1-Methylxanthine enhances the radiosensitivity of tumor cells

Purpose:?To determine the efficacy of a caffeine derivative 1-methylxanthine (1-MTX) in increasing radiosensitivity of cancer cells and elucidate the underlying mechanisms in vitro.

Materials and methods:?RKO human colorectal cancer cells carrying wild type protein 53 kDa (p53) were incubated with 3 mM 1-MTX for 30 min, exposed to 4 Gy ionizing radiation, and further incubated with 1-MTX for three days. The clonogenic cell death was determined, and the cell cycle distribution and apoptosis were studied with flow cytometry at different times after irradiation. The DNA double strand break (DNA DSB) was examined using phosphorylated Histone2A (γ-H2AX) foci formation, and the expression/activity of checkpoint 2 kinase (Chk2), cell division cycle 25 (Cdc25) phosphatase and cyclin B1/Cdc2 kinase were also investigated using western blotting and in vitro kinase assays.

Results:?The treatment with 3 mM 1-MTX increased the radiation-induced clonogenic and apoptotic cell death. The radiation-induced phosphorylation of Chk2 and Cdc25c and the radiation-induced increase in the cyclin B1/Cdc2 kinas activity were little affected by 1-MTX. The radiation-induced G2/M arrest was only slightly shortened and the expression of radiation-induced γ-H2AX was markedly prolonged by 1-MTX.

Conclusions:?1-MTX significantly increased the radiosensitivity of RKO human colorectal cancer cells carrying wild type p53 mainly by inhibiting the repair of radiation-induced DNA DSB without causing significant alteration in radiation-induced G2/M arrest. Such a radiosensitization occurred at 1-MTX concentrations almost non-toxic to the target tumor cells.     

DNA-dependent protein kinase: effect on DSB repair,G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2?Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1?h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24?h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4?Gy, but decreased both at higher doses of 20?Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.     

Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells

Purpose: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs.

Materials and methods: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed.

Results: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8?Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs.

Conclusions: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.     

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells

Abstract

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy.

Material and methods: Cells were exposed to EMFs (25, 50 and 100?Hz; 8 and 10?mT). In the co-treatment, cells were first exposed to EMFs (50?Hz/10?mT) for 30?min and then to ionizing radiation (IR) (2?Gy) 4?h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay.

Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4?h after exposure to 50?Hz/10?mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24?h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells.

Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.     

Biological effects of three types of ionizing radiation on creeping bentgrass

Abstract

Purpose: Gamma-rays and carbon ions are frequently used for mutation breeding in diverse plant species, whereas proton ions have been rarely used for this purpose. This study assessed the potential of proton ions for plant mutation breeding.

Materials and methods: We compared the effects of radiation on creeping bentgrass seeds with γ-rays, proton ions, and carbon ions on seed germination, plant growth parameters, and DNA fragmentation.

Results and conclusions: The lethal dose 50 (LD₅₀) doses based on seed germinability were 115.9?Gy (γ-rays), 225.1?Gy (proton ions), and 57.7?Gy (carbon ions). Threshold doses for survival were 150?Gy (γ-rays), 150?Gy (proton ions), and 25?Gy (carbon ions). Suppression of plant growth was displayed at 100?Gy (γ-rays), 25?Gy (proton ions), and 25?Gy (carbon ions). Similar patterns of decreasing head DNA percentage were observed for γ-rays and proton ions. Carbon ions induced the lowest frequency of DNA fragmentation. The biological effects of the ionizing radiation types on creeping bentgrass are summarizable as follows: germination, carbon ions (C)>γ-rays (G)>proton ions (P); survival, C?>?P?=?G; growth, C?≥?P?>?G; DNA fragmentation, G?≥?P?>?C. These results indicate that proton ions are useful as a physical mutagen in plant mutation breeding.     

Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line

Purpose: Lepidopteran insect cells are known to exhibit very high radioresistance. Although very effective DNA excision–repair has been proposed as a contributing factor, a detailed understanding of insect cell radiation responses has not yet been obtained. Therefore, the study was carried out to understand the in vitro radiation responses of Sf9 lepidopteran cells.

Materials and methods: Exponentially growing asynchronous Sf9 cells (derived from ovaries of Spodoptera frugiperda) were exposed to gamma‐radiation doses of 2–200?Gy. Cell survival, growth inhibition, cell cycle progression delay, alterations in cell morphology as well as induction of DNA damage, micronuclei and apoptosis were studied at various post‐irradiation time intervals.

Results: Biphasic survival response curves were obtained with D₀ rising from 20?Gy (at doses ≤60?Gy) to 85?Gy (between 60 and 200?Gy), corroborating earlier reports on lepidopteran cells. An additional downward deviation at 2?Gy indicated a hypersensitive response. Dose‐dependent growth inhibition with a transient G2 delay starting 12?h and extending up to 48–96?h was observed at doses of 10–200?Gy, while a brief G1/S transition delay was observed only at higher doses (≥100?Gy). Significant DNA damage was detected only at 20?Gy and higher doses, in contrast with human cells that showed similar damage at 2?Gy. Interestingly, micronuclei were not induced at any of the doses tested, although spontaneous micronucleation was evident in <1% of cells. Lack of micronucleus induction even at doses that induced significant DNA damage and a transient G2 block (20–50?Gy) strongly indicated a role of holocentric lepidopteran chromosomes. Apoptosis was detected only in a small proportion of cells (3%) exposed to 200?Gy, and cell/nucleus size and granularity increased by 72–96?h post‐irradiation in a dose‐dependent manner. Sf9 nucleoids extracted at 2?M NaCl showed higher compactness than the nucleoids prepared from human cells.

Conclusions: It is clearly shown that lepidopteran cells are highly resistant to the induction of DNA damage and micronuclei, and display very low induction of apoptosis at doses up to 200?Gy. While the lack of micronucleus induction seems to be primarily due to the holocentric nature of their chromosomes, certain unique signalling pathways might be responsible for the low induction of apoptosis. Factors causing protection of Sf9 cellular DNA from radiation‐induced damage are presently being investigated.     

Role of Nrf2-antioxidant in radioprotection by root extract of Inula racemosa

Abstract

Purpose: Inula racemosa, a Trans-Himalayan plant is an important medicinal herb. In this study, the radio-modulatory efficacy of aqueous root extract of I. racemosa was investigated.

Materials and methods: Normal Kidney Epithelium cells were treated with extract (50–200?μg/ml) and exposed to 3?Gy of γ-radiation, while C57BL/6 mice were administered with extract (300–600?mg/kg BW) intraperitoneally prior to exposure to 7.5?Gy of γ-radiation to assess radiation modulatory efficacy.

Results: The administration of extract (30?min and 1?h) prior to radiation exposure improved the survival of NKE cells (as measured by proliferation), restored MMP and ROS levels as compared to radiation-exposed alone cells. These cells showed up-regulated Nrf2 protein levels at 7?h and increased expression of HO-1 and NOQ1 protein at 24?h In mice, the 30 days whole body survival study demonstrated that extract pre-treatment increases survival or delays the onset of radiation-induced mortality as against 70% mortality of 7.5?Gy of γ-radiation.

Conclusions: The aqueous extract of roots of I. racemosa enhanced the survival of irradiated NKE cells and rescued C57BL/6 mice against WBI-induced mortality. The radiation modulation efficacy was mediated through cumulative activation of HO-1 and NQO1 downstream of Nrf2 translocation in NKE cells.

