BRD4 Short Isoform Is a Myelodysplasia Prognostic Marker Involved in Inappropriate DNA Damage Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3234-3234
Author(s):  
Fernando V Pericole ◽  
Fernanda Marconi Roversi ◽  
Adriana Silva Santos Duarte ◽  
Bruna Palodetto ◽  
Flavia Adolfo Corrocher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Lower Risk ◽  
De Novo ◽  
Leukemia Cell ◽  
Cd34 Cells ◽  
Damage Response ◽  
De Novo Aml

Abstract INTRODUCTION: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. BRD4 is a BET member of bromodomain-containing proteins, known as epigenome readers, exerting key roles in chromatin remodeling and transcriptional regulation. BRD4 gene encodes two major isoforms, short (BRD4S) and long (BRD4L). Recently, Floyd SR et al (Nature, 2013) described BRD4S as an endogenous inhibitor of the DNA damage response (DDR). Under normal conditions, damaged DNA induces histone phosphorylation of H2AX at Ser 139 (γ-H2AX) and activates a protective signaling network that blocks cell cycle and recruits DNA repair factors. Despite having been described as a therapeutic target in acute myeloid leukemia (AML) by Zuber J et al (Nature, 2011), BRD4 has never been studied in myelodysplastic syndrome (MDS) and its role in the pathogenesis is currently unknown. AIMS: To evaluate BRD4L and BRD4S expression in MDS and AML patients, correlating with clinical data, progression and survival. We also explored the BRD4 role in DDR signaling using human leukemia cell line models. MATERIAL AND METHODS: Diagnostic total bone marrow (BM) samples from 24 healthy donors (HD) and 99 patients, including 48 MDS (22 higher-risk MDS and 36 lower-risk MDS) and 51 AML (16 of them with MDS-related changes, AML-MRC), were collected. We also isolated CD34+ cells from 7 HD, 5 de novo AML, 4 AML-MRC and 14 MDS (6 higher-risk and 8 lower-risk MDS). BRD4L and BRD4S gene expressions were assessed through q-PCR and expressed as median (minimum-maximum). MDS patients were stratified according IPSS, WHO classification, R-IPSS and cytogenetic risk. Samples were age-adjusted when significant differences were observed, using ANOVA and Tukey’s test. Progression-free and overall survival curves were estimated by Kaplan-Meier method and were analyzed by the Wilcoxon´s test and Cox regression. JQ1, a specific BRD4 inhibitor, was kindly provided by James Bradner. A panel of human myeloid leukemia cell lines (KG1a, HEL, HL60, U937) in exponential growth was treated with increasing doses of JQ1 for 48 hours and cell growth (MTT colorimetric assay), apoptosis (annexin-V/PI) and cell cycle (flow cytofluorometric analysis detecting nuclear PI incorporation) were evaluated. We also determined the expression of p-γH2AX (DDR signaling) by western blot, after 12 hours of JQ1 treatment. RESULTS: A higher expression of BRD4S was observed in total BM cells from AML (4.01 [0.33-2.58], P=.01) and MDS patients (4.21 [0.01-56.17], P=.01) compared with HD (2.11 [0.04-10.32], P=.01). When stratified according WHO classification, AML-MRC (4.5 [0.33-25.22], P=.04) and higher-risk MDS (4.66 [0.17-56.17], P=.04) subgroups showed higher BRD4S expression. In CD34+ cells, BRD4S expression was increased in de novo AML (0.28 [0.21-0.45]) compared with lower-risk MDS (0.02 [0.00-0.44], P=.01). BRD4L mRNA expression was not modulated in total BM and CD34+ cells from any subgroup. With median follow-up time of 34.4 months, we found that higher BRD4S gene expression was a worse prognostic factor for MDS transformation and survival, along with IPSS, R-IPSS, low hemoglobin (less than 10g/dL) and higher BM blast percentage. After multivariate analysis, BRD4S gene expression and higher-risk (very high, high and intermediate) R-IPSS remained as independent prognostic factors for MDS progression and overall survival. KG1a and U937 cells showed greater resistance to JQ1, with lower apoptosis rate and proliferation (IC50 not reached, over 3000nM), whereas HEL and HL60 were more responsive (IC50 under 800nM in both cell lines). JQ1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and apoptosis and caused a progressive increase in histone phosphorylation of γ-H2AX, indicating activation of DDR signaling. CONCLUSIONS: MDS is a clonal myeloid neoplasm characterized by profound epigenetic modifications. Our data establishes BRD4S as a novel MDS prognostic factor, related to aggressive phenotype, higher progression rate and shorter survival. Biologically, BRD4S plays a role in the inappropriate DNA Damage Response of MDS, favoring the disease towards genetic instability and clonal evolution. Disclosures No relevant conflicts of interest to declare.

