Dataset Information

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

ABSTRACT:

Background

The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR.

Methods

Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging.

Results

ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells.

Conclusions

Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

SUBMITTER: Johnson GG

PROVIDER: S-EPMC4096176 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

Johnson Guyla G GG White Misti C MC Wu Jian-He JH Vallejo Matthew M Grimaldi Maurizio M

Neuro-oncology 20140224 8

<h4>Background</h4>The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR.<h4>Methods</h4>Several glioma cell lines and pr ...[more]

PMID: 24569545

Similar Datasets

Project description:Purpose: To explore the pathway cross-talk of endoplasmic reticulum (ER) stress response and cellulases synthesis in Neurospora crassa. Methods: To identify ER stress response targets(ESRTs), conidial suspensions of WT were obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium (1x vogel’s salts , 1% w/v crystalline cellulose (Avicel PH-101; Sigma-Aldrich), 0.2% w/v NH4NO3 ,pH 7.0 )for 36 hours (25ºC, 200 rpm, constant light) followed by the addition of dithiothreitol (DTT, final concentration of 0.1mM for mild stress level while 5mM for acute stress level) or tunicamycin (TM, final concentration of 5ug/mL for mild stress level while 40ug/mL for acute stress level) and growth for 1 more hour.To identify which ESRTs regulated by IRE-1/Hac-1 mediated UPR pathway,conidial suspensions of the Ire-1 and Hac-1 KO mutants as well as their parental strains (FGSC#2489 and FGSC#9720, respectively) were obtained and 1 x 10^6 cells/ml incubated in minimal medium containing 2% sucrose for 16 hours, then young hyphae was harvested and transferred into cellulases induction medium as mentioned above culture for 4 hours followed by the addition of DTT (final concentration of 5mM)(or H2O as mock)and growth for 1 more hour.For Hac-1 KO mutant and FGSC#9720,cultures were plus with final concentration of 100ug/ml L-histidine.To explore the function of transcription factors RES-1, RES-2 and RRG-2 in response to ER stress and cellulases synthesis, each conidial suspension of the three strains was obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium for 36 hours followed by the addition of dithiothreitol (DTT, final concentration of 5mM ) (or H2O as mock)and growth for 1 more hour.Cells were harvested by vacuum filtration with Whatman filter paper, frozen immediately in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. An additional clean-up including the on-column DNase I treatment was performed by using the RNeasy mini kit (Qiagen).The thirteen 49-nt single-end RNA-seq libraries were generated commercially at Beijing Genomics Institute (BGI, Shenzhen, China) while eight 90-nt paired-end RNA-seq libraries were generated at Genewiz Co. Ltd (Suzhou) by using the HiSeq™ platform.Sequencing data was handled essentially with Bowtie2, Tophat2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: Transcriptional profiling of ER stress during cellulase synthesis in N.crassa uncovered a set of 766 genes was identified as the ER stress response target genes (ESRTs). Of these 766 genes, canonical UPR components IRE-1 and HAC-1 regulated 223 and 186 genes, respectively. In screening 527 available ESRT mutants, we found 249 that exhibited DTT sensibility, including 100 targets with unknown function. Disruption of ire-1 or hac-1 renders N. crassa cells seriously deficient in cellulase secretion. Further investigation of the 39 transcription factors (TFs) that showed differential expression under ER stress uncovered three novel TFs (RES-1, RES-2, RRG-2) that markedly affected cellulase secretion. Conclusions: Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.

Project description:BACKGROUND: Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer. METHODS: Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry. RESULTS: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers. CONCLUSION: These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

Dataset Information

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

Background

Methods

Results

Conclusions

Publications

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure