Hyper-radiosensitivity Research Articles

Effect of ATM on low-dose hyper-radiosensitivity in A549 cells synchronized at G2 phase

Objective To investigate the low-dose hyper-radiosensitivity (HRS)/induced radioresistance (IRR) in A549 cells synchronized at G2 phase and the role of ATM kinase in the process. Methods Human lung adenocarcinoma cell line A549 was synchronized at G2 phase by aphidicolin. The ATM-specific activator and inhibitor, chloroquine and KU55933, were used to regulate the activity of ATM. The colony formation assay was used to evaluate cell survival. Flow cytometry was used to determine the cell cycle of radiation-exposed A549 cells synchronized at G2 phase. Immunofluorescence was used to observe the dynamics of γ-H2AX fluorescence and evaluate the efficiency of DNA repair in A549 cells synchronized at G2 phase. Western blot was used to detect the expression of phosphorylated ATM (Ser1981) and ATM. Results A549 cells synchronized at G2 phase had substantially enhanced HRS than non-synchronized cells. The dose-induced transition from HRS to IRR was in accordance with the dose-response pattern of early G2/M checkpoint. However, with the same threshold dose, the activation of early checkpoint occurred earlier and lasted longer than normal. The activation of ATM kinase inhibited HRS and enhanced DNA repair, while the inhibition of ATM kinase enhanced HRS and hindered DNA repair. Conclusions ATM kinase-mediated early G2+ M checkpoint is a molecular switch for HRS in synchronized A549 cells. Low-dose irradiation with G2-phase synchronization and ATM inhibitor can enhance the low-dose radiosensitivity. Key words: Low-dose radiation sensitivity/increased radiation resistance; Early G2/M checkpoint; Cell cycle

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients.

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1-2Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.

Abstract 1805: DUOX2: The key player for hyper-radiosensitivity in gastric cancer cells with low dose fractionation radio therapy (LDFRT)

Abstract The most conventional therapy for solid tumors is radiotherapy. Still there is a challenge when it comes to managing highly disseminated gastrointestinal cancers because of the toxicity issues. Earlier, low dose fractionated radiotherapy (LDFRT) was considered to be ineffective for tumor removal, rather believed to cause cancer. There are few improvements with the use of novel approach in few cases of solid malignancy. This paradigm exploits the advantage of LDFRT with the combination of chemotherapy. Because now it's known that LDFRT causes hyper-radiosensitivity (HRS). The aim of our study is to determine the efficacy of LDFRT in combination with modified regimen of docetaxel, cisplatin and 5′-fluorouracil (mDCF) and their mechanism of action in gastric cancer cells. Our data indicate that consecutive three days radiation with daily fraction of 0.15 Gy produced HRS in gastric cancer cells and potentiated the effect of chemotherapeutic drugs (mDCF). Colony survival assays showed only 10% of gastric cancer cell survival when LDFRT was combined with mDCF while ten times higher dose (1.35 Gy) of radiation alone was required to achieve the same results. RT2-PCR profiler array analysis showed marked upregulation of dual oxidase-2 (DUOX2), an enzyme of NADPH- oxidase family, without inducing the genes involved in DNA repair. This was evident from 3.5 fold increase in reactive oxygen species (ROS) in cells exposed to LDFRT and mDCF. Further, this was confirmed by increase in cancer cell survival hence radioresistance with downregulation of DUOX2 expression. In addition to this inhibition of DUOX2 activity also abrogated the efficacy of the combination therapy. Taken together these data suggest that chemopotentiation by LDFRT in gastric cancer cells may be partly due to increased ROS production by DUOX2 without inducing the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT as a chemopotentiator for advanced and metastatic gastric cancers. (* = Equally Contributed) Citation Format: Duc Nguyan, Palak R. Parekh, Elizabeth T. Chang, Navesh Sharma, France Carrier. DUOX2: The key player for hyper-radiosensitivity in gastric cancer cells with low dose fractionation radio therapy (LDFRT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1805. doi:10.1158/1538-7445.AM2015-1805

Contribution of Dual Oxidase 2 (DUOX2) to Hyper-Radiosensitivity in Human Gastric Cancer Cells.