Abrogation of the DNA-Damage Response in De Novo AML Versus MDS Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2649-2649
Author(s):  
Simone Boehrer ◽  
Lionel Ades ◽  
Claire Fabre ◽  
Pierre Fenaux ◽  
Guido Kroemer
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Line ◽  
Cell Lines ◽  
Dna Damage Response ◽  
De Novo ◽  
Apoptotic Cells ◽  
Damage Response ◽  
De Novo Aml ◽  
Gamma Irradiated

Abstract Background: The increasing evidence that activation of the DNA-damage-response (DDR) pathway in preneoplastic lesions of solid neoplasms hinders tumorigenesis prompted us to evaluate the same hypothesis in cell line models representing different subtypes of MDS or AML. Methods: myeloid cell lines corresponding to different subgroups of MDS, having or not progressed to AML (p39, MOLM-13) and de novo AML (MV4-11, and KG-1) were assessed for their capacity to induce a DDR. Using the quintessential inducer of a DDR, the cell lines were gamma-irradiated (2, 5 and 10Gy) and their ability to arrest the cell cycle in G2/M and to undergo apoptosis were assessed. Results: in contrast to the de novo AML-derived KG-1 cells - the irradiated MDS/AML-derived cell lines P39 and MOLM-13 exhibited an earlier and more pronounced arrest of the cell cycle in G2/M, as well as a higher percentage of apoptotic cells. Whereas 10 Gy induced a G2/M-arrest in about 30% of KG-1 cells, this percentage increased to about 60% in P39 and MOLM-13 cells. Comparably, whereas KG-1 cells exhibited about 15% of apoptotic cells following irradiation with 10Gy, the percentage reached 30% in P39 and MOLM-13 cells. Assessing the underlying molecular determinants we demonstrated that irradiated P39 and MOLM-13 cells retained the ability to activate crucial mediators of the DDR, whereas this ability was decreased in the de novo AML cell line KG-1. In particular, in comparison to KG-1 cells, irradiated P39 and MOLM-13 cells exhibited a more pronounced upregulation of the crucial cell cycle/checkpoint regulators Chk1-P-Ser317 and Cdk1-P-Tyr15. Noteworthy, MDS cell lines with an increased G2/M-arrest exhibited a higher expression of nucleophosmin - which serves as a shuttling protein between the nucleus and the cytoplasm - and were able to retain this increased expression upon irradiation. Moreover, elevated expression of NPM in MDS cell lines corresponded to higher PCNA-expression, which serves as an essential component of the DNA-repair machinery. To further investigate the functionality of the DDR-pathway, the different cell lines were gamma-irradiated in the presence of the ATM-inhibitor KU-55933 and the Chk1 inhibitor UCN-01. Whereas inhibition of Chk1 (and to a lesser extent inhibition of ATM) was able to hinder G2/M-progression in irradiated MDS cells, it remained without effect in the AML cell lines KG-1 and MV4-11. Noteworthy, whereas irradiated P39/MDS cells hindered to activate Chk1 decrease expression of Polo-like-kinase-1 (Plk-1) - which not only governs mitotic entry and exit, but also functions as an oncogene - KG-1 cells maintained a high expression of Plk-1, comparable to that observed in non-irradiated cells. Conclusion: we demonstrated the different capacities of MDS (or AML post MDS) and de novo AML cell lines to activate the DDR and thus provide evidence that the DDR-pathway is abrogated in de novo AML as compared to MDS cells.

