Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration.
Ontology highlight
ABSTRACT:
SUBMITTER: Sheard MA
PROVIDER: S-EPMC8219348 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The Fas-associated death domain (FADD) has long been regarded as a crucial adaptor protein in the extrinsic apoptotic pathway. Despite the non-apoptotic function of FADD is gradually being discovered and confirmed, its corresponding physiological and pathological significance is still unclear. Based on the database of GWAS catalog and GTEx Portal, 17 SNPs associated with leukemia susceptibility were found to be linked to FADD expression. We then investigated a regulatory role of FADD in T-acute lymphoblastic leukemia (T-ALL) using Jurkat cells as a model. Jurkat cells stably depleted of FADD (FADD-/- Jurkat) expression exhibited dampened proliferation, hypersensitivity to Etoposide-induced intrinsic apoptosis whereas near total resistance to TRAIL-induced extrinsic apoptosis. Comparison between wild type and FADD-/- Jurkat cells using iTRAQ-based proteomics revealed considerably altered expression spectrum of genes, and led us to focus on metabolic pathways. Investigation of glycolytic and mitochondrial pathways and relevant enzymes revealed that FADD knockout triggered a metabolic shift from glycolysis to mitochondrial respiration in Jurkat cells. Re-expression of FADD in FADD-/- Jurkat cells partially rescued glycolytic capacity. FADD loss triggers global metabolic reprogramming in Jurkat cells and therefore remains as a potential druggable target for ALL treatment.
| S-EPMC9134909 | biostudies-literature
Project description:High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.
| S-EPMC9099058 | biostudies-literature
Project description:Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a poor clinical outcome. Previously, we showed that overproduction of reactive oxygen species (ROS), arising from constitutive activation of NOX2 oxidase, occurs in >60% of patients with AML and that ROS production promotes proliferation of AML cells. We show here that the process most significantly affected by ROS overproduction is glycolysis. Whole metabolome analysis of 20 human primary AML showed that blasts generating high levels of ROS have increased glucose uptake and correspondingly increased glucose metabolism. In support of this, exogenous ROS increased glucose consumption while inhibition of NOX2 oxidase decreased glucose consumption. Mechanistically, ROS promoted uncoupling protein 2 (UCP2) protein expression and phosphorylation of AMPK, upregulating the expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Overexpression of PFKFB3 promoted glucose uptake and cell proliferation, whereas downregulation of PFKFB3 strongly suppressed leukemia growth both in vitro and in vivo in the NSG model. These experiments provide direct evidence that oxidase-derived ROS promotes the growth of leukemia cells via the glycolytic regulator PFKFB3. Targeting PFKFB3 may therefore present a new mode of therapy for this disease with a poor outcome. SIGNIFICANCE: These findings show that ROS generated by NOX2 in AML cells promotes glycolysis by activating PFKFB3 and suggest PFKFB3 as a novel therapeutic target in AML.
| S-EPMC7611211 | biostudies-literature
Project description:BACKGROUND:Drug-resistant cell lines, established from drug-sensitive cell lines by drug exposure in vitro, are the most useful cancer models in studies on the mechanism of chemoresistance. However, the success rate of the traditional approaches to construct such cell lines is low because a long time is required for the addition of drugs. METHODS:A cell culture technique was used to establish the drug-resistant cell lines from their parental cells. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, and DNA fingerprinting analysis were used to characterize the drug-resistant cell lines. Nude mice were used for xenograft studies. RESULTS:We established novel glucocorticoid (GC)-resistant cell lines from 3 GC-sensitive acute lymphoblastic leukemia (ALL) cell lines. First, we established a novel GC-resistant T-ALL cell line, CEM-C7/HDR, by mimicking the microenvironment of the bone marrow and culturing GC-sensitive CEM-C7-14 cells under hypoxia for 5?weeks with a single dexamethasone (Dex) treatment. The CEM-C7/HDR cells had been cultured continuously in drug-free medium under normoxia for 1?year. The IC50 and resistance index (RI) to Dex were maintained at 60~70??M and 1500~1800, respectively, which is in consistent with the IC50 and RI of GC-resistant CEM-C1-15 cells. To clarify the reliability of the method, we subcloned CEM-C7-14 cells, and obtained Dex-resistant cell lines, CEM-C7-SC2/HDR and CEM-C7-SC14/HDR, from 2 monoclonal cells of CEM-C7-14 by the same method. Moreover, we obtained two additional Dex-resistant B-ALL cell lines, NALM-6/HDR and HXEX-ALL1/HDR, from NALM-6 and HXEX-ALL1 cells with the same approach. CONCLUSIONS:CEM-C7/HDR, NALM-6/HDR and HXEX-ALL1/HDR cell lines may serve as useful GC-resistant ALL models for both in vitro and in vivo studies. Culturing under hypoxic condition with a single Dex treatment is a novel and convenient approach for generating stable GC resistant cell lines.