Whole-abdominal radiotherapy (WART) is a primary method for managing gastrointestinal cancers that have disseminated into intra-abdominal tissues. While effective, this approach is limited because of the increased toxicity to normal tissue associated with combined WART and full-dose chemotherapy regimens. Recent studies have demonstrated a survival advantage in a novel treatment paradigm that allows for the safe use of full-dose systemic chemotherapy in combination with low-dose fractionated radiotherapy (LDFRT). Traditionally, radiation doses greater than 120 cGy have been used in radiotherapy because lower doses were thought to be ineffective for tumor therapy. However, we now know that LDFRT can produce hyper-radiosensitivity (HRS), a phenomenon where cells undergo apoptosis at radiation doses as low as 15 cGy, in a number of proliferating cells. The objectives of our current study were to determine whether LDFRT can induce HRS in gastrointestinal cancer cells and to identify biomarkers of chemopotentiation by LDFRT. Our data indicate that three consecutive daily fractions of 15 cGy produced HRS in gastric cancer cells and potentiated a modified regimen of docetaxel, cisplatin and 5'-fluorouracil (mDCF). Colony survival assays indicated that 15 cGy was sufficient to kill 90% of the cells when LDFRT was combined with mDCF whereas a dose almost 10 times higher (135 cGy) was needed to achieve the same rate when using conventional radiotherapy alone. RT(2) PCR Profiler™ array analysis indicated that this combined regimen upregulated dual oxidase 2 (DUOX2), an enzyme functioning in the production of hydrogen peroxide, without upregulating genes involved in DNA repair. Moreover, downregulation of DUOX2 increased radioresistance at every radiation dose tested. In addition, our data indicate that reactive oxygen species (ROS) increase up to 3.5-fold in cells exposed to LDFRT and mDCF. Furthermore, inhibition of NADPH oxidase abrogated the killing efficiency of this combined regimen. Taken together these data suggest that chemopotentiation by LDFRT in gastric cancer cells may be due, at least in part, to increased ROS production (DUOX2) without upregulation of the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT, such as in WART, as a chemopotentiator for advanced and metastatic gastric cancers.

ATR signaling cooperates with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.

Little work has been done on the mechanism of low dose hyper-radiosensitivity (HRS) and later appeared radioresistance (termed induced radioresistance (IRR)) after irradiation with medium and high linear energy transfer (LET) particles. The aim of this study was to find out whether ATR pathway is involved in the mechanism of HRS induced by high LET radiation. GM0639 cells and two ATM deficient/mutant cells, AT5BIVA and AT2KY were irradiated by carbon ion beam. Thymidine block technique was developed to enrich the G2-phase population. Radiation induced early G2/M checkpoint was quantitatively assess with dual-parameter flow cytometry by detecting the cells positive for phospho-histone H3. The involvement of ATR pathway in HRS/IRR response was detected with pretreatment of specific inhibitors prior to carbon ion beam. The link between the early G2/M checkpoint and HRS/IRR under carbon ion beam was first confirmed in GM0639 cells, through the enrichment of cell population in G2-phase or with Aurora kinase inhibitor that attenuates the transition from G2 to M phase. Interestingly, the early G2/M arrest could still be observed in ATM deficient/mutant cells with an effect of ATR signaling, which was discovered to function in an LET-dependent manner, even as low as 0.2Gy for carbon ion radiation. The involvement of ATR pathway in heavy particles induced HRS/IRR was determined with the specific ATR inhibitor in GM0639 cells, which affected the HRS/IRR occurrence similarly as ATM inhibitor. These data demonstrate that ATR pathway may cooperate with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.

Association of ATM activation and DNA repair with induced radioresistance after low-dose irradiation.

