scholarly journals Mutant nucleophosmin1 (NPM1) Links Protein Translation with Oncogenic Metabolism in Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2727-2727
Author(s):  
Marisa Juntilla ◽  
Alina Garbuzov ◽  
Natalie Ortiz ◽  
Julia Eberhard ◽  
Caitlin Roake ◽  
...  
Keyword(s):  
Cell Size ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Aerobic Glycolysis ◽  
Leukemia Cell ◽  
Lactate Production ◽  
Protein Translation ◽  
Metabolic Reprogramming ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Nucleophosmin 1 (NPM1) is the most common gene mutated in acute myeloid leukemia (AML). Several putative mechanisms for the role of NPM1 in the generation of AML have been proposed, including acting as a tumor suppressor by promoting the degradation of p53 and sequestration of wild-type NPM1. However, the specificity of the mutation- most commonly a 4 base pair duplication in the C-terminus- suggests that it may be a gain- of- function mutation conferring an unidentified pro-leukemic effect. Using a novel set of leukemia cell lines generated with CRISPR/Cas9 technology in combination with studies in primary hematopoietic cells, we show that mutant NPM1 may promote leukemia by deregulating protein translation with downstream effects on cellular metabolism. Thus, mutant NPM1 may serve as a critical lynchpin to match metabolic precursors and cellular energetics to ribosomal output. The implications of our results suggest that combined metabolic and protein translation inhibition may be a previously unidentified vulnerability in AML with mutated NPM1. CRISPR/Cas9 technology was used to generate a set of isogenic cell lines derived from the heterozygous OCI-AML3 line. Transcriptomic analysis of clones with either parental allele configuration (wild-type and mutant, WTAM) vs. inactivation of the mutant allele (wild-type only, WTO) confirmed that mutant NPM1 regulates HOX genes, as shown in both mouse models and patient samples. Next, the effects of mutant NPM1 on ribosome biogenesis and protein translation were assayed with pulse- chase and static experiments and showed both ribosomal RNA and protein translation were increased in WTAM clones vs. WTO clones. During these investigations, cell size was noted to be increased in WTAM cells. Cell size can be a consequence of the metabolic activity of a cell. Analysis of the rate of glucose consumption and lactate production showed that WTAM cells have increased aerobic glycolysis compared with WTO cells. To test if mutant NPM1 is sufficient to drive both protein translation and aerobic glycolysis in a normal cell, these studies were replicated in normal human hematopoietic stem cells stably expressing either wild-type NPM1 or mutant NPM1. In summary, our results suggest that mutant NPM1 acts through dysregulation of protein translation and participates in metabolic reprogramming required to drive oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

DOCK2 Interacts with FLT3 and Modulates the Survival of FLT3-Expressing Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3584-3584
Author(s):  
Min Wu ◽  
Max Hamaker ◽  
Li Li ◽  
Donald Small ◽  
Amy S. Duffield
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Wild Type ◽  
Knock Down ◽  
Leukemia Cell Lines ◽  
Significant Difference ◽  
Flt3 Expression

