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Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

ABSTRACT: Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation.

SUBMITTER: Warters RL

PROVIDER: S-EPMC2734195 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

Warters Raymond L RL Packard Ann T AT Kramer Gwen F GF Gaffney David K DK Moos Philip J PJ

Radiation research 20090701 1

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered ...[more]

PMID: 19580510

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Project description:BACKGROUND:Exposure to ionizing radiation induces complex stress responses in cells, which can lead to adverse health effects such as cancer. Although a variety of studies investigated gene expression and affected pathways in human fibroblasts after exposure to ionizing radiation, the understanding of underlying mechanisms and biological effects is still incomplete due to different experimental settings and small sample sizes. Therefore, this study aims to identify the time point with the highest number of differentially expressed genes and corresponding pathways in primary human fibroblasts after irradiation at two preselected time points. METHODS:Fibroblasts from skin biopsies of 15 cell donors were exposed to a high (2Gy) and a low (0.05Gy) dose of X-rays. RNA was extracted and sequenced 2?h and 4?h after exposure. Differentially expressed genes with an adjusted p-value <?0.05 were flagged and used for pathway analyses including prediction of upstream and downstream effects. Principal component analyses were used to examine the effect of two different sequencing runs on quality metrics and variation in expression and alignment and for explorative analysis of the radiation dose and time point of analysis. RESULTS:More genes were differentially expressed 4?h after exposure to low and high doses of radiation than after 2?h. In experiments with high dose irradiation and RNA sequencing after 4?h, inactivation of the FAT10 cancer signaling pathway and activation of gluconeogenesis I, glycolysis I, and prostanoid biosynthesis was observed taking p-value (<?0.05) and (in) activating z-score (?2.00 or???-?2.00) into account. Two hours after high dose irradiation, inactivation of small cell lung cancer signaling was observed. For low dose irradiation experiments, we did not detect any significant (p?<?0.05 and z-score???2.00 or???-?2.00) activated or inactivated pathways for both time points. CONCLUSIONS:Compared to 2?h after irradiation, a higher number of differentially expressed genes were found 4?h after exposure to low and high dose ionizing radiation. Differences in gene expression were related to signal transduction pathways of the DNA damage response after 2?h and to metabolic pathways, that might implicate cellular senescence, after 4?h. The time point 4?h will be used to conduct further irradiation experiments in a larger sample.

Project description:Cancer-associated fibroblasts (CAFs) are acknowledged key determinants in the progression of cancer and thereby represent important targets for cancer therapies. Given the increase attention that ablative radiotherapy is gaining in the clinics, in this study we have aimed at identifying the transcriptional responses occurring in primary CAFs exposed to high-dose irradiation. Established primary cancer-associated fibroblasts (CAFs) obtained from non-small-cell lung cancer (NSCLC) patient material were irradiated with a single dose of 18 Gy and total RNA was isolated 24 hrs after treatment. Radiation-induced transcriptional alterations were investigated by gene expression analysis using genome-wide microarrays. Obtained results were verified by qRT-PCR of relevant genes. To confirm the data achieved by microarrays, diverse functional assays were performed including DNA damage response foci assay, measurements of reactive oxygen species (ROS) by flow cytometry and senescence-associated beta-galactosidase assays were applied. Irradiation resulted in differential expression of 680 genes of which 553 were up- and 127 down-regulated. 153 genes were differentially expressed with a fold-change greater than 1 and an adjusted p-value less than 0.05 across different comparisons (non-irradiated vs. irradiated). Expression patterns revealed profound changes in biological functions and processes involved in DNA repair, apoptosis, p53 pathway, autophagy, senescence, ROS production and immune response. Conclusions: CAFs display pro- and anti-tumorigenic effects after having received a single high-dose radiation. The effects might have an impact on the tumor microenvironment in respect to tumor growth and metastasis. The complexity of the observed differential gene expression patterns and implicative biological effects are interesting future objectives for further elucidation. Established primary cancer-associated fibroblasts (CAFs) obtained from non-small-cell lung cancer (NSCLC) patient material were irradiated with a single dose of 18 Gy and total RNA was isolated 24 hrs after treatment.

Project description:Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Project description:Fibroblasts (Fbs) effectively promote Schwann cells (SCs) migration, proliferation, and neurite regeneration. Whether Fbs express different motor and sensory phenotypes that regulate the cell behavior and peripheral nerve function has not been elucidated. The present study utilized the whole rat genome microarray analysis and identified a total of 121 differentially expressed genes between the primary cultured motor and sensory Fbs. The genes with high expression in sensory Fbs were related to proliferation, migration, chemotaxis, motility activation, protein maturation, defense response, immune system, taxis, and regionalization, while those with high expression in motor Fbs were related to neuron differentiation, segmentation, and pattern specification. Thus, the significant difference in the expression of some key genes was found to be associated with cell migration and proliferation, which was further validated by quantitative real-time PCR (qPCR). The cell proliferation or migration analysis revealed a higher rate of cell migration and proliferation of sensory Fbs than motor Fbs. Moreover, the downregulated expression of chemokine (C-X-C motif) ligand 10 (CXCL10) and chemokine (C-X-C motif) ligand 3 (CXCL3) suppressed the proliferation rate of sensory Fbs, while it enhanced that of the motor Fbs. However, the migration rate of both Fbs was suppressed by the downregulated expression of CXCL10 or CXCL3. Furthermore, a higher proportion of motor or sensory SCs migrated toward their respective (motor or sensory) Fbs; however, few motor or sensory SCs co-cultured with the other type of Fbs (sensory or motor, respectively), migrated toward the Fbs. The current findings indicated that Fbs expressed the distinct motor and sensory phenotypes involved in different patterns of gene expression, biological processes, and effects on SCs. Thus, this study would provide insights into the biological differences between motor and sensory Fbs, including the role in peripheral nerve regeneration.

Dataset Information

Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

Publications

Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

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