Transcription profiling of human glioblastoma cell lines after treatment with two ubiquitin-proteasome system inhibitors to characterize the molecular signals of cell death by necrosis
ABSTRACT: The regulation of necrotic death and its relevance in anti-cancer therapy are largely unknown. Here we have investigated the pro-apoptotic and pro-necrotic activities of two ubiquitin-proteasome system inhibitors (UPSIs): bortezomib and G5. The present study points out that the glioblastoma cell lines U87MG and T98G are useful models to study the susceptibility to apoptosis and necrosis in response to UPSIs. U87MG cells are resistant to apoptosis induced by bortezomib and G5 but susceptible to necrosis induced by G5. On the opposite T98G cells are susceptible to apoptosis induced by both inhibitors but show some resistance to G5-induced necrosis. By comparing the transcriptional profiles of the two cell lines, we have found that the resistance to G5-induced necrosis could arise from differences in glutathione synthesis/utilization and in the microenvironment. In particular collagen IV, which is highly expressed in T98G cells, and fibronectin, whose adhesive function is counteracted by tenascin-C in U87MG cells, can restrain the necrotic response to G5. Collectively, our results provide an initial characterization of the molecular signals governing cell death by necrosis in glioblastoma cell lines. Experiment Overall Design: Gene expression profiling was evaluated from 3 replicates each of T98G and U87MG cells.
Project description:The regulation of necrotic death and its relevance in anti-cancer therapy are largely unknown. Here we have investigated the pro-apoptotic and pro-necrotic activities of two ubiquitin-proteasome system inhibitors (UPSIs): bortezomib and G5. The present study points out that the glioblastoma cell lines U87MG and T98G are useful models to study the susceptibility to apoptosis and necrosis in response to UPSIs. U87MG cells are resistant to apoptosis induced by bortezomib and G5 but susceptible to necrosis induced by G5. On the opposite T98G cells are susceptible to apoptosis induced by both inhibitors but show some resistance to G5-induced necrosis. By comparing the transcriptional profiles of the two cell lines, we have found that the resistance to G5-induced necrosis could arise from differences in glutathione synthesis/utilization and in the microenvironment. In particular collagen IV, which is highly expressed in T98G cells, and fibronectin, whose adhesive function is counteracted by tenascin-C in U87MG cells, can restrain the necrotic response to G5. Collectively, our results provide an initial characterization of the molecular signals governing cell death by necrosis in glioblastoma cell lines. Keywords: cell line comparison Overall design: Gene expression profiling was evaluated from 3 replicates each of T98G and U87MG cells.
Project description:The WWOX gene is known for its tumor suppressor character and seems to be one of the main cellular regulators of cell cycle and apoptosis. The aim of the study was to identify cellular pathways and biological processes influenced by WWOX in glioblastoma cells. Our experiment showed that in this type of cancer WWOX, beside cell cycle and apoptosis control, may be involved in metabolism, cytoskeleton structure and differentiation. T98G glioblastoma cells were stably transfected with WWOX cDNA. T98G cells transfected with an empty vector served as a control. Total mRNA was isolated to look for gene-expression differences induced by the WWOX overexpression.
Project description:To extend our understanding of systemic necrosis in susceptible potato tubers infected with the necrotic strain of Potato virus Y (PVYNTN) gene expression was compared between healthy and infected non-necrotic and necrotic (both non-necrotic and necrotic tissue) potato tubers.
Project description:This study investigated the biological function of CD133 by ectopic expression of CD133 in U87MG cell line. Although CD133 is widely used as a cancer stem cell marker, there are a few studies that examined its own biological functions. While a number of loss-of-function studies about CD133 have shown that CD133 have effects on cancer progression, there are few gain-of-function studies about functions of CD133. Thus, we identified the potential function of CD133 by its overexpression in U87MG glioblastoma cell line. Though there were no significant changes in cell growth and sphere forming ability, elevated IL-1β and its downstream chemokines (CCL3, CXCL3, CXCL5) may function as chemoattractants which affect recruitment of Ly6G+ neutrophils surrounding necrotic regions in vivo and migration of neutrophil-like dHL-60 cells. Taken together, this results imply that CD133 can regulate IL-1β signaling, and promotes the environmental change. Overall design: A Glioblastoma cell line, U87MG, was transfected with pCDH-CMV-MCS-EF1-Puro (U87MG-control) or pCDH-CMV-CD133-EF1-Puro (U87MG-CD133). Plasmids for CD133 overexpression (NM_001145847) were gifted by Dr. Young-Gyu Ko (Korea University). The 3 biological replicate of RNA extracts in these cells was used for transcriptome analysis.