Gene Expression Changes in CD34+ Cells Precede Development of Therapy-Related Leukemia (t-MDS/AML) After Autologous Hematopoietic Cell Transplantation (aHCT) for Hodgkin (HL) or Non-Hodgkin Lymphoma (NHL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 677-677
Author(s):  
Liang Li ◽  
Min Li ◽  
Can-Lan Sun ◽  
Melanie D. Sabado ◽  
Liton Francisco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Oxidative Phosphorylation ◽  
Dna Damage Response ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Gene Set ◽  
Gene Sets ◽  
Cd34 Cells ◽  
Damage Response

Abstract Abstract 677 t-MDS/AML is a lethal complication of aHCT for HL and NHL. The pathogenesis of t-MDS/AML is unclear. We are addressing this gap by conducting a prospective study of patients undergoing aHCT for HL/ NHL at City of Hope, with serial collection of biospecimens from pre-aHCT to 5 years after aHCT (Figure). In this report we describe alterations in gene expression (analyzed using Affymetrix HG-U133 plus 2.0 arrays) in CD34+ hematopoietic stem and progenitor cells (HSPC) associated with development of t-MDS/AML. Patients who developed t-MDS/AML after aHCT (”cases”) were compared with patients who did not develop t-MDS/AML after aHCT (“controls”: matched for primary diagnosis, age, race/ethnicity, and time since aHCT). CD34+ cells were selected from specimens obtained at the following time points: 1) pre-aHCT: peripheral blood stem cell (PBSC) product; and 2) at time of t-MDS/AML for cases and comparable time for controls: bone marrow (BM). Conditional logistic model was used to identify differences in gene expression between cases and controls in: 1) PBSC samples (18 patients who subsequently developed t-MDS/AML [cases]; 37 who did not [controls]); 2) BM samples at time of t-MDS/AML (12 cases; 21 controls); and 3) changes in gene expression from PBSC to t-MDS/AML (Figure). Gene Set Enrichment Analysis (GSEA) showed that PBSC from patients that later developed t-MDS/AML showed significant downregulation of gene sets related to mitochondria and oxidative phosphorylation, ribosomes, tRNA synthesis, proteasome, late progenitors and cell cycle (FDR<10-5for each) and upregulation of genes related to G-protein coupled receptors (GPCR, FDR=0.003) and hematopoietic regulation (CEBP, HOX, Hedgehog, FDR=0.003, 0.04, 0.06 respectively). These pathway alterations were confirmed by Ingenuity and Gene Ontology analysis. BM CD34+ cells obtained at t-MDS/AML showed downregulation of gene sets for cell cycle, p21 and p53 signaling and late progenitors (FDR<10-5 for each) and upregulation of gene sets for GPCR and cell communication/adhesion (FDR<10-5). Analysis of changes in gene expression from PBSC to t-MDS/AML revealed that compared to the controls, the cases demonstrated increased expression of mitochondrial and ribosomal gene sets and reduced expression of DNA damage response and cell cycle regulation gene sets. These results show that specific abnormalities in gene expression associated with t-MDS/AML are present in HSPC long before the development of clinical disease, and that other gene expression abnormalities occur later in the course of disease development. Indeed 22 of the top 50 upregulated and 20 of the top 50 downregulated gene sets at t-MDS/AML were represented within the top 50 up- and down-regulated sets prior to aHCT in the PBSC sample. A gene set comprised of genes differentially expressed between cases and controls at t-MDS/AML was significantly enriched amongst genes differentially expressed in PBSC (NES= -1.74, P = 0.003, FDR=0.024), further demonstrating that gene expression abnormalities associated with t-MDS/AML were present at the time of PBSC collection. To explore the hypothesis that reduced mitochondrial oxidative phosphorylation may lead to enhanced generation of reactive oxygen species (ROS) and oxidative damage, we measured ROS levels in PBSC in untreated cells and after VP16 exposure. ROS level were increased at baseline in PBSC CD34+ cells from t-MDS/AML cases compared to controls (P=0.003) and ROS elimination after VP16 exposure was reduced (P=0.05, 2 hours after VP-16 treatment). The observed gene expression changes support a model for t-MDS/AML development in which 1) reduced mitochondrial oxidative phosphorylation leads to increased ROS levels and increased damage to HSPC following therapeutic exposures, and 2) subsequent loss of DNA damage response and cell cycle regulatory mechanisms in damaged HSPC leads to the emergence of t-MDS/AML. Disclosures: No relevant conflicts of interest to declare.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

