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Transcriptional responses in B-cell lymphomas by the alpha-emitting radioimmunoconjugate Bi-213-CD20-rituximab

ABSTRACT: The possibility to deliver radioisotopes directly to tumor cells by using monoclonal antibodies has become a promising concept for the elimination of small tumor nodules or single disseminated tumor cells. The alpha-particle emitter Bismut-213 which has a high linear energy transfer and a very short path length appears to be able to kill cells by only few nuclear hits. Bismut-213 based alpha-immunotherapy treatment is currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma. Because little is known about the biological effects of alpha radiation we examined the molecular effects of the alpha emitter Bismut-213 in malignant and normal B-cells. In this study, we used the cell line Karpas 422 (K422) which had been derived from a malignant B-cell Non-Hodgkin’s lymphoma as well as normal primary CD19+ B-cells which had been selected immunomagnetically from peripheral blood. For the delivery and binding of the alpha emitter we used the monoclonal antibody rituximab which targets the CD20 receptor of malignant and normal B-cells. The lymphoma cell line K422 was incubated in vitro with Bismut-213-CD20 with 100 µCi and 200 µCi respectively, for 46 min (1 half life time of the alpha emitter) or 24 h. Immunomagnetically isolated CD19+ cells from healthy donors were incubated in vitro with Bismut-213-CD20 with 100 µCi or 200 µCi, for 46 min. Gene expression profiles of irradiated cells were analysed using cDNA oligonucleotide arrays (Affymetrix Human Genome Focus Arrays) comprising 8,793 genes and compared with those from untreated control cells. Following normalization significantly altered genes were identified by using the variance stabilization normalization (VSN) method and the significance analysis of microarrays (SAM) algorithm. Differentially expressed genes were defined to exhibit a fold change of 1,4 and higher or 0,7 and lower and a q-value of 5% or lower compared to the genes of the untreated control cells. Irradiated K422 cells with 100 µCi showed 42 differential expressed genes after 46 minutes (one half life time) and 451 deregulated genes after exposure of 24 hours. A similar response could be observed at an exposure of 200 µCi (50 differentially expressed genes after 46 min; 485 genes after 24 h). Hence, a severe radiation induced response could not be detected before 24 hours. 200 µCi irradiated CD19+ cells showed 67 differentially expressed genes after 46 minutes compared to the untreated CD19+ cells (for 100 µCi: 42 differentially expressed genes). By performing cluster analyses the irradiated cells exhibited a distinct homogenous molecular phenotype in comparison to not irradiated cells. Alpha irradiated cells showed transcriptional activation of DNA repair genes. Furthermore, both examined cell types showed up-regulated genes which are known to play a role in general and oxidative stress response. Surprisingly, up-regulation of interferon-stimulated genes in K422 cells could be observed. Because of the transcriptional activation of genes regulating the G1-S- cell cycle transition the supposition came up that the irradiated K422 do progress into the S-phase of the cell cycle and arrest in the S- or G2/M-phase. In contrast to the K422 cells the primary CD19+ B-cells revealed transcriptional activation of cell cycle inhibitors which is an already known reaction following ionizing radiation but no apoptosis associated genes could be observed. In spite of the massive cytotoxic damages which are assumed referring to the gene expression data the K422 cells appear to be able to activate several defense mechanisms in a precise manner. Hence, the malignant K422 cells exhibit a larger amount as well as more various responses than the CD19+ cells. This might be due to the tumorigenic transformation of the malignant cells that exhibit probably more effective defense strategies than the CD19+ cells. The balance between the therapeutic effect of alpha radiation on malignant cells and the damaging effect on normal cells is an essential issue and it is of important clinical relevance concerning radioimmunotherapy with alpha radiation. Keywords: time course and dose dependency

