radiosensitization did occur with dinitrobenzonitrile at higher concentrations and after longer periods of exposure to the drug. The altered radiation response, caused by either nitrotoluene or dinitrobenzonitrile, agreed fairly well with the theoretical prediction of the radiation response as a function of oxygen utilization. A third anoxic s...

Effect of irradiation on tumor microvascular permeability to macromolecules.

In C3H mouse Gardner lymphosarcoma, we have demonstrated an increase in the extravascular albumin space 48 hours after delivering 3,000 rads of irradiation. This observation has implications for organ radiobiology in that the microvascular effect could alter radiation response in tumors. This effect may be time dependent and thus may be an extremely important consideration in dose fractionation which is not necessarily dependent on tumor cell cytokinetics. The second ramification of this observation concerns the effect of irradiation on radiotracer techniques designed to identify tumors; following irradiation, an increased macromolecular transport into the interstitial space of a tumor would be anticipated to cause increased accumulation of scanning agents which are bound to protein (e.g., gallium).

Quantitative Studies of Relationships between Tumor Cell Ploidy and Dose Response to Ionizing Radiation in Vivo: Modification of Radiation Response in a Previously Irradiated Tumor

ggradiation was demonstrated in vivo between two closely related murine lymphocytic leukemias, one diploid and the other tetraploid. Dose-response curves were determined under oxygenated and anoxic conditions; in either condition the slopes of the dose-response curves were similar, but the extrapolation number for the tetraploid tumor exceeded that for the diploid tumor by a factor of approximately 2. A tumor line of diploid origin was subjected to 1000 rads of x irradiation at each of fifty sequential passages in vivo with 10/ sup 6/ tumor cells transplanted at each passage to minimize the chance of selection from small numbers of surviving celis. During the fifty irradiated passages, this tumor line was noted to have altered radiation response with a greater proportion of tumor cells surviving any given radiation dose, increased nucleic acid content per cell was seen, and tetraploid and aneuploid cells appeared in the population. The application of these findings to the acquired radioresistance reported by radiotherapists in the treatment of human cancer is discussed. (auth)