2019 ◽  
Vol 105 (3) ◽  
pp. 839-853
Author(s):  
Aglaia Kyrilli ◽  
David Gacquer ◽  
Vincent Detours ◽  
Anne Lefort ◽  
Frédéric Libert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Iodide Uptake ◽  
Transcriptomic Response ◽  
Uptake Inhibition ◽  
Γ Radiation ◽  
Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

Pathogenesis of Mantle-Cell Lymphoma: All Oncogenic Roads Lead to Dysregulation of Cell Cycle and DNA Damage Response Pathways

2005 ◽  
Vol 23 (26) ◽  
pp. 6364-6369 ◽  
Author(s):  
Veronica Fernàndez ◽  
Elena Hartmann ◽  
German Ott ◽  
Elias Campo ◽  
Andreas Rosenwald
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response ◽  
Cell Cycle Machinery

Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and has a median survival of 3 to 4 years. The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and upregulation of Cyclin D1, an important regulator of the G1 phase of the cell cycle. This genetic event is present in virtually all cases of MCL, whereas additional genetic alterations that occur in subsets of MCL have been described. Most of these alterations appear to disturb the cell cycle machinery/interfere with the cellular response to DNA damage, thus making MCL a paradigm for cell cycle and DNA damage response dysregulation in cancer in general. In particular, Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase (CDK) -4, deletions of the CDK inhibitor p16INK4a and overexpression of BMI-1, a transcriptional repressor of the p16INK4a locus, are associated with dysregulation of the cell cycle machinery in MCL. The DNA damage response pathway is affected by frequent alterations of the ataxia-telangiectasia mutated (ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and CHK2 that are kinases that act downstream of ATM in response to detection of DNA damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent gene expression profiling study defined the proliferation signature, a quantitative measure of gene expression of proliferation-associated genes as the strongest survival predictor available to date allowing the definition of prognostic MCL subgroups that differ in median survival by more than 5 years.

scholarly journals BRCA1 Effects on the Cell Cycle and the DNA Damage Response Are Linked to Altered Gene Expression

2000 ◽  
Vol 275 (4) ◽  
pp. 2777-2785 ◽  
Author(s):  
Timothy K. MacLachlan ◽  
Kumaravel Somasundaram ◽  
Magda Sgagias ◽  
Yelena Shifman ◽  
Ruth J. Muschel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Altered Gene Expression ◽  
Damage Response ◽  
Altered Gene

Coordinate Changes In RNA Expression, Transcript Splicing, and DNA Methylation In Pediatric AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3761-3761
Author(s):  
Jason Farrar ◽  
Michael Ochs ◽  
David W Lee ◽  
C.C. Talbot ◽  
Jonathan Buckley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Alternative Splicing ◽  
Dna Damage Response ◽  
Cell Cycle Progression ◽  
Expression Patterns ◽  
Cycle Progression ◽  
Damage Response ◽  
Pediatric Aml