Abbreviations:

ARE: Antioxidant Response Element; FITC: Fluorescein isothiocyanate; GCS: Glutamylcysteine synthase; HO-1: Heme oxygenase-1; LPS: Lipopolysacharide; MRP: Multidrug Resistance-Associated Proteins; NQO1: NAD(P)H Quinone Dehydrogenase 1; NRH: Quinone Oxidoreductase 2 (NQO2); PBS: Phosphate Buffer Saline; PKA: Protein Kinase A; PKC: Protein Kinase C; PI3-kinase: Phosphatidylinositol 3-Kinase; SRB: Sulforhodamine B; UV: Ultra-Violet radiation     

Feasibility study on the use of gold nanoparticles in fractionated kilovoltage X-ray treatment of melanoma

Purpose: Despite the high radioresistance of melanoma, unresectable lesions can be subjected to radiation treatment with the use of gold nanoparticles (AuNPs) as a dose-enhancing agent preferentially loaded on these lesions. The modality of single high-dose treatment has been investigated to confirm its therapeutic efficiency for AuNP-treated melanoma cells. This study explores the feasibility of utilizing AuNPs in fractionated radiation therapy of melanoma for further therapeutic gain.

Materials and methods: The responses of human skin melanoma cells to 150-kVp X-ray exposure at 2 and 4?Gy were assessed by quantify gamma-H2AX expression and clonogenic survival, with or without 320 μM of 50?nm AuNP treatment in a culture medium. The influence of AuNPs on cell cycle distribution was observed before irradiation and during 3 d period after irradiation.

Results: The AuNP treatment of melanoma cells influenced the cellular response to kilovoltage X-rays to similar extents in terms of the percentage of gamma-H2AX-positive cells and the fractional loss of clonogenicity. Without radiation exposure, AuNPs reduced the portion of melanoma cells at the G₂/M phase from 11 to 7%. After irradiation, the progression of the melanoma cells treated with AuNPs toward the G₂/M phase was more rapid than that of the AuNP-free cells, and the release of the former from the G₂/M phase was slower than that of the latter. At 24?h after irradiation with AuNPs, the cell cycle was rearranged in a pattern that increased the vulnerability of the cells to radiation damage.

Conclusions: In addition to the benefit of AuNP treatment to the control of melanoma in single high-dose treatment, further therapeutic gain is expected through fractionated X-ray treatment that involves daily exposure. The AuNP-treated melanoma cells of an increased portion in the radiosensitive G₂/M phase following a fractionated dose delivery would respond to the next treatment with an enhanced chance of clonogenic death.     

In vitro radiosensitization by pentoxifylline does not depend on p53 status

Abstract

Purpose: The mode by which the xanthine derivative, pentoxifylline, induces a radiosensitizing effect in cell cultures is a key and controversial radiobiological issue and requires further elucidation.

Materials and methods: Six human glioblastoma cell lines were tested for the effect of pentoxifylline treatment at maximum G2/M block on the basis of cell survival, mitotic activity, and micronucleus formation after exposure to gamma radiation. Cell survival was measured by the colony-forming assay. Micronucleus formation (an indicator of DNA damage) and the proportion of binucleated cells (a representation of mitotic activity) were determined using the cytokinesis-block assay.

Results: Remarkably, exposure to a single dose of 4 Gy produced strong G2/M blocks in both p53 mutant and wild-type cells. Addition of pentoxifylline at the peak of radiation-induced G2/M blocks resulted in a p53-independent reduction in cell survival in all cell lines. This radiosensitization was strongly correlated with the magnitude of the radiation-induced G2/M block. The changes observed in mitotic activity and micronucleus yield were also p53-independent.

Conclusions: These results are at variance with the view that pentoxifylline preferentially sensitizes p53 mutant cells, and that sensitization occurs only when cells are irradiated in the presence of the drug. The data suggest that the effectiveness of pentoxifylline as radiosensitizer depends on the proportion of cells that are arrested in the G2/M phase transition following exposure to ionizing radiation. These findings can assist in the identification of cancers that may benefit from therapies using G2/M checkpoint abrogators.     

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines

Purpose: To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure.

Materials and methods: Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells.

Results: We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1?Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5?Gy.

Conclusion: These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.     

Effects of CKMT1 on radiosensitivity of nasopharyngeal carcinoma cells

Abstract

Purpose: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear. The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity.

Material and methods: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2. Biological changes were detected after cells exposing to different doses of X-ray to determine the role of CKMT1 on NPC cell radiosensitivity.

Results: CKMT1 promotes proliferation and migration in NPC cell lines CNE-1 and CNE-2. Overexpression of CKMT1 in CNE-1 cells enhanced colony formation rates, reduced G2/M phase cell cycle arrest, lowered apoptosis rate and c-PARP level, and elevated STAT3 phosphorylation level after radiation treatment. While knocking out CKMT1 using the CRISPR/Cas9 system in CNE-2 cells lowered colony formation rates, increased G2/M phase cell cycle arrest, apoptosis rates, and c-PARP levels, and decreased STAT3 phosphorylation in response to radiation treatment.

Conclusions: NPC cells with higher CKMT1 exhibited lower radiosensitivity through promoting phosphorylation of STAT3. Our findings suggest that CKMT1 may be an alternative radiotherapeutic target in NPC therapy.     

Parallel comparison of pre-conditioning and post-conditioning effects in human cancers and keratinocytes upon acute gamma irradiation

Abstract

Purpose: To determine and compare the effects of pre-conditioning and post-conditioning towards gamma radiation responses in human cancer cells and keratinocytes.

Material and methods: The clonogenic survival of glioblastoma cells (T98G), keratinocytes (HaCaT), and colorectal carcinoma cells (HCT116 p53^+/+ and p53^?/?) was assessed following gamma ray exposure from a Cs-137 source. The priming dose preceded the challenge dose in pre-conditioning whereas the priming dose followed the challenge dose in post-conditioning. The priming dose was either 5 mGy or 0.1?Gy. The challenge dose was 0.5–5?Gy.

Results: In both pre- and post-conditioning where the priming dose was 0.1?Gy and the challenge dose was 4?Gy, RAR developed in T98G but not in HaCaT cells. In HCT116 p53^+/+, pre-conditioning had either no effect or a radiosensitizing effect and whereas post-conditioning induced either radiosensitizing or radioadaptive effect. The different observed outcomes were dependent on dose, the time interval between the priming and challenge dose, and the time before the first irradiation. Post-conditioning effects could occur with a priming dose as low as 5 mGy in HCT116 p53^+/+ cells. When HCT116 cells had no p53 protein expression, the radiosensitizing or radioadaptive response by the conditioning effect was abolished.

Conclusions: The results suggest that radiation conditioning responses are complex and depend on at least the following factors: the magnitude of priming/challenge dose, the time interval between priming and challenge dose, p53 status, cell seeding time prior to the first radiation treatment. This work is the first parallel comparison demonstrating the potential outcomes of pre- and post-conditioning in different human cell types using environmentally and medically relevant radiation doses.     

Combined gamma‐irradiation and subsequent cisplatin treatment in human squamous carcinoma cell lines sensitive and resistant to cisplatin

Purpose: To investigate the effects of combined radiation and subsequent cisplatin treatment on the human squamous carcinoma cell line SCC‐25 and its cisplatin‐resistant derivative SCC‐25/CP.

Materials and methods: SCC‐25 and SCC‐25/CP cells were treated with various gamma‐ray doses (5?cGy–7?Gy) followed 60?min later by cisplatin treatment and subsequently assayed for survival using a conventional colony assay. For SCC‐25, the subsequent cisplatin treatment was 0.1, 1, 10 and 20?µM for 1?h. For the more cisplatin‐resistant SCC‐25/CP cells, the subsequent cisplatin treatment was 10 and 50?µM for 1?h.

