ABSTRACT: Genome-wide microarray analysis of immortalized human fibroblasts in response to alpha-particle radiation and the radiation induced bystander effect
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from unirradiated cells to the irradiation of their neighbors. The bystander effect can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in noncancerous human cell lines. In this study we have used a genome-wide microarray approach to investigate transcriptional responses in irradiated and bystander immortalized human fibroblasts following 0.1 Gy α-particle irradiation.
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from unirradiated cells to the irradiation of their neighbors. The bystander effect can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in noncancerous human cell lines. In this study we have used a genome-wide microarray approach to investigate transcriptional responses in irradiated and bystander immortalized human fibroblasts following 0.1 Gy ?-particle irradiation. Total RNA was isolated from F11hTERT fibroblasts irradiated with 0.1 Gy ?-particles and bystander fibroblasts receiving medium from control (sham irradiated) and irradiated cells (0.1 Gy). RNA was isolated 4, 8 and 26 h after irradiation.
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from non-irradiated cells to the irradiation of their neighbors. The bystander effect is predominant at low doses and can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in normal human cell lines. In this study, we have monitored transcriptional responses to γ-radiation in irradiated and bystander normal fibroblasts simultaneously using a genome-wide microarray approach. Bystander fibroblasts incubated in medium from irradiated cells, showed transient enrichment (less than 1.5 fold) in ribosome and oxidative phosphorylation pathways, and neurodegenerative disease pathways associated with mitochondrial dysfunctions. Bystander fibroblasts did not, however, display increases in oxidative stress, a phenomenon often linked with the radiation induced bystander effect. Total RNA was isolated from normal human fibroblasts irradiated with 2.0 Gy and fibroblasts incubated with medium from sham irradiated and irradiated cells 2 h after irradiation. RNA was isolated 4, 8 and 26 h after irradiation and there are 4 replicates for each sample for a total of 36 samples.
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from non-irradiated cells to the irradiation of their neighbors. The bystander effect can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in normal human cell lines. In this study, we have monitored transcriptional responses to γ-radiation in irradiated and bystander normal fibroblasts simultaneously using a genome-wide microarray approach. In this study we have investigated the transcriptional response in F11hTERT fibroblasts irradiated with 2 Gy, 2 hours after irradiation.
Project description:To investigate the mechanism of radiation induced bystander effect, we explored miRNAs expression in supernatant of human skin fibroblasts after culturing for 24h post UV irradiation. Primary human skin fibroblasts were obtained from healthy volunteers by means of a foreskin circumcision. Human skin fibroblasts was irradiated with 20J/cm2 UVA or 60mJ/cm2 UVB. Expression of miRNAs were tested by microarray between radiation and control samples.
Project description:The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. It raises concerns for the interpretation of risks from exposure to low doses of ionizing radiation. Sparse data exists about the bystander signaling mechanisms and the ability to transmit damaging effects both spatially and temporally. To understand early signaling and cellular changes in bystanders, we have measured global gene expression 30 minutes after direct and bystander exposure to alpha particle in primary human lung fibroblasts. Gene ontology and pathway analyses suggested that the earliest measured changes at 30 minutes after treatment are in cell structure, motility and adhesion categories and a significant number of genes belong to the category of inflammation and cell-to-cell communication. We investigated time course gene expression profiles of matrix metalloproteinases 1 and 3 (MMP1 and MMP3), chemokine ligands 2, 3 and 5 (CXCL2, CXCL3 and CXCL5), interleukins 1a, 1b, 6 and 33 (IL1A, IL1B, IL6 and IL33) growth differentiation factor 15 (GDF15) and superoxide dismutase2 (SOD2) by real time quantitative PCR. These encode proteins involved in cellular signaling via the NFkappaB pathway and time course of mRNA levels revealed an increased response at 30 minutes after irradiation followed by another wave at 4 to 6 hours. We also investigated protein modifications in the AKT-GSK-3 signaling pathway and found that in irradiated cells AKT and GSK3beta are hyper-phosphorylated at 30 minutes and this effect is maintained until 4 hours after exposure. In bystanders there is a similar response with a delay of 30 minutes. In irradiated cells, inactivated GSK3beta led to decreased phosphorylation of beta-catenin. Our results are the first to show that the radiation induced bystander signal can induce a widespread gene expression response as early as 30 minutes after exposure and that these changes are accompanied by protein modification of signaling modules such as AKT and GSK3beta. There are 12 total samples, 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C), irradiated (alpha=A) and bystander (B), cells were harvested 0.5 hr after treatment
Project description:Background: The radiation bystander response is an important component of the overall response of cells to radiation and critical to understanding health risks of radiation exposure to humans. The mechanism of radiation response includes inter-cellular signaling and intra-cellular communication by which the bystander signal is propagated. Methods: We measured the bystander response to 1Gy a-particle radiation in Mrad9-/- mouse stem cells and H1299shRAD9 cells, using chromosomal aberration and micronucleus formation as DNA damage endpoints. In the H1299 model we used whole genome microarray analyses to profile the transcriptome of irradiated and bystander cells. Results: We investigated the role of RAD9 in the bystander response and showed that depletion or mutation of RAD9 had an effect of increasing chromosomal structural damage as well as micronucleus formation in bystander cells. The enhancement of the damage effect correlated strongly with a transcriptomic response in critical pathways. RAD9 depletion affected many pathways in the cell, including the UV-MAPK pathway, involving p38MAPK members, STAT1 and PARP1 at the mRNA levels. There was an overall reduction of RNA biogenesis of gene members of this pathway suggesting that perhaps these signaling pathways do not function optimally after RAD9 depletion. Using network analysis we found there may be differential activation of transcriptional regulators between the irradiated and bystander cells involving the SP1 and NUPR1 transcription factors. Network analysis also suggested that HIF1a (Hypoxia induced factor 1a) activation could be a negative predictor of the bystander effect and perhaps that local hypoxic stress observed by cells that are directly exposed to radiation may predict whether or not they will elicit a bystander response. Gene expression in H1299 cells was measured at 4 hours after exposure to 1 Gy a-particles. There were two groups based on RAD9 status, RAD9 normal and RAD9 depleted by siRNA. In each of these groups, sham irradiated, direct irradiated cells for positive bystanders, positive bystanders, direct irradiated cells for negative bystanders and negative bystanders; were identified based on micronucleus responses. Five biological replicates were analyzed for each experimental group.
Project description:We have recently investigated the response of irradiated and bystander fibroblasts to heavy ions in confluent cultures exposed randomly to broadbeam and selectively to precision microbeam that target a predefined fraction of cells with counted number of particle(s), respectively, and found the difference in the time course of apoptosis induction and p53 phosphorylation between irradiated and bystander cells [Hamada MR2008]. In the present investigation, to further scrutinize the underpinnings of their temporally distinct responses, we set out to examine the gene expression changes in irradiated and bystander fibroblasts at a genome-wide level with the microarray analysis under the same experimental condition. We demonstrate that the gene expression profiles of bystander cells are substantially different from those of heavy ion-irradiated cells. Keywords: bystander effect, hour-time course cell number dependency, irradiation-dose response