Abstract While mutations of splicing and epigenetic factors have been reported in adult AML and related myeloid disorders, relatively few such changes have been identified in pediatric AML. We previously identified a chromatin remodeling helicase, PASG (SMARCA6, HELLS, LSH), by down-regulation in AML cell lines following cytokine withdrawal and identified an alternatively spliced variant lacking a highly conserved (STRAGGLG) domain. To assess the prevalence of this splicing variant (PASGΔ75) in pediatric AML, we tested 167 diagnostic specimens from the TARGET-AML cohort for fractional PASGΔ75 expression (PASGΔ75/PASG) using a discriminatory RT-qPCR assay. These studies demonstrated a broad, continuous distribution of PASGΔ75 with right skew (mean PASGΔ75: 26%, interquartile range: 9% – 41%) that was not significantly associated with cytogenetic class (inv16, t(8;21), MLL, normal) or FAB subtype. For further comparison, specimens were quantized by PASGΔ75 quartile. Given reported associations between loss of PASG function and abnormalities of genomic methylation, we tested 48 AML specimens at the extremes of the PASGΔ75 distribution for total 5-methylcytosine (5-mC) content by liquid chromatography/tandem mass spectrometry. The mean total methylation was significantly lower in the high compared to the low PASGΔ75 groups (mean 5-mC 3.95% vs. 4.22% of cytosine, p=0.015 Mann-Whitney). To identify specific regions of altered methylation, we used high-throughput sequencing of DNA enriched by pull-down with the methyl binding domain fragment of MBD2 (MBD-Seq). Comparison of summed methylation signal across regions flanking RefSeq transcriptional start sites (TSS) showed the expected decrease in methylation just upstream of the TSS in both groups. However, methylation more distal to the TSS was proportionally lower in PASGΔ75 high than PASGΔ75 low samples (Fig 1a). To evaluate methylation at CpG islands (CGI), UCSC CGI were scaled to 500 bp and MBD-Seq data were summed across 20Kb flanking CGI. While both groups showed the anticipated increase in methylation signal on CGI, methylation in the shore regions immediately flanking CGI was proportionally lower in high PASGΔ75 compared to low PASGΔ75 samples (Fig 1b), further suggesting epigenetic differences between these sample groups. Because we were unable to identify sequence variants in PASG intron 18 or flanking exons that might explain alternative splicing, we asked whether expression of PASGΔ75 was associated with global changes in transcript splicing. We evaluated gene expression patterns on the Affymetrix HuGene array, with assessment of alternative splicing using Partek software alternative splicing (altsplice) algorithm for quartile-grouped samples. In contrast to comparison of adjacent quartile groups, which showed modest changes in expression and relatively few transcripts with significant altsplice scores, comparison of the highest and lowest quartile samples showed marked changes in gene expression and a large number of alternatively spiced transcripts as assessed by significance of the altsplice score (Fig 2). In addition to splicing changes, these analyses suggested marked differences in gene expression patterns of AML specimens grouped by PASGΔ75 quartile, with clear separation of Q1 and Q4 samples by principal components analysis. Using a conservative Wilcoxon gene sets test and limiting ourselves to small, curated Biocarta pathways, we found expression patterns associated with high deletion variant expression strongly linked to overlapping pathways involving DNA repair, replication, and cell cycle progression. Table Pathways (Biocarta) Pathway Class Benjamini Hochberg Corrected p-Value ATR/BRCA1/BRCA2 DNA Damage Response 1.3E-6 RB/DNA Damage DNA Damage Response 3.5E-3 p27 Phosphorylation Cell Cycle Progression 3.5E-3 G2/M Checkpoint Cell Cycle Progression 3.5E-3 G1/S Checkpoint Cell Cycle Progression 4.1E-3 PLK3 Cell Cycle Progression 4.1E-3 MEF2D Apoptosis 0.01 SRC/PTPa Cell Cycle Progression 0.02 Mitochondrial Acetyl-Co Shuttle Metabolism 0.02 p53 Signaling DNA Damage Response 0.04 E2F-1 Cell Cycle Progression 0.04 ATM Signaling DNA Damage Response 0.04 These data suggest the existence of a previously unrecognized AML subclass characterized by widespread and coordinated changes in RNA expression, alternative transcript splicing, and epigenetic modifications. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Transcriptomic profiling and pathway analysis of cultured human lung microvascular endothelial cells following ionizing radiation exposure