Results: The cisplatin‐resistant SCC‐25/CP cells were not cross‐resistant to gamma‐irradiation. Subsequent treatment with an LD₅₀ concentration of cisplatin (10 and 50?µM for SCC‐25 and SCC‐25/CP, respectively) resulted in radiosensitization for SCC‐25/CP but not for SCC‐25 cells. Gamma‐irradiation of SCC‐25/CP cells followed by treatment with 10 and 50?µM cisplatin for 1?h resulted in radiation survival curves displaying a significant low‐dose hypersensitive region followed by increased radioresistance at higher doses. A total of 10?µM cisplatin resulted in radiosensitization confined to the low‐dose region (0.05 and 0.25?Gy), whereas the higher cisplatin treatment of 50?µM resulted in the appearance of a hypersensitive region together with a reduction of the increased radioresistance region. In contrast, cisplatin treatment (0.1, 1, 10 and 20?µM for 1?h) of SCC‐25 cells had no significant effect on survival following 2.5 or 7.0?Gy and actually resulted in an increased low‐dose radiation survival (0.05, 0.25 and 1?Gy) when survival was corrected for cisplatin treatment (p<0.01 for all cisplatin concentrations tested).

Conclusions: The significant radiosensitization for SCC‐25/CP given subsequent treatment with 50?µM cisplatin indicates cisplatin can inhibit the increased radioresistance response in SCC‐25/CP cells. In contrast, the subsequent cisplatin treatment of SCC‐25 cells can enhance their survival following low radiation doses.     

Low-dose hyper-radiosensitivity of progressive and regressive cells isolated from a rat colon tumour: Impact of DNA repair

Purpose: To ask whether highly metastatic sublines show more marked low-dose hyper-radiosensitivity (HRS) response than poorly metastatic ones.

Materials and methods: The progressive (PRO) subline showing tumourigenicity and metastatic potential and the regressive (REG) subline showing neither tumourigenicity nor metastatic potential were both isolated from a parental rat colon tumour. Clonogenic survival, micronuclei and apoptosis, cell cycle distribution, DNA single- (SSB) and double-strand breaks (DSB) induction and repair were examined.

Results: HRS phenomenon was demonstrated in PRO subline. Before irradiation, PRO cells show more spontaneous damage than REG cells. After 0.1 Gy, PRO cells displayed: (i) More DNA SSB 15 min post-irradiation, (ii) more unrepaired DNA DSB processed by the non-homologous end-joining (NHEJ) and by the RAD51-dependent recombination pathways, (iii) more micronuclei, than REG cells while neither apoptosis nor p53 phosphorylation nor cell cycle arrest was observed in both sublines.

Conclusions: HRS response of PRO subline may be induced by impairments in NHEJ repair that targets G₁ cells and RAD51-dependent repair that targets S-G₂/M cells. The cellular consequences of such impairments are a failure to arrest in cell cycle, the propagation of damage through cell cycle, mitotic death but not p53-dependent apoptosis. Tumourigenic cells with high metastatic potential may preferentially show HRS response.     

Radioprotective and radiomitigative effects of BP-C2, a novel lignin-derived polyphenolic composition with ammonium molybdate,in two mouse strains exposed to total body irradiation

Purpose: There remains an unmet medical need for radioprotective and mitigative agents. BP-C2 is a novel lignin-derived polyphenolic composition with ammonium molybdate, developed as radioprotector/radiomitigator.

Objectives: The present study evaluated BP-C2 for the mitigation of acute radiation syndrome (ARS).

Methods: A total-body irradiation mouse model (TBI, 4.0–8.0?Gy) was used in the study.