Project description:Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to such treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and -5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased of cell proliferation, and increased cell cycle arrest and apoptosis in comparison to only irradiated cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation and cell cycle regulation. To our knowledge, this is the first study to describe role of miR-338-5p in GBM and its potential to improve sensitivity of GBM to radiation. Study was performed on three glioblastoma multiforme cell lines A172, T98G and U87MG. This experiment was performed on Affymetrix GeneChip Human Gene ST 1.0 to elucidate the targets of miRNA-338-5p. Cell lines were seeded 24 hours prior transfection. After transfection with pre-miR338-5p or negative control cell were cultured for 24 hours and harvested. RNA was isolated using MirVana miRNA Isolation Kit (Ambion, USA) and checked for RNA integrity by Bioanalyzer 2100 and purity by ratios 260/280>1.8 and 260/230>1.8 by Nanodrop2000.
Project description:Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Additional cellular activities, unrelated to cell death seem to be influenced by these enzymes. Identification of genes co-regulated with caspases could help to ascertain new biological roles for these proteases.To identify genes and pathways under the influence of caspase-2 we silenced its expression in U87MG glioblastoma cell line. Transcriptional expression profiles of cells transfected with caspase-2 siRNA or control siRNA were compared. Gene expression profiling was evaluated from 2 replicates of U87MG cells transfected with CASP2-siRNA, or with Control-siRNA.
Project description:The human glioma cell lines T98G and U87MG were cultured in media alone, with cannabidiol (CBD), tetrahydrocannabinol (THC) or a combination of CBD and THC at different ratios for 4 hours. Cells were then harvested and RNA isolated by Trizol. Total RNA was labelled and hybridised to Illumina Human HT-12v4 arrays.
Project description:U87MG is a glioblastoma cell line that shows substantial heterogeneity despite long-term passaging. We used microarrays to identify variations in gene expression that are associated with phenotypic differences among subclones derived from U87MG. Overall design: U87MG was plated dilutely such that individual colonies arising from single cells could be readily isolated and propagated as subclones.
Project description:Wollbold2014 - Effects of reactive oxygen
This model is described in the article:
Anti-inflammatory effects of
reactive oxygen species ¿ a multi-valued logical model
validated by formal concept analysis.
Wollbold J, Jaster R, Müller S,
Rateitschak K, Wolkenhauer O.
BMC Syst Biol 2014 Sep; 8(1): 101
BackgroundRecent findings suggest that in pancreatic acinar
cells stimulated with bile acid, a pro-apoptotic effect of
reactive oxygen species (ROS) dominates their effect on
necrosis and spreading of inflammation. The first effect
presumably occurs via cytochrome C release from the inner
mitochondrial membrane. A pro-necrotic effect ¿ similar to
the one of Ca2+ ¿ can be strong opening of mitochondrial
pores leading to breakdown of the membrane potential, ATP
depletion, sustained Ca2+ increase and premature activation of
digestive enzymes. To explain published data and to understand
ROS effects during the onset of acute pancreatitis, a model
using multi-valued logic is constructed. Formal concept
analysis (FCA) is used to validate the model against data as
well as to analyze and visualize rules that capture the
dynamics.ResultsSimulations for two different levels of bile
stimulation and for inhibition or addition of antioxidants
reproduce the qualitative behaviour shown in the experiments.
Based on reported differences of ROS production and of ROS
induced pore opening, the model predicts a more uniform
apoptosis/necrosis ratio for higher and lower bile stimulation
in liver cells than in pancreatic acinar cells. FCA confirms
that essential dynamical features of the data are captured by
the model. For instance, high necrosis always occurs together
with at least a medium level of apoptosis. At the same time,
FCA helps to reveal subtle differences between data and
simulations. The FCA visualization underlines the protective
role of ROS against necrosis.ConclusionsThe analysis of the
model demonstrates how ROS and decreased antioxidant levels
contribute to apoptosis. Studying the induction of necrosis via
a sustained Ca2+ increase, we implemented the commonly accepted
hypothesis of ATP depletion after strong bile stimulation.
Using an alternative model, we demonstrate that this process is
not necessary to generate the dynamics of the measured
variables. Opening of plasma membrane channels could also lead
to a prolonged increase of Ca2+ and to necrosis. Finally, the
analysis of the model suggests a direct experimental testing
for the model-based hypothesis of a self-enhancing cycle of
cytochrome C release and ROS production by interruption of the
mitochondrial electron transport chain.
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