ORGANISM(S): Homo sapiens

PROVIDER: GSE7291 | GEO | 2007/07/01

SECONDARY ACCESSION(S): PRJNA104065

REPOSITORIES: GEO

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Project description:The possibility to deliver radioisotopes directly to tumor cells by using monoclonal antibodies has become a promising concept for the elimination of small tumor nodules or single disseminated tumor cells. The alpha-particle emitter Bismut-213 which has a high linear energy transfer and a very short path length appears to be able to kill cells by only few nuclear hits. Bismut-213 based alpha-immunotherapy treatment is currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma. Because little is known about the biological effects of alpha radiation we examined the molecular effects of the alpha emitter Bismut-213 in malignant and normal B-cells. In this study, we used the cell line Karpas 422 (K422) which had been derived from a malignant B-cell Non-Hodgkin’s lymphoma as well as normal primary CD19+ B-cells which had been selected immunomagnetically from peripheral blood. For the delivery and binding of the alpha emitter we used the monoclonal antibody rituximab which targets the CD20 receptor of malignant and normal B-cells. The lymphoma cell line K422 was incubated in vitro with Bismut-213-CD20 with 100 µCi and 200 µCi respectively, for 46 min (1 half life time of the alpha emitter) or 24 h. Immunomagnetically isolated CD19+ cells from healthy donors were incubated in vitro with Bismut-213-CD20 with 100 µCi or 200 µCi, for 46 min. Gene expression profiles of irradiated cells were analysed using cDNA oligonucleotide arrays (Affymetrix Human Genome Focus Arrays) comprising 8,793 genes and compared with those from untreated control cells. Following normalization significantly altered genes were identified by using the variance stabilization normalization (VSN) method and the significance analysis of microarrays (SAM) algorithm. Differentially expressed genes were defined to exhibit a fold change of 1,4 and higher or 0,7 and lower and a q-value of 5% or lower compared to the genes of the untreated control cells. Irradiated K422 cells with 100 µCi showed 42 differential expressed genes after 46 minutes (one half life time) and 451 deregulated genes after exposure of 24 hours. A similar response could be observed at an exposure of 200 µCi (50 differentially expressed genes after 46 min; 485 genes after 24 h). Hence, a severe radiation induced response could not be detected before 24 hours. 200 µCi irradiated CD19+ cells showed 67 differentially expressed genes after 46 minutes compared to the untreated CD19+ cells (for 100 µCi: 42 differentially expressed genes). By performing cluster analyses the irradiated cells exhibited a distinct homogenous molecular phenotype in comparison to not irradiated cells. Alpha irradiated cells showed transcriptional activation of DNA repair genes. Furthermore, both examined cell types showed up-regulated genes which are known to play a role in general and oxidative stress response. Surprisingly, up-regulation of interferon-stimulated genes in K422 cells could be observed. Because of the transcriptional activation of genes regulating the G1-S- cell cycle transition the supposition came up that the irradiated K422 do progress into the S-phase of the cell cycle and arrest in the S- or G2/M-phase. In contrast to the K422 cells the primary CD19+ B-cells revealed transcriptional activation of cell cycle inhibitors which is an already known reaction following ionizing radiation but no apoptosis associated genes could be observed. In spite of the massive cytotoxic damages which are assumed referring to the gene expression data the K422 cells appear to be able to activate several defense mechanisms in a precise manner. Hence, the malignant K422 cells exhibit a larger amount as well as more various responses than the CD19+ cells. This might be due to the tumorigenic transformation of the malignant cells that exhibit probably more effective defense strategies than the CD19+ cells. The balance between the therapeutic effect of alpha radiation on malignant cells and the damaging effect on normal cells is an essential issue and it is of important clinical relevance concerning radioimmunotherapy with alpha radiation. Keywords: time course and dose dependency