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Roxane M. Bouten ◽  
Clifton L. Dalgard ◽  
Anthony R. Soltis ◽  
John E. Slaven ◽  
Regina M. Day
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Endothelial Cells ◽  
Dna Damage Response ◽  
Human Lung ◽  
Microvascular Endothelial Cells ◽  
Mesenchymal Transition ◽  
Damage Response ◽  
Endothelial To Mesenchymal Transition

AbstractThe vascular system is sensitive to radiation injury, and vascular damage is believed to play a key role in delayed tissue injury such as pulmonary fibrosis. However, the response of endothelial cells to radiation is not completely understood. We examined the response of primary human lung microvascular endothelial cells (HLMVEC) to 10 Gy (1.15 Gy/min) X-irradiation. HLMVEC underwent senescence (80–85%) with no significant necrosis or apoptosis. Targeted RT-qPCR showed increased expression of genes CDKN1A and MDM2 (10–120 min). Western blotting showed upregulation of p2/waf1, MDM2, ATM, and Akt phosphorylation (15 min–72 h). Low levels of apoptosis at 24–72 h were identified using nuclear morphology. To identify novel pathway regulation, RNA-seq was performed on mRNA using time points from 2 to 24 h post-irradiation. Gene ontology and pathway analysis revealed increased cell cycle inhibition, DNA damage response, pro- and anti- apoptosis, and pro-senescence gene expression. Based on published literature on inflammation and endothelial-to-mesenchymal transition (EndMT) pathway genes, we identified increased expression of pro-inflammatory genes and EndMT-associated genes by 24 h. Together our data reveal a time course of integrated gene expression and protein activation leading from early DNA damage response and cell cycle arrest to senescence, pro-inflammatory gene expression, and endothelial-to-mesenchymal transition.

scholarly journals Cell Cycle Regulation and DNA Damage Response Networks in Diffuse- and Intestinal-Type Gastric Cancer

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5786
Author(s):  
Shihori Tanabe ◽  
Sabina Quader ◽  
Ryuichi Ono ◽  
Horacio Cabral ◽  
Kazuhiko Aoyagi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Cycle Regulation ◽  
Intestinal Type ◽  
Diffuse Type ◽  
Damage Response ◽  
Canonical Pathways ◽  
Cycle Regulation

Dynamic regulation in molecular networks including cell cycle regulation and DNA damage response play an important role in cancer. To reveal the feature of cancer malignancy, gene expression and network regulation were profiled in diffuse- and intestinal-type gastric cancer (GC). The results of the network analysis with Ingenuity Pathway Analysis (IPA) showed that the activation states of several canonical pathways related to cell cycle regulation were altered. The G1/S checkpoint regulation pathway was activated in diffuse-type GC compared to intestinal-type GC, while canonical pathways of the cell cycle control of chromosomal replication, and the cyclin and cell cycle regulation, were activated in intestinal-type GC compared to diffuse-type GC. A canonical pathway on the role of BRCA1 in the DNA damage response was activated in intestinal-type GC compared to diffuse-type GC, where gene expression of BRCA1, which is related to G1/S phase transition, was upregulated in intestinal-type GC compared to diffuse-type GC. Several microRNAs (miRNAs), such as mir-10, mir-17, mir-19, mir-194, mir-224, mir-25, mir-34, mir-451 and mir-605, were identified to have direct relationships in the G1/S cell cycle checkpoint regulation pathway. Additionally, cell cycle regulation may be altered in epithelial-mesenchymal transition (EMT) conditions. The alterations in the activation states of the pathways related to cell cycle regulation in diffuse- and intestinal-type GC highlighted the significance of cell cycle regulation in EMT.

