Medium Transfer Technique Research Articles

Radiation-Induced Bystander Effect via GRID Radiotherapy and Medium Transfer in the A-375 Human Melanoma Cancer Cell Line: An In-vitro Study

Purpose: The goal of this research was to investigate the bystander effect in the A-375 cell line under the GRID therapy technique. In GRID therapy, due to direct and indirect cell damage after high-dose radiation, evaluation of Radiation-Induced Bystander Effects (RIBE) is of the most importance for investigating the risk of therapy.
 Materials and Methods: The potential role of RIBE was evaluated with different doses of 6 MeV electron radiation and different incubation times after irradiation using two methods; GRID therapy and medium transfer. Colony Formation Assay (CFA) and MTT test were used to detect the mentioned effects. Alpha and beta parameters were calculated from the cell survival curve by the quadratic-linear model.
 Results: The result showed that the survival fraction significantly decreases by increasing the radiation dose for both bystander and irradiated cells. However, a decrease in the number of colony-forming cells caused by electron radiation greater than 4MeV to target cells was significantly increased compared with bystander cells (P < 0.05). While increasing the incubation time after exposure to an electron beam, it had no significant effect on cell survival fraction (P > 0.05). Furthermore, the RIBE level in non-target cells increased up to a dose of 4Gy, but decreased significantly at doses higher than 4Gy. This result in high doses confirmed that a negative feedback mechanism was responsible for reducing the RIBE response.
 Conclusion: Based on the results, we can state there are classic radiation-induced bystander effects in A-375 monolayer exposed by GRID therapy and medium transfer technique, which can play an important role in pre-clinical and clinical studies.

Bystander Effect of Therapeutic Ultrasound in the Presence of Cisplatin: An in Vitro Study on Human Melanoma Cells.

In the bystander effect, non-irradiated cells receive biological signals from adjacent irradiated cells and undergo a variety of alterations, considered recently in non-ionizing irradiation like ultrasound waves. In this study, the bystander effect of therapeutic ultrasound exposure alone and in combination with cisplatin was determined. This study aims to determine the bystander effect caused by ultrasound and cisplatin. This experimental study was conducted on the human melanoma cell line including two groups of target and bystander cells. The target cell group was divided into three sub-groups of ultrasound irradiation alone, cisplatin alone, and ultrasound irradiation in the presence of cisplatin that the culture medium of these three groups of cells was transferred to the bystander cell group using the medium transfer technique. Then, apoptotic bystander cells and the expression of P53 and HO-1 in target and bystander groups were measured. The results of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptosis assay showed that cell death in target and bystander groups receiving the ultrasound with cisplatin is higher than in the ultrasound without cisplatin. PCR (the polymerase chain reaction) results in the target and bystander groups receiving treatments with increased expression of the P53 gene. Target and bystander groups receiving the ultrasound without cisplatin showed a decrease in HO-1 gene expression, while the ultrasound with cisplatin showed an increase in the HO-1 gene compared to the control group. Combining ultrasound with ultrasound and without it can transfer bystander signals to the cells that are not directly treated.

Bystander Effect Induced in Breast Cancer (MCF-7) and Human Osteoblast Cell Lines (hFOB 1.19) with HDR-Brachytherapy.

Background: Radiation induced bystander effects (RIBEs) occurs in unirradiated cells exhibiting indirect biological effect as a consequence of signals from other irradiated cells in the population.Objective: In this study, bystander effects in MCF-7 breast cancer cells and hFOB 1.19 normal osteoblast cells irradiated with gamma emitting HDR Brachytherapy Ir-192 source were investigated.Material and Methods: In this in-vitro study, bystander effect stimulation was conducted using medium transfer technique of irradiated cells to the non-irradiated bystander cells. Cell viability, reactive oxygen species (ROS) generation and colony forming assay was employed to evaluate the effect.Results: Results indicate that the exposure to the medium irradiated MCF-7 induced significant bystander killing and decreased the survival fraction of bystander MCF-7 and hFOB from 1.19 to 81.70 % and 65.44 %, respectively. A significant decrease in survival fraction was observed for hFOB 1.19 bystander cells (p < 0.05). We found that the rate of hFOB 1.19 cell growth significantly decreases to 85.5% when added with media from irradiated cells. The ROS levels of bystander cells for both cell lines were observed to have an increase even after 4 h of treatment. Our results suggest the presence of bystander effects in unirradiated cells exposed to the irradiated medium. Conclusion: These data provide evidence that irradiated MCF-7 breast cancer cells can induce bystander death in unirradiated MCF-7 and hFOB 1.19 bystander cells. Increase in cell death could also be mediated by the ROS generation during the irradiation with HDR brachytherapy.