| S-EPMC6585113 | biostudies-literature
Project description:The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBP?, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1?. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1? was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBP? expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1?.
| S-EPMC6039343 | biostudies-literature
Project description:IntroductionMethotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use.MethodsTo better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX.ResultsMTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested.ConclusionAltogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.
| S-EPMC9751399 | biostudies-literature
Project description:Despite numerous efforts aiming to characterise glioblastoma pathology (GBM) and discover new therapeutic strategies, GBM remains one of the most challenging tumours to treat. Here we propose the optimisation of in vitro culturing of GBM patient-derived cells, namely the establishment of GBM-derived cultures and their maintenance at oxygen tension mimicking oxygenation conditions occurring within the tumour. To globally analyse cell states, we performed the transcriptome analysis of GBM patient-derived cells kept as spheroids in serum-free conditions at the reduced oxygen tension (5% O2), cells cultured at atmospheric oxygen (20% O2), and parental tumour. Immune cells present in the tumour were depleted, resulting in the decreased expression of the immune system and inflammation-related genes. The expression of genes promoting cell proliferation and DNA repair was higher in GBM cell cultures when compared to the relevant tumour sample. However, lowering oxygen tension to 5% did not affect the proliferation rate and expression of cell cycle and DNA repair genes in GBM cell cultures. Culturing GBM cells at 5% oxygen was sufficient to increase the expression of specific stemness markers, particularly the PROM1 gene, without affecting neural cell differentiation markers. GBM spheroids cultured at 5% oxygen expressed higher levels of hypoxia-inducible genes, including those encoding glycolytic enzymes and pro-angiogenic factors. The genes up-regulated in cells cultured at 5% oxygen had higher expression in parental GBMs compared to that observed in 20% cell cultures, suggesting the preservation of the hypoxic component of GBM transcriptome at 5% oxygen and its loss in standard culture conditions. Evaluation of expression of those genes in The Cancer Genome Atlas dataset comprising samples of normal brain tissue, lower-grade gliomas and GBMs indicated the expression pattern of the indicated genes was specific for GBM. Moreover, GBM cells cultured at 5% oxygen were more resistant to temozolomide, the chemotherapeutic used in GBM therapy. The presented comparison of GBM cultures maintained at high and low oxygen tension together with analysis of tumour transcriptome indicates that lowering oxygen tension during cell culture may more allegedly reproduce tumour cell behaviour within GBM than standard culture conditions (e.g., atmospheric oxygen tension). Low oxygen culture conditions should be considered as a more appropriate model for further studies on glioblastoma pathology and therapy.
| S-EPMC9564145 | biostudies-literature
Project description:BackgroundThe clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.MethodsSensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.ResultsLoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ?3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.ConclusionsThese studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.
| S-EPMC7325652 | biostudies-literature
Project description:t(17;19)(q21-q22;p13), responsible for TCF3-HLF fusion, is a rare translocation in childhood B-cell precursor acute lymphoblastic leukemia(BCP-ALL). t(1;19)(q23;p13), producing TCF3-PBX1 fusion, is a common translocation in childhood BCP-ALL. Prognosis of t(17;19)-ALL is extremely poor, while that of t(1;19)-ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3-HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)-ALL case, while TCF3-PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)-ALL cases. Using 4 t(17;19)-ALL and 16 t(1;19)-ALL cell lines, drug response profiling was analyzed. t(17;19)-ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)-ALL cell lines. Sensitivities to three (Pred, VCR, and l-asparaginase [l-Asp]), four (Pred, VCR, l-Asp, and DNR) and five (Pred, VCR, l-Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)-ALL cell lines than for t(1;19)-ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P-glycoprotein (P-gp) were higher in t(17;19)-ALL cell lines than in t(1;19)-ALL cell lines. Inhibitors for P-gp sensitized P-gp-positive t(17;19)-ALL cell lines to VCR and DNR. Knockout of P-gp by CRISPRCas9 overcame resistance to VCR and DNR in the P-gp-positive t(17;19)-ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P-gp-positive t(17;19)-ALL cell line. These findings indicate involvement of P-gp in resistance to VCR and DNR in Pgp positive t(17;19)-ALL cell lines. In all four t(17;19)-ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)-ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)-ALL.
| S-EPMC6718581 | biostudies-literature
Project description:For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.
| S-EPMC8929001 | biostudies-literature