Results: In a 30-day survival study, performed in CBA mice, BP-C2, at a dosage of 81.0?mg/kg, improved survival (dose reduction factor (DRF)?=?1.1) and increased the formation of endogenous spleen colony-forming units (CFU). In C57BL/6 mice, BP-C2, when administered daily for 7 days, starting 24?hours after TBI, also improved survival. In animals irradiated with 5.0?Gy, BP-C2 increased the number of CFUs (6.7?±?5.1) compared to the 5.0?Gy placebo group (2.3?±?2.3, p?=?.0245). The number of surviving intestinal crypts was maintained in the 5.0?Gy BP-C2 group (133.7?±?13.9), in contrast to the 5.0?Gy placebo group (124.2?±?10.5, p?Conclusion: BP-C2 mitigates radiation-induced damage in mid-lethal range of radiation doses. Effects are mediated by enhancement of extramedullar hematopoiesis in the spleen and a protective effect on the intestinal epithelium.     

Radiochemotherapy with paclitaxel: Synchronization effects and the role of p53

Purpose

We have studied the interaction of paclitaxel (Taxol) and radiation in V79 cells and human lymphoblasts with special emphasis on cell cycle effects and the role of p53.

Material and Methods

V79 cells in log- and plateau-phase and human lymphoblasts (p53wt TK6 and p53mut WTK1) were used. Paclitaxel was given for 2 hours. Survival was determined using clonogenic assays. Cell cycle analysis was done using DNA flow cytometry.

Results

In V79 cells there was a dose dependent delay of colony formation after paclitaxel. The LD₅₀ was about 0.4 μM with a 2-hour exposure. In exponentially growing cells, there was an accumulation of 40% of cells in G2/M 6 hours after paclitaxel. The dose modification factor was about 3.9 when radiation was given 6 hours after 0.3 μM paclitaxel for 2 hours. Synchronization experiments using serum starvation and induction showed that synchronization was not sufficient to induce a comparable dose modification factor. Human lymphoblasts with mutated p53 (WTK1, LD₅₀=75 μM) were more resistant to paclitaxel than wild type p53 cells (TK6, LD₅₀=25 μM).

Conclusion

The radiosensitization induced by paclitaxel was critically dependent on the timing of irradiation and chemotherapy, although synchronization alone was not sufficient to explain the dose modification. Lymphoblasts with mutated p53 were less sensitive than wild type p53 cells.

     

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

Abstract

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).     

Evaluation of the premature chromosome condensation scoring protocol after proton and X-ray irradiation of human peripheral blood lymphocytes at high doses range

Purpose of the study: One of the main difficulties in radiation dose assessment is cells inability to reach mitosis after exposure to acute radiation. Premature chromosome condensation (PCC) has become an important method used in biological dosimetry in case of exposure to high doses. Various ways to induce PCC including mitotic cells fusion, chemical stimulation with calyculin A or okadaic acid give wide spectrum of application. The main goal of this study was to evaluate the utility of drug-induced PCC scoring procedure by testing 2 experimental modes where 150 and 75 G2/M-PCC phase cells were analyzed after exposure to high dose proton and X-ray radiation. Another aim is to determine the differences in cellular response induced by proton and photon radiation using a HPBL in vitro model as a further extension of our previous studies involving doses up to 4.0?Gy.

Materials and methods: Total body exposure was simulated by irradiating whole blood collected from a healthy donor. Whole blood samples were exposed to two radiation types: 60?MeV protons and 250 kVp X-rays in the dose range of 5.0–20.0?Gy, the dose rate for protons was 0.075 and 0.15?Gy/s for X-rays. Post 48?h of human peripheral blood lymphocytes (HPBL) culture, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers in the G2/M-PCC phase. In order to check the consistency of obtained results all scorers followed identical scoring criteria. Additionally, PCC index kinetics was evaluated for first 500 cells scored.

Conclusions: Here we provide a different method of results analysis. Presented dose-response curves were obtained by calculating the value of counted excess chromosome fragments. The results indicated that obtained dose estimates as adequate in the high dose range till 18.0?Gy for both studied radiation types, giving an opportunity to further improve PCC assay procedure and shorten the analysis time i.e. in case of partial-body exposure. Moreover, the study presents preliminary results of HPBL cellular response after proton irradiation at high doses range showing differences of PCC index kinetics for different cell classes and cell distribution.