Mammalian cells often exhibit a hyper-radiosensitivity (HRS) to radiation doses <20 cGy, followed by increased radioresistance (IRR) at slightly higher doses (∼20-30 cGy). Here, the influence of DNA double-strand break repair (DSBR) on IRR was examined. The failure of Ataxia telangiectasia (AT) cells to undergo IRR reported by others was confirmed. Flow cytometric analysis indicated that normal cells fail to show a measurable increase in serine 1981 phosphorylated AT-mutated (ATM) protein after 10 cGy up to 4 h post irradiation, but a two- to fourfold increase after 25 cGy. Similarly, more proficient reduction of phosphorylated histone H2AX was observed 24 h after 25 cGy than after 10 cGy, suggesting that DSBR is more efficient during IRR than HRS. A direct examination of the consequences of inefficient DNA repair per se (as opposed to ATM-mediated signal transduction/cell cycle responses), by determining the clonogenic survival of cells lacking the DNA repair enzyme polynucleotide kinase/phosphatase, indicated that these cells have a response similar to AT cells, i.e. HRS but no IRR, strongly linking IRR to DSBR.

Connexin-43 downregulation in G2/M phase enriched tumour cells causes extensive low-dose hyper-radiosensitivity (HRS) associated with mitochondrial apoptotic events

Enrichment of tumour cells in G2/M phases in vitro is known to be associated with low-dose hyper-radiosensitivity (HRS). These cell cycle phases also involve reduced expression of adhesion protein connexin-43 (Cx43). Therefore, we investigated the role of Cx43 in HRS. Asynchronous or G2/M enriched tumour cells (U87, BMG-1, HeLa) and normal primary fibroblasts (HDFn) were γ-irradiated at varying doses, with an asynchronous group separately subjected to Cx43-knockdown prior to irradiation. Cx43 level, gap junctional activity, clonogenic cell survival, cell growth/viability, mitochondrial alterations and other apoptosis-regulating events were studied. G2/M enrichment reduced Cx43 level by ~50% and caused considerable HRS at doses 10 cGy–30 cGy in all tumour cell lines. Cx43-knockdown to the same level (~60%) also elicited prominent HRS response in these cells. Quite important, radiosensitivity of primary HDFn cells remained unaltered by all these treatments. In Cx43-knockdown tumour cells, low-dose irradiation caused significant growth inhibition and apoptosis involving loss of MMP, cytochrome-c release and caspase-3 activation, thereby demonstrating the important cytoprotective role of Cx43. Therefore, this study significantly shows that Cx43 downregulation (a constitutive feature of G2/M phase) selectively renders tumour cells hypersensitive to low-dose radiation, and presents connexins as potential therapeutic targets.

Effects of low oxygen levels on G2-specific cytogenetic low-dose hyper-radiosensitivity in irradiated human cells.

Low-dose hyper-radiosensitivity (HRS) has been reported in normal human lymphoblastoid cell lines for exposures at ≤ 20 cGy, but the cytogenetic effects of oxygen (O2 ) levels in tissue culture medium on HRS have not been evaluated. We asked whether HRS was lost in G2-irradiated cells grown in atmospheres of 2.5% or 5% O2 , compared to responses by cells cultured in ambient O2 (21%). The results indicate a loss of HRS when cells are cultured and irradiated either in 2.5% or 5% O2 . We then evaluated whether low O2 levels either before or after exposure were responsible for the loss of HRS. For cells irradiated in 5% O2 , subsequent immediate re-oxygenation to ambient O2 levels restored the HRS effect, while cells cultured and irradiated at ambient O2 levels and then transferred to 5% O2 exhibited little or no HRS, indicating that ambient O2 levels after, but not before, radiation substantially affect the amounts of cytogenetic damage. HRS was not observed when cells were irradiated in G1. At doses of 40-400 cGy there was significantly less cytogenetic damage when cells were recovering from radiation at low O2 levels than at ambient O2 levels. Here we provide the first cytogenetic evidence for the loss of HRS at low O2 levels in G2-irradiated cells; these results suggest that at low O2 levels for all doses evaluated there is either less damage to DNA, perhaps because of lower amounts of reactive oxygen species, or that DNA damage repair pathways are activated more efficiently.

