scholarly journals Early Increase of Radiation-induced γH2AX Foci in a Human Ku70/80 Knockdown Cell Line Characterized by an Enhanced Radiosensitivity

2010 ◽  
Vol 51 (6) ◽  
pp. 633-641 ◽  
Author(s):  
Veerle VANDERSICKEL ◽  
Julie DEPUYDT ◽  
Bram VAN BOCKSTAELE ◽  
Gianpaolo PERLETTI ◽  
Jan PHILIPPE ◽  
...  
Keyword(s):  
Cell Line ◽  
Knockdown Cell ◽  
Γh2ax Foci ◽  
Early Increase ◽  
Radiation Induced ◽  
Knockdown Cell Line

BCL-Xl Contributes to Decreased Cellular Respiration During Erythroid Differentiation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3201-3201
Author(s):  
Cori Abikoff ◽  
Daciana Margineantu ◽  
David Hockenbery
Keyword(s):  
Oxygen Consumption ◽  
Western Blot ◽  
Cell Line ◽  
Control Cell ◽  
Erythroid Differentiation ◽  
Cellular Respiration ◽  
Knockdown Cell ◽  
Bcl2 Family ◽  
Control Cell Line ◽  
Knockdown Cell Line

Abstract Abstract 3201 The BCL2 family of proteins are well known for their ability to both positively and negatively regulate mitochondrial mechanisms of apoptosis. The anti-apoptotic members of this family can decrease mitochondrial outer membrane permeability and cytochrome c release. By stabilizing the cell against apoptosis, these proteins allow cell survival even in states of low energy. However, despite this intimate link between the BCL2 family proteins and mitochondria, their direct effect on metabolism is less clearly understood. It is generally expected that metabolic changes induced by the BCL2 family of proteins will further impact cell survival as murine hepatoma and cancer cells overexpressing Bcl-xL are sensitive to Bcl-xL inhibition1 but BCL-xL is also known to be essential for erythroid differentiation and more recently was linked specifically to heme synthesis2. We therefore set out to investigate whether there was a connection between BCL-xL induced changes in cellular respiration and erythroid differentiation. Murine erythroleukemia (MEL) cells were differentiated by exposure to 2% DMSO for 5 days and then real time oxygen consumption was measured on the Seahorse extracellular flux analyzer (XFA). DMSO induced differentiation yielded a 4-fold decrease in oxygen consumption. Western blot analysis revealed that BCL-xL was induced during differentiation. We then generated cell lines in which BCL-xL was knocked down with small hairpin RNA (shRNA). As differentiation has previously been reported to be fatal in MEL cells without BCL-xL activity, both parental cells and BCL-xL knockdowns were infected with a vector over expressing BCL2. Differentiation over 5 days with 2% DMSO was performed on these new cell lines. Erythroid differentiation was confirmed using Benzidine staining. While the control cell line showed high rates of Benzidine staining after exposure to DMSO, the BCL-xL knockdown cell line consistently showed <5% benzidine positivity. Western blot analysis confirmed the absence of BCL-xL induction by DMSO exposure in the knockdown cell line. Using the Seahorse XFA the control cell line was shown to have significant decrease in oxygen consumption when exposed to DMSO, while DMSO exposed BCL-xL knockdown cells showed less than half this drop in oxygen consumption. However, both control and BCL-xL knockdowns have limited respiratory reserve as the response to CCCP, an uncoupler of electron transport, is diminished after DMSO exposure as compared to their undifferentiated counterparts. Our results suggest that erythroid differentiation is associated with a significant decrease in cellular respiration. Although, not the only contributor to the decreased dependence on oxidative phosphorylation of cells undergoing erythroid differentiation, BCL-xL expression is clearly a necessary factor. Our data is able to connect BCL-xL expression to both erythroid differentiation and this distinct metabolic phenotype. As BCL-xL's role in erythroid differentiation has previously been reported to be associated with heme synthesis, future work will focus on identifying oxidative metabolic pathways associated with BCL-xL expression and heme synthesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Disease modeling of core pre-mRNA splicing factor haploinsufficiency

2019 ◽  
Vol 28 (22) ◽  
pp. 3704-3723 ◽  
Author(s):  
Katherine A Wood ◽  
Charlie F Rowlands ◽  
Wasay Mohiuddin Shaikh Qureshi ◽  
Huw B Thomas ◽  
Weronika A Buczek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Cell Line ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Rna Seq ◽  
Knockdown Cell ◽  
Er Stress Response ◽  
Knockdown Cell Line

Abstract The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.

Dose-dependant radiation-induced apoptosis in a cochlear cell-line

APOPTOSIS ◽  
2006 ◽  
Vol 11 (12) ◽  
pp. 2127-2136 ◽  
Author(s):  
Wong-Kein Low ◽  
Michelle G. K. Tan ◽  
Li Sun ◽  
Alvin W. C. Chua ◽  
Lip-Kai Goh ◽  
...  
Keyword(s):  
Cell Line ◽  
Radiation Induced ◽  
Induced Apoptosis

scholarly journals Involvement of APOBEC3B in mutation induction by irradiation

2020 ◽  
Vol 61 (6) ◽  
pp. 819-827
Author(s):  
Yohei Saito ◽  
Hiromasa Miura ◽  
Nozomi Takahashi ◽  
Yoshikazu Kuwahara ◽  
Yumi Yamamoto ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Risk ◽  
Cell Line ◽  
Human Cancer ◽  
Spontaneous Mutation ◽  
Mutation Induction ◽  
Parent Cell ◽  
Induced Mutations ◽  
Parent Cell Line ◽  
Radiation Induced

Abstract To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

Elevated radiation-induced γH2AX foci in G2 phase heterozygous BRCA2 fibroblasts

2011 ◽  
Vol 101 (1) ◽  
pp. 46-50 ◽  
Author(s):  
Andrea Beucher ◽  
Dorothee Deckbar ◽  
Eik Schumann ◽  
Andrea Krempler ◽  
Marlis Frankenberg-Schwager ◽  
...  
Keyword(s):  
Γh2ax Foci ◽  
Radiation Induced ◽  
G2 Phase
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