Assessment of The Dose-Response Relationship of Radiation-Induced Bystander Effect in Two Cell Lines Exposed to High Doses of Ionizing Radiation (6 and 8 Gy).

The dose-response relationship of radiation-induced bystander effect (RIBE) is controversial at high dose levels. The aim of the present study is to assess RIBE at high dose levels by examination of different endpoints. This experimental study used the medium transfer technique to induce RIBE. The cells were divided into two main groups: QU-DB cells which received medium from autologous irradiated cells and MRC5 cells which received medium from irradiated QU-DB cells. Colony, MTT, and micronucleus assays were performed to quantify bystander responses. The medium was diluted and transferred to bystander cells to investigate whether medium dilution could revive the RIBE response that disappeared at a high dose. The RIBE level in QU-DB bystander cells increased in the dose range of 0.5 to 4 Gy, but decreased at 6 and 8 Gy. The Micronucleated cells per 1000 binucleated cells (MNBN) frequency of QU-DB bystander cells which received the most diluted medium from 6 and 8 Gy QU-DB irradiated cells reached the maximum level compared to the MNBN frequency of the cells that received complete medium (P<0.0001). MNBN frequency of MRC5 cells which received the most diluted medium from 4 Gy QU-DB irradiated cells reached the maximum level compared to MNBN frequency of cells that received complete medium (P<0.0001). Our results showed that RIBE levels decreased at doses above 4 Gy; however, RIBE increased when diluted conditioned medium was transferred to bystander cells. This finding confirmed that a negative feedback mechanism was responsible for the decrease in RIBE response at high doses. Decrease of RIBE at high doses might be used to predict that in radiosurgery, brachytherapy and grid therapy, in which high dose per fraction is applied, normal tissue damage owing to RIBE may decrease.

High dose radiotherapy can be preserved normal tissues from bystander effects of irradiated tumors

Introduction Radiation induced bystander effect (RIBE) is dependent to several parameters including total dose, dose rate, radiation quality, tissue type and volume of tissue irradiated in radiotherapy. Purpose The present study was designed to assess uncertainties in high dose RIBE. Materials and methods In this study, we used QU-DB and MRC5 cell lines. One hour following irradiation the medium transfer technique was used to induce bystander effect. Three end point was used in this study are MTT assay (to evaluate percentage of survival), colony assay (to evaluate survival fraction) and MN assay (for assessing chromosome aberrations). Results RIBE in both MN and survival fractions was decreased in doses higher than 4 Gy in lung tumoral cell line. After these two tests, we ensured that bystander effect has actually decreased at doses above 4 Gy. In other words high doses of ionizing radiation can produce a “negative feedback”. We did the dilution assay to evaluate for finding the main reason of RIBE reduction. In this test, by reducing the dilution of transferred media, the bystander effect (MN formation) was recreated. Conclusions This observation verified the negative feedback theory and reject activation of repair systems by more biological signals induced in target cells (as another theory). According to our results and because the dose range we used in the present study are as same as doses used in high dose radiotherapy techniques (e.g. Radiosurgery, brachytherapy etc.), we concluded it is promising that normal tissues can be preserved during high dose radiotherapy from dead signals generated by irradiated tumors. Disclosure There is not any relationship that might lead to a conflict of interest. Mashhad University of Medical Sciences (MUMS) has financially supported this work.

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells.

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

MRC5 and QU-DB bystander cells can produce bystander factors and induce radiation bystander effect.

Radiation damages initiated by radiation-induced bystander effect (RIBE) are not limited to the first or immediate neighbors of the irradiated cells, but the effects have been observed in the cells far from the irradiation site. It has been postulated that bystander cells, by producing bystander factors, are actively involved in the propagation of bystander effect in the regions beyond the initial irradiated site. Current study was planned to test the hypothesis. MRC5 and QU-DB cell lines were irradiated, and successive medium transfer technique was performed to induce bystander effects in two bystander cell groups. Conditioned medium extracted from the target cells was transferred to the bystander cells (first bystander cells). After one hour, conditioned medium was substituted by fresh medium. Two hours later, the fresh medium was transferred to a second group of non-irradiated cells (second bystander cells). Micronucleated cells (MC) were counted to quantify damages induced in the first and second bystander cell groups. Radiation effect was observed in the second bystander cells as well as in the first ones. Statistical analyses revealed that the number of MC in second bystander subgroups was significantly more than the corresponding value observed in control groups, but in most cases it was equal to the number of MC observed in the first bystander cells. MRC5 and QU-DB bystander cells can produce and release bystander signals in the culture medium and affect non-irradiated cells. Therefore, they may contribute to the RIBE propagation.