scholarly journals PIG-a Gene Expression Deficiency Association with Reduced DNA Damage Checkpoint Response and Activation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3875-3875
Author(s):  
Jeffrey J Pu ◽  
Shasha Lu ◽  
Matthew Nemesure ◽  
Emmanuel Kwame Teye ◽  
Emily Schleicher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Comet Assay ◽  
Gene Mutation ◽  
Dna Damage Response ◽  
Leukemia Cell ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Damage Response ◽  
Repair Activity

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disease caused by mutation in the PIG-A gene. When expressed, it encodes a protein that is crucial in glycophosphatidylinositol (GPI) anchor biosynthesis. The mutation of this gene causes plasma membrane GPI anchor biosynthesis deficiency, cell membrane structure alteration, signal transduction pathway blockage and vulnerability to complement complex attack. Despite these deficiencies, PNH cells have a clonal advantage and this dominant expansion is the hallmark of PNH progression. The mechanism of this growth, however, is still unknown. Recently, multiple studies demonstrated that malignant cells harboring the PIG-A mutation appear to have a higher degree of genomic instability and disease progression. This led to the proposal of using the PIG-A gene mutation frequency as the biomarker of mutagenesis to evaluate the transgenic animal mutation or mutagenicity of test compounds. The PIG-A gene mutation assay is even considered as a valuable tool for quantifying mutational events in vivo and in vitro despite the mechanism remaining unknown. This study investigated the impacts PIG-A mutation in genomic stability, DNA damage response, and DNA damage checkpoint activity, which as a measure of cellular stability as well as a possible mechanism for clonal expansion. Methods: To investigate the relationship between PIG-A gene mutation status and DNA damage checkpoint activity we looked at 45 high-risk MDS/AML patients, 3 classic PNH patients and 12 healthy controls. Additionally, the TF-1 leukemia cell line was used to evaluate PIG-A wild-type vs PIG-A CRISPR knockout vs PIG-A siRNA transient suppression. H2AX/ɣH2AX, ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins and RNA levels were used as a quantitative indicator of DNA damage repair activity via qTR-PCR and western blot. The comet assay and fiber combing assay were further applied to explore the DNA damage and cellular replication events. RNAseq analysis was used to explore the impact of PIG-A mutation in global gene expression. Results: The in vitro results from the leukemia cell lines with various PIG-A mutation statuses showed that the PIG-A mutation decreases ɣH2AX expression. PIG-A mutation was also associated with down-regulated expression of ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins. The decreased levels of those gene expressions indicate that there is increased genomic stability and less repair activity at the DNA damage response checkpoint. The results of the comet assay support this data with the PIG-A mutated cells exhibiting significantly (p<0.01) shorter tail lengths than the wild-type counterparts. The fiber-combing assay showed a reduced helicase activity for DNA replication in PIG-A mutated cells. In patient samples, subjects with PNH showed significantly lower levels of ɣH2AX expression when compared to both healthy controls and AML/MDS patients. The comet assay revealed that PNH patients had decreased DNA double strand breaks and fiber combing assay showed a reduced DNA repair activity when compared to normal controls and AML patients with intact PIG-A gene expression (p<0.01). Additionally, RNAseq analysis reveals that the expression of 10/10 genes involved in the DNA repair mechanism were down-regulated in PIG-A mutated cells when compared to wild-type control in both cell lines and patient samples. Conclusion: The above-mentioned results showed that there were decreased expressions of both DNA damage response checkpoint genes and repair genes in both the PIG-A CRISPR knockout leukemia cell lines as well as in the PNH patients. PIG-A mutation is globally associated with reduced DNA damage response capability and increased cellular stability. Our finding explains, at least partially, why PIG-A gene mutation status could be seen as a biomarker of mutagenesis and how PNH cells dominantly expend via clonal escape. Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukaemia in Elderly: Expression Analysis of DNA Damage Response (DDR) and Anti-Apoptosis Genes Identify Novel Targets for Potential "Synergistic Lethality"