Project description:Alpha (α)-radiation is a ubiquitous environmental agent with epigenotoxic effects. Human exposure to α-radiation at potentially harmful levels can occur repetitively over the long term via inhalation of naturally occurring radon gas that accumulates in enclosed spaces, or as a result of a single exposure from a nuclear accident. Alterations in epigenetic DNA methylation (DNAm) have been implicated in normal aging and cancer pathogenesis. Nevertheless, the effects of aberrations in the methylome of human lung cells following exposure to single or multiple α-radiation events on these processes remain unexplored. Here, we performed genome-wide DNAm profiling of human embryonic lung fibroblasts from control and irradiated cells using americium-241 α-sources. Cells were α-irradiated in quadruplicates to seven doses using two exposure regimens, a single-fraction (SF) where the total dose was given at once, and a multi-fraction (MF) method, where the total dose was equally distributed over 14 consecutive days. Our results revealed that SF irradiations were prone to a decrease in methylation levels, while MF irradiations mostly increased methylation. The analysis also showed that the gene body (i.e. exons and introns) was the region mostly altered by both the SF hypomethylation and the MF hypermethylation. Additionally, the MF irradiations induced the highest number of differentially methylated regions in genes associated with DNAm biomarkers of aging, carcinogenesis, and cardiovascular disease. The DNAm profile of the oncogenes and tumour suppressor genes suggested that the fibroblasts elicited a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure including changes in methyl levels in mitochondria dysfunction-related genes were mainly identified in the MF groups. However, these alterations were under-represented, indicating that the mitochondria undergo adaptive mechanisms, aside from methylation, in response to radiation-induced oxidative stress. In this study, we identified a contrasting methylomic profile in the lung fibroblasts α-irradiated to SF compared with MF exposures. These findings demonstrate that the methylome response of the lung cells to α-radiation is highly dependent on both the total dose and the exposure regimen. They also provide novel insights into potential biomarkers of α-radiation which may lead to the development of innovative approaches to detect, prevent and treat α-particle related diseases.

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:Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap, 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10, 30, or 80 cGy of densely ionizing, 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance, grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive, pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18), while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this, K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However, K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells, stroma, and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative, K18-positive tumor, whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts, 9 from 10 cGy Si-irradiated hosts, 10 from 30 cGy Si-irradiated hosts, and 8 from irradiated hosts.

Project description:In plants, an increase in resource allocation to growth (primary metabolism) associated with the presence of neighbors is likely to reduce defense-related production (secondary metabolism), making plants more vulnerable to herbivory. Even though there is increasing evidence supporting this “trade-off hypothesis”, the underlying mechanisms are still unclear. Far red (FR) radiation reflected from plant tissues serves as an early warning signal of future competition, triggering a suite of plastic morphological adjustments that improve plant’s ability to compete for light in crowded populations. Recent evidence from our lab showed that, when competition signals are present, plant defenses are severely reduced. Besides direct effects of herbivory and competition signals on target plants, second order effects occurs on neighboring plants through plant volatiles (PVs) communication. PVs play a key role in plant-plant and plant-insect interactions, changing its content and composition in response to environmental conditions. To increase our understanding of the molecular mechanisms underlying those interacting signaling webs, we performed a field study with tomato plants (cv Moneymaker), in which plants of EMITTER plots (six plants plot-1) were subjected to herbivory (nine larvae of Spodoptera eridania plant-1) and competition signals (increased FR radiation) in a factorial design. Light treatment started 28 days after sowing (DAS), and herbivory treatment and volatiles conduction started 34 DAS. Volatiles were conducted from EMITTER to RECEIVER plots (five plants plot-1) using a 5 inch, 1.4 m long tube fitted with a computer-type fan. 40 and 45 DAS, larval performance was measured on EMITTER plots as well as naturally-occurring insect colonization on RECEIVER plots. Finally (46 DAS), samples for bulk phenolic content were taken on every plot, and plant material from 4th and 5th leaves was collected for microarray analysis. There were three real biological replicates. Keywords: Reference design

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Transcriptional responses in B-cell lymphomas by the alpha-emitting radioimmunoconjugate Bi-213-CD20-rituximab

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