Radiation dose and time after exposure mediate two types of bystander expression in a single cell line

AbstractPurpose: Cells receiving ionising radiation influence the survival of non‐irradiated neighbouring cells through cellular communication (Bystander effects). The aim of this study is to identify conditions for bystander expressions at different doses and times after exposure to ionising radiation of therapeutic energies, using a medium transfer technique. Methods: Donor flasks plated with non‐small lung cancer cells, NCI‐H460, were exposed to 0 (control), 1, 4, 8 Gy using a 6 MV photon beam. Donor flasks were incubated for designated time periods, then the irradiated conditioned culture medium (ICM) was transferred to non‐irradiated receiver cells. Receiver cells were incubated for 5–7 days to form colonies, which were then counted. The data were reported in the form of survival fraction (SF). Results: Proliferative bystander effects, corresponding to bystander effect Type II, were observed at 1 Gy and 8 Gy. At 8 Gy and 1 hour following irradiation a 21% increase in survival fraction was observed. At 4 Gy the ICM has a cytotoxic effect on the receiver cells, corresponding to bystander effect Type I, exhibiting a maximum response at 15 minutes after irradiation. Conclusion: Two types of bystander expression were established in a single cell line. The results provide in vitro evidence to suggest that when lethal doses of radiation are administered to a tumour volume, peripheral non‐irradiated regions that contain subclinical disease may show a proliferative response.

Evaluation of the cytotoxicity and the genotoxicity induced by α radiation in an A549 cell line

Exposure to radon and its progenies represents one of the greatest risks of ionizing radiation from natural sources. Nowadays, these risks are assessed by the extrapolation of biological effects observed from epidemiological data. In the present study, we made a dose response curve, to evaluate the in vitro response of A549 human lung cells to a-radiation resulting from the decay of a 210 Po source, evaluated by the cytokinesis blocked micronuclei assay. The clonogenic assay was used to measure the survival cell fraction. As expected, the results revealed an increase of cellular damage with increased doses made evident from the increased number of micronuclei (MN) per binucleated cell (BN). Besides this study involving the biological effects induced by direct irradiation, and due to the fact that radiation-induced genomic instability is thought to be an early event in radiation carcinogenesis, we analyzed the genomic instability in early and delayed untargeted effects, by using the medium transfer technique. The obtained results show that unirradiated cells exposed to irradiated medium reveal a higher cellular damage in earlier effects when compared to the delayed effects. The obtained results may provide clues for the biodosimetric determination of radon dose to airway cells at cumulative exposures.

Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect

Purpose: To clarify the experimental conditions that might influence the release of cytokines in the culture medium and give some basic input for building a model for cytokine (e.g., Interleukin-6, IL-6) regulation in the case of ‘sham irradiation’ and after ionising radiation exposure.Materials and methods: The influence of cell type, cell density, medium volume, medium storage temperature and other methodological aspects on IL-6 and Interleukin-8 (IL-8) release were investigated. In addition, the effects over the time of different doses of gamma irradiation on the clonogenic survival of bystander cells and on the secretion of these cytokines were studied.Results: We observed significant decreases of clonogenic survival in AG01522 and T98G cells after the transfer of medium collected 5 and 20 h after low doses of gamma irradiation. Concerning the Interleukins' measurements, our experiments showed that the aggregate removal modalities tested, and up to 10 freeze-thaw cycles, do not have significant influence on the measurements of IL-6 concentration in the medium. We also observed that the IL-6 accumulated in the medium of human fibroblasts is not degraded when maintained at 37°C. Sets of experiments demonstrated that cell density or medium volume do not influence the release of IL-6. On the contrary, our results showed that IL-8 released by glioblastoma cells strongly depends on the amount of medium. Finally, the exposure of fibroblasts to gamma irradiation has influence on the release kinetics of both IL-6 and IL-8 with peculiar features.Conclusions: This study solved some of the methodological doubts concerning the study of bystander effects by means of the medium transfer technique; moreover it also highlighted some experimental aspects that need to be considered when approaching this sort of experiments.