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Hassan Rizwan ◽  
Hamza Kamran ◽  
Ariz Akhter ◽  
Ghaleb Elyamany ◽  
Meer-Taher Shahbani-Rad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Age Related ◽  
Elderly Aml ◽  
Damage Response ◽  
Treatment Naïve ◽  
Apoptotic Genes ◽  
Acute Myeloid

Background: Genetic instability and heterogeneity are fundamental to AML biology and pivotal to its prognosis. Acute myeloid leukaemia (AML) is most frequent in older adults (&gt;60 years) and a significant majority are considered "treatment-naïve" given medical comorbidities, high-risk disease biology, and poor tolerance to chemotherapy. These patients receive low-intensity regimens, mostly hypomethylating agents due to impaired tolerance to induction chemotherapy, resulting in low overall survival. Thus, there is a critical need to develop targeted therapies capable of rapidly inducing a high rate of clinical response, with better tolerability and durable responses for these elderly AML patients. DNA damage response (DDR) is a specialized and highly orchestrated signalling cascade to maintain genetic stability and is closely linked to the cell cycle. The cell cycle checkpoints result in arrest and the resumption of cell cycle progression when DNA damage has been repaired. However, the DNA repair failure will direct cells towards cellular senescence or apoptosis. Hence, DDR and apoptosis are intricately related physiological processes. In AML, mutations in key regulators of gene expression and/or chromatin structure, such as p53, K-RAS, and isocitrate dehydrogenase 1 and 2 (IDH1/2) results in defective DDR. Defects in DDR are also age-related; therefore, resulting in an exponential increase in the incidence of cancer with age. The emerging treatment regimens for elderly AML are focusing on synergistic lethality using a combination of DDR inhibitors (PARP1) and/ or anti-apoptotic inhibitors (BCL2) in combination with low dose anthracycline and additional targeted therapies like (IDH2/DNTM etc.). However, the wider implication and influence of DDR genes either alone or in combination with anti-apoptotic genes remains uncharacterized in elderly AML. In this pilot study, we screened a large cohort of AML samples for mRNA expression for a series of DDR and apoptotic genes to investigate the age-related variation in expression of these molecules. We observed distinct differences in DDR and anti-apoptotic gene expression between elderly AML compared to paediatric patients. We anticipate, that these preliminary observations will pave the pathway for future comprehensive studies to expand the employment of synergistic lethality strategy for elderly AML patients. Methods: We employed RNA extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic tissuesamples in 100 AML patients. Age defined the categorization of patients into three groups; a, &lt;18 years (n=34); b,18-60 years (n=32) and c, &gt;60 years (n=34). nCounter (NanoString Technologies) platform was used for the quantification of mRNA. Qlucore Omics Explorer software was employed with defined criteria (fold change &gt;2.0; p&lt;0.01 and q &lt;0.05) for statistical analysis. Results:We noted that several DDR mediators (BRIP1, POLD1, XRCC4) are differentially up-regulated in the AML samples linked with elderly patient group, alongside anti-apoptotic genes (IDH2, IKBKB) when compared with samples from patient &lt;18 years (Figure 1). Moreover, DDR effectors (BRCA1& ATM) were up-regulated in the elderly cohort relative to paediatric AML samples, suggesting potential targets for treating elderly AML (Figure2). Conclusions:This pilot study identifies several additional and novel DDR targets; which can be exploited to enhance chemotherapeutic efficacy in "treatment fit" as well as "treatment naïve" elderly AML patients. Besides, we also report numerous novel anti-apoptotic molecules up-regulated in elderly AML. Taken together the preliminary data, presented here, expand the repertoire for targeted therapy in elderly AML with a specific focus on synergistic lethality. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document