Profiles of global gene expression in ionizing radiation-damaged human diploid fibroblasts
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE6902 | GEO | 2007/02/01
SECONDARY ACCESSION(S): PRJNA98149
REPOSITORIES: GEO
Similar Datasets
Project description:Cell cycle arrest and transcriptional responses to ionizing radiation (IR)-induced DNA damage were quantified in telomerase-expressing human diploid fibroblasts. Assays of clonal expansion established 1.5 Gy IR as the D0 dose in three fibroblast lines and this dose was used in all subsequent analyses. Fibroblasts exhibited >90% arrest of progression from G2 to M at 2 h and from G1 to S at 6 and 12 h post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 h after irradiation caused the S and M compartments to empty by over 70% at 24 h. Microarray monitored global gene expression in IR-treated cells and a new microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, like CDKN1A, GADD45, BTG2 and PLK3, were significantly up-regulated at 2 h post-IR while many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 h post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile of gene expression 24 h after IR with similarity to that of G0 growth quiescence. These results demonstrate a highly stereotypic pattern of response to IR that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. Experiment Overall Design: Logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâs Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScan® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-15 | E-GEOD-6902 | biostudies-arrayexpress
Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Experiment Overall Design: Normal human fibroblasts were synchronized and harvested at G0, G1, S G2 and M cell cycle phase, and logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâ??s Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScan® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-15 | E-GEOD-7075 | biostudies-arrayexpress
Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Keywords: Microarray, Cell cycle, Ionizing radiation, Human fibroblasts, EPIG
2007-08-01 | GSE7075 | GEO
Project description:The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. The observation of attenuated and not ablated checkpoint responses in AT cells supports a hypothesis that the ATM- and rad3-related checkpoint kinase ATR also responds to IR-induced DNA damage and complements ATM signaling. Keywords: ATM, cell cycle checkpoint, ionizing radiation, microarray
2007-07-01 | GSE6971 | GEO
Project description:Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wildtype N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. Experiment Overall Design: Normal human melanocyte and melanoma cell lines w/wo mutations in B-raf or N-ras were treated with 1.5 Gy IR irridiartion for G1 and G2 checkpoint determination. RNA was isolated from exponentially growing cultures and applied for microarray hybridizaton with Agilent 44 K (G4112A) array.
2008-06-15 | E-GEOD-7469 | biostudies-arrayexpress
Project description:The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. The observation of attenuated and not ablated checkpoint responses in AT cells supports a hypothesis that the ATM- and rad3-related checkpoint kinase ATR also responds to IR-induced DNA damage and complements ATM signaling. Experiment Overall Design: Logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâs Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScan® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-15 | E-GEOD-6971 | biostudies-arrayexpress
Project description:Heldt2018 - Proliferation-quiescence decision
in response to DNA damage
This model is described in the article:
A comprehensive model for
the proliferation-quiescence decision in response to endogenous
DNA damage in human cells.
Heldt FS, Barr AR, Cooper S, Bakal
C, Novák B.
Proc. Natl. Acad. Sci. U.S.A. 2018 Feb;
:
Abstract:
Human cells that suffer mild DNA damage can enter a
reversible state of growth arrest known as quiescence. This
decision to temporarily exit the cell cycle is essential to
prevent the propagation of mutations, and most cancer cells
harbor defects in the underlying control system. Here we
present a mechanistic mathematical model to study the
proliferation-quiescence decision in nontransformed human
cells. We show that two bistable switches, the restriction
point (RP) and the G1/S transition, mediate this decision by
integrating DNA damage and mitogen signals. In particular, our
data suggest that the cyclin-dependent kinase inhibitor p21
(Cip1/Waf1), which is expressed in response to DNA damage,
promotes quiescence by blocking positive feedback loops that
facilitate G1 progression downstream of serum stimulation.
Intriguingly, cells exploit bistability in the RP to convert
graded p21 and mitogen signals into an all-or-nothing
cell-cycle response. The same mechanism creates a window of
opportunity where G1 cells that have passed the RP can revert
to quiescence if exposed to DNA damage. We present experimental
evidence that cells gradually lose this ability to revert to
quiescence as they progress through G1 and that the onset of
rapid p21 degradation at the G1/S transition prevents this
response altogether, insulating S phase from mild, endogenous
DNA damage. Thus, two bistable switches conspire in the early
cell cycle to provide both sensitivity and robustness to
external stimuli.
This model is hosted on
BioModels Database
and identified by:
MODEL1703030000.
To cite BioModels Database, please use:
Chelliah V et al. BioModels: ten-year
anniversary. Nucl. Acids Res. 2015, 43(Database
issue):D542-8.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
2017-03-01 | BIOMD0000000700 | BioModels
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186086 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186085 | GEO
Project description:Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. We performed BrdU IP-Seq to examine the DNA replication profile genome-wide. These data revealed that the initiation of replication was delayed and fewer origins were active when G0 cells entered the cell cycle compared to the entry of G1 cells into S phase. We found that both the transcript and protein levels of these replication factors were significantly diminished during the development of proliferating cells into quiescence. The levels of these factors, including MCM2-7 helicase, increased as G0 cells re-entered the cell cycle, suggesting that the replication program is re-established de novo. Consistent with these results, Mcm4 ChIP-seq analysis showed fewer and reduced binding at a number of origins during the re-entry of G0 cells into the cell cycle. In support of this evidence, the chromatin context surrounding inactive origins of G0 cells exhibits greater increased nucleosome occupancy and reduced periodicity of nucleosome positioning. Altogether, these observations provide insights into the important role of chromatin context that determines the ability of replication origin to accrue limiting replication factors during the the re-entry of quiescent cells into the cell cycle.
2020-08-07 | GSE155831 | GEO