Lack of Bystander Effects from High-LET Radiation for Early Cytogenetic End Points

The aim of this work was to study radiation-induced bystander effects for early cytogenetic end points in various cell lines using the medium transfer technique after exposure to high- and low-LET radiation. Cells were exposed to 20 MeV/ nucleon nitrogen ions, 968 MeV/nucleon iron ions, or 575 MeV/nucleon iron ions followed by transfer of the conditioned medium from the irradiated cells to unirradiated test cells. The effects studied included DNA double-strand break induction, gamma-H2AX focus formation, induction of chromatid breaks in prematurely condensed chromosomes, and micronucleus formation using DNA repair-proficient and -deficient hamster and human cell lines (xrs6, V79, SW48, MO59K and MO59J). Cell survival was also measured in SW48 bystander cells using X rays. Although it was occasionally possible to detect an increase in chromatid break levels using nitrogen ions and to see a higher number of gamma-H2AX foci using nitrogen and iron ions in xrs6 bystander cells in single experiments, the results were not reproducible. After we pooled all the data, we could not verify a significant bystander effect for any of these end points. Also, we did not detect a significant bystander effect for DSB induction or micronucleus formation in these cell lines or for clonogenic survival in SW48 cells. The data suggest that DNA damage and cytogenetic changes are not induced in bystander cells. In contrast, data in the literature show pronounced bystander effects in a variety of cell lines, including clonogenic survival in SW48 cells and induction of chromatid breaks and micronuclei in hamster cells. To reconcile these conflicting data, it is possible that the epigenetic status of the specific cell line or the precise culture conditions and medium supplements, such as serum, may be critical for inducing bystander effects.

Effects of low and high LET radiations on bystander human lung fibroblast cell survival

Purpose: This investigation is aimed to determine the role of low LET (linear energy transfer, γ-rays) and high LET (α-particles) radiations on bystander effect of using the same type of cells and its implications on colony-forming efficiency from a single cell.Materials and methods: Normal human fetal lung (MRC-5), immortalized repair deficient ataxia telangiectasia mutated (ATM) (GM5849C) and normal (GM637H) fibroblast cells were used. Colony-forming efficiency in bystander cells (GM637H) was studied using the medium transfer technique from the two donor (MRC-5 and GM5849C) cells and the procedure followed for bystander treatment is presented schematically in . Evidence of change in colony formation in bystander cells, was assessed by scavenging nitric oxide (NO).Figure 1. Schematic representation of the technical procedure followed for the sham-treated control and bystander cells.Results: Enhancement of 10 – 30% in colony-forming efficiency was observed in bystander GM637H cells treated with irradiated conditioned medium (ICM) from MRC-5 cells collected 1 h after different doses of either γ-rays (1, 2.5, 5 and 10 Gy) or α particles (0.25, 0.5, 1 and 2.5 Gy) irradiation. Similar results were obtained when ICM derived from the ATM (GM5849C) cells. However, the stimulation was not dose dependent. Furthermore, we also show that the increase in dilutions of ICM (1:1, 1:5 and 1:10) showed an inverse correlation with cloning efficiency. Treatment of MRC-5 cells with PTIO (2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide) a NO scavenger, 1 h prior to irradiation reduced the enhancement of ICM mediated cell survival.Conclusions: In the present study, though both the low and high LET radiations enhanced the clonogenic potential of the bystander recipient cells, medium from the ATM defective (GM5849C) cells after γ-irradiation showed less stimulating effect than the medium from the normal (MRC-5) cells. However, after α-irradiation an inverse effect was seen. NO may play an important role in enhancing the growth potential in these bystander cells.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

Induction of spontaneous and UV-induced mutations during commitment to meiosis in Saccharomyces cerevisiae

Inductions of reversions of nonsense, missense and frameshift-type mutations were investigated in a diploid cell population of Saccharomyces cerevisiae during commitment to meiosis, by using the medium-transfer technique from sporulation medium to vegetative medium. The yields of spontaneous reverse mutations obtained from the cells that were committed to different stages during meiosis were rather constant irrespective of the alleles tested, although the yields of both intergenic and intragenic recombinations markedly increased. The susceptibilities to UV-induced reverse mutations examined during commitment to meiosis were not changed appreciably. It is concluded that induction of base-change-type mutations in meiosis is not essentially different from that in mitosis.