Cytogenetic Low-Dose Hyperradiosensitivity Is Observed in Human Peripheral Blood Lymphocytes

The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

TGF-B3 Dependent Modification of Radiosensitivity in Reporter Cells Exposed to Serum From Whole-Body Low Dose-Rate Irradiated Mice.

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.

Abstract 844: Contribution of dual oxidase 2 (DUOX2) to hyper radiosensitivity in human gastric cancer cells

Abstract Whole abdominal radiotherapy (WART) is a primary method for managing gastrointestinal cancers that have disseminated into intra-abdominal tissues. While effective, this approach is limited since combination of WART with full-dose chemotherapy regimens increase toxicity to normal tissue. Recent studies have demonstrated a survival advantage in a novel treatment paradigm that allows for the safe use of full-dose systemic chemotherapy in combination with Low Dose Fractionated Radiotherapy (LDFRT). Traditionally, radiotherapy used doses greater than 120 cGy because it was thought that lower radiation doses would be ineffective for tumor therapy. We now know that LDFRT can produce hyper-radiosensitivity (HRS), a phenomenon where cells undergo apoptosis at radiation doses as low as 15 cGy, in a number of proliferating cells. Here we aimed at determining whether LDFRT can induce HRS in gastrointestinal cancer cells and at identifying biomarkers of chemopotentiation by LDFRT. Our data indicate that three consecutive daily fractions of 15 cGy produced HRS in gastric cancer cells and potentiated a modified regimen of Docetaxel, Cisplatin, and 5’-fluorouracil (mDCF). Colony survival assays indicated that 15 cGy was sufficient to kill 90% of the cells when LDFRT was combined with mDCF whereas an almost ten times higher dose (135 cGy) was needed to achieve the same rate when using conventional radiotherapy alone. RT2 PCR Profiler Array analysis indicated that this combined regimen up-regulated dual oxidase 2 (DUOX2), an enzyme functioning in the production of hydrogen peroxide, without up-regulating genes involved in DNA repair. Moreover, down regulation of DUOX2 increased radiation resistance at every radiation doses tested. In addition, a subset of genes known to be activated by Reactive Oxygen Species were specifically upregulated by LDFRT when combined with mDCF. Taken together these data suggest that chemopotentiation by LDFRT in gastric cells may be due, at least in part, to increased ROS production (DUOX2) without upregulation of the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT, such as in WART, as chemopotentiator for advanced and metastatic gastric cancers. Citation Format: Duc Nguyen, Elizabeth Chang, Navesh Sharma, France Carrier. Contribution of dual oxidase 2 (DUOX2) to hyper radiosensitivity in human gastric cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 844. doi:10.1158/1538-7445.AM2014-844

Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells

The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0–400cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures.

Pulsed Low-Dose-Rate External Beam Radiation Therapy: An in-vivo Study on Prostate Cancer

Although hyper radio-sensitivity has been demonstrated at low doses below 0.4 Gy for various tumor cell lines in vitro, there was no report on in vivo studies for human prostate cancer cell lines. In our previous in vivo studies, we performed total body irradiation of nude mice with both pulsed low dose rate (PLDR) and conventional radiation therapy (CRT) techniques. The PLDR treated animal group showed significantly lower level of radiation toxicity to normal tissues than CRT, which leads to much longer survival of the irradiated animals.

The role of interleukin-13 in the removal of hyper-radiosensitivity by priming irradiation.

It has previously been demonstrated that the presence of fetal bovine serum is necessary for TGF-β3 (transforming growth factor beta 3)-dependent elimination of low-dose hyper-radiosensitivity (HRS) in cells by 1 h of low-dose-rate γ-irradiation (0.2–0.3 Gy/h). The purpose of the present study was to identify the serum constituent involved. Two human HRS-positive (T-47D, T98G) cell lines were used. The effects of different pretreatments on HRS were investigated using the colony assay. Total inducible nitric oxide synthase (iNOS) levels were measured using a cell-based ELISA assay. The serum factor was identified as interleukin-13 (IL-13). In order for low dose-rate irradiation to eliminate HRS through the TGF-β3-dependent mechanism, the cells must be exposed to IL-13 first. Inhibiting receptor IL-13Rα2 showed that this receptor is involved in the response. Adding IL-13 to serum-free medium restored the properties of full medium but not when an inhibitor of proprotein convertase activity was added together with IL-13. The presence of IL-13 resulted in upregulation of total iNOS protein levels. Thus, this study indicates that IL-13 interacts with the cells though receptor IL-13Rα2 and induces upregulation of iNOS and activation of one or more furin-like proprotein convertases.

Dynamics of Cellular Responses to Radiation

Understanding the consequences of exposure to low dose ionizing radiation is an important public health concern. While the risk of low dose radiation has been estimated by extrapolation from data at higher doses according to the linear non-threshold model, it has become clear that cellular responses can be very different at low compared to high radiation doses. Important phenomena in this respect include radioadaptive responses as well as low-dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR). With radioadaptive responses, low dose exposure can protect against subsequent challenges, and two mechanisms have been suggested: an intracellular mechanism, inducing cellular changes as a result of the priming radiation, and induction of a protected state by inter-cellular communication. We use mathematical models to examine the effect of these mechanisms on cellular responses to low dose radiation. We find that the intracellular mechanism can account for the occurrence of radioadaptive responses. Interestingly, the same mechanism can also explain the existence of the HRS and IRR phenomena, and successfully describe experimentally observed dose-response relationships for a variety of cell types. This indicates that different, seemingly unrelated, low dose phenomena might be connected and driven by common core processes. With respect to the inter-cellular communication mechanism, we find that it can also account for the occurrence of radioadaptive responses, indicating redundancy in this respect. The model, however, also suggests that the communication mechanism can be vital for the long term survival of cell populations that are continuously exposed to relatively low levels of radiation, which cannot be achieved with the intracellular mechanism in our model. Experimental tests to address our model predictions are proposed.

The roles of TGF-β3 and peroxynitrite in removal of hyper-radiosensitivity by priming irradiation

Purpose: To investigate the mechanisms inducing and maintaining the permanent elimination of low dose hyper-radiosensitivity (HRS) in cells given a dose of 0.3 Gy at low dose-rate (LDR) (0.3 Gy/h).Materials and methods: Two human HRS-positive cell lines (T-47D, T98G) were used. The effects of pretreatments with transforming growth factor beta (TGF-β) neutralizers, TGF-β3 or peroxynitrite scavenger on HRS were investigated using the colony assay. Cytoplasmic levels of TGF-β3 were measured using post-embedding immunogold electron microscopic analysis.Results: TGF-β3 neutralizer inhibited the removal of HRS by LDR irradiation. Adding 0.001 ng/ml TGF-β3 to cells removed HRS in T98G cells while 0.01 ng/ml additionally induced resistance to higher doses. Cytoplasmic levels of TGF-β3 were higher in LDR-primed cells than in unirradiated cells. The presence of the peroxynitrite scavenger uric acid inhibited the effect of LDR irradiation. Furthermore, the permanent elimination of HRS in LDR-primed cells was reversed by treatment with uric acid. The removal of HRS by medium from hypoxic cells was inhibited by adding TGF-β3 neutralizer to the medium before transfer or by adding hypoxia inducible factor 1 (HIF-1) inhibitor chetomin to the cell medium during hypoxia.Conclusions: TGF-β3 is involved in the regulation of cellular responses to small doses of acute irradiation. TGF-β3 activation seems to be induced by low dose-rate irradiation by a mechanism involving inducible nitric oxide (iNOS) and peroxynitrite, or during cycling hypoxia by a mechanism most likely involving HIF-1. The study suggests methods to turn resistance to doses in the HRS-range on (by TGF-β3) or off (by TGF-β3 neutralizer or by peroxynitrite inhibition).

Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis.

Suppression of low-dose hyper-radiosensitivity in human lung cancer cell line A549 by radiation-induced autophagy

This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.

Three-Times Daily Ultrafractionated Radiation Therapy, A Novel and Promising Regimen for Glioblastoma Patients

Glioblastomas are considered to be one of the most radio resistant tumors. Despite new therapies, the prognosis of this disease remains dismal. Also, the mechanisms of radiation resistance in mammalian cells are more complex than once believed. Experimental studies have indicated that some human cell lines are sensitive to low radiation doses of <1 Gy. This phenomenon has been termed low-dose hyper-radio-sensitivity (HRS), and is more apparent in radio resistant cell lines, such as glioblastoma cells. Sensitivity may result from the inability of low dose radiation to efficiently induce repair mechanisms, whereas higher doses cause enough damage to trigger repair responses for radio resistance. In vitro studies have demonstrated this phenomenon using various human malignant glioma cell lines: (1) daily repeated irradiation of cells with low doses compared to irradiation using a single biologically equivalent dose resulted in significantly higher cell killing; (2) experiments conducted on glioma xenografts demonstrated that repeated irradiation with low doses was more effective for inhibiting tumor growth than a single dose. In order to confirm and validate these promising studies on HRS, a few phase II trials were developed. For translating the experimental observations into the clinic, ultra fractionation protocols (with three daily doses) were tested in glioblastoma patients. Tolerance and toxicity were the primary endpoints, with overall survival as a secondary endpoint. These protocols were initiated before concomitant radio chemotherapy became the standard of care. For these trials, patients with an unfavorable clinical prognostic factor of newly unresectable GBM were included. When comparing the results of these trials with international literature using multivariate analysis for both progression free survival and overall survival, ultra fractionated irradiation showed superiority over radiotherapy alone. In addition, it was found to be equivalent to treatment using radiotherapy and temozolomide. Therefore, ultra fractionated protocols may prolong survival of glioblastoma patients. In this review, we describe the main experimental data regarding low-dose hypersensitivity as well as the findings of clinical trials that have investigated this new radiotherapy regimen.

Determining if low dose hyper-radiosensitivity (HRS) can be exploited to provide a therapeutic advantage: A cell line study in four glioblastoma multiforme (GBM) cell lines

Purpose: To determine if ultra-fractionation using repeated pulses of radiation (10 × 0.2 Gray [Gy]) would be more cytotoxic than continuously-delivered radiation to the same total dose (2 Gy) in four glioma cell lines.Materials and methods: Human T98G, U373, U87MG and U138MG cells were conventionally X-irradiated with 0.1–8 Gy and clonogenic survival assessed. Next, cells were treated with either a single dose of 2 Gy or 10 pulses of 0.2 Gy using a 3-min inter-pulse interval and DNA (Deoxyribonucleic acid) repair (pHistone H2A.X), G2-phase cell cycle checkpoint arrest (pHistone H3) and apoptosis (caspase-3) compared between the two regimens. A dose of 0.2 Gy was selected as this reflects the hyper- radiosensitivity (HRS)/increased radioresistance (IRR) transition point of the low-dose cell survival curve.Results: T98G, U87MG and U138MG exhibited distinct HRS responses and survival curves were well-described by the Induced Repair model. Despite the prolonged delivery time, ultra-fractionation (10 × 0.2 Gy) was equally effective as a single continuously-delivered 2 Gy dose. However, ultra-fractionation was more effective when given for five consecutive days to a total dose of 10 Gy. The increased effectiveness of ultra-fractionation could not be attributed directly to differences in DNA damage, repair processes or radiation-induced apoptosis.Conclusions: Ultra-fractionation (10 × 0.2 Gy) is an effective modality for killing glioma cell lines compared with standard 2 Gy dosing when multiple days of treatment are given.