Abstract The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying FLT3/ITD mutations have a poor prognosis. Despite continuing progress in the development of more effective FLT3 inhibitors, long-term success in inhibition of FLT3 activity in AML patients is still elusive. In order to achieve a better understanding of FLT3 biology and more effective strategies for the inhibition of FLT3 activity, a screen was performed on leukemia cell lines to search for FLT3-interacting proteins. One of the proteins identified in the screen was dedicator of cytokinesis 2 (DOCK2). The DOCK family of proteins acts as guanine nucleotide exchange factors (GEFs) for Rho family of GTPases, which includes Rac GTPases. DOCK2 expression is limited to hematopoietic cells, and is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells, and cell-cell adhesion and bone marrow homing of various immune cells. We first verified that DOCK2 is expressed in primary AML samples from patients, and co-immunoprecipitation experiments showed that DOCK2 interacts with both wild-type FLT3 and FTL3/ITD in these cells. Co-immunoprecipitation experiments using leukemia cell lines demonstrated that DOCK2 interacts with FLT3, FLT3/ITD, FLT3/D835Y and FLT3/D835H, and that it predominantly interacts with the unphosphorylated form of FLT3. Knock-down of DOCK2 by shRNA did not significantly affect the growth of cell lines that lack expression of FLT3, but greatly reduced growth of cell lines expressing amplified wild type FLT3 (Sem K2), FLT3/D835H (HB11;19) and FLT3/ITD (MV4;11). Accordingly, colony formation assays revealed that cell lines with elevated expression of wild type or mutant FLT3 produced fewer, smaller and more compact colonies when the expression of DOCK2 was decreased, while colonies from cell lines lacking FLT3 expression showed no significant difference in response to the knock-down of DOCK2. Furthermore, an Annexin V binding assay indicated that reduction in DOCK2 expression level greatly sensitized cells with elevated FLT3 activity (MV4;11 and Sem K2) to cytarabine, resulting in increased apoptosis, but no significant sensitization was observed in cell lines that lack FLT3 expression. These findings demonstrate that DOCK2 interacts with FLT3 in leukemia cell lines, and suggest that this interaction has important roles in regulating the survival of leukemia cells with elevated FLT3 activity, both alone and in combination with conventional anti-leukemic agents. Therefore, DOCK2 is a potential therapeutic target for AML treatment, and better understanding of the interaction between DOCK2 and FLT3 may contribute to the development of novel strategies to effectively inhibit FLT3 activity in AML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals L-Asparaginase Causes Metabolic Reprogramming in ALL Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 922-922
Author(s):  
Ivana Hermanova ◽  
Karel Valis ◽  
Hana Nuskova ◽  
Meritxell Alberich-Jorda ◽  
Amaia Arruabarrena Aristorena ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Protein Translation ◽  
Metabolic Reprogramming ◽  
Leukemic Cells ◽  
Wild Type ◽  
Cell Respiration ◽  
Pyrimidine Synthesis ◽  
Mtorc1 Pathway ◽  
De Novo Pyrimidine Synthesis

Abstract Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. Our aim is to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients´ response. We have generated ASNase-resistant subclones through chronic exposure to the enzyme. Pathway analysis of gene expression profiles of the cell lines (REH;TEL/AML1-positive, NALM-6; TEL/PDGFRB1-positive and their resistant counterparts) and primary samples (sensitive and resistant to ASNase; Holleman et al. (NEJM, 2004)) revealed that ASNase affects the translation machinery and metabolism of leukemic cells. The key nutrient sensor positively regulating protein synthesis, pyrimidine synthesis, glycolysis and lipid synthesis is mTORC1. Since ASNase depletes glutamine that is essential for mTORC1 activity, we hypothesized that the effect of ASNase is driven through mTORC1 signaling. Main aim of the study was to explore the effect of ASNase on downstream mTORC1 targets in leukemic cells. ASNase treatment inhibited protein synthesis, displayed by dephosphorylation of p-P70SK6 and p-S6. ASNase also decreased de-novo DNA synthesis as shown by dephosphorylation of p-CAD. This result was confirmed by analysis of de-novo pyrimidine synthesis intermediates, uridine monophosphate and uridine, measured by UPLC-ToF-MS. Both were decreased upon ASNase treatment. Except experiments done on ALL cell lines we also detected dephosphorylation of p-S6, p-CAD in primary samples. Regarding the effect on glycolysis we observed inhibition of glucose uptake and decrease of lactate production in cells treated with ASNase. We also detected the decrease of protein levels of c-Myc, the activator of glucose and glutamine catabolism, and glucose transporter 1 (Glut-1). On the contrary, ASNase increased fatty acid oxidation (FAO) followed by the elevation of the capacity of cell respiration and NAD+/NADH ratio in both cell lines. Next, we wanted to elucidate whether ASNase inhibits mTORC1 targets through RagB, the key protein mediating response to general amino acid deprivation. We established a RagB mutant leukemic cell line with constitutive activation of mTORC1 pathway despite deprivation of amino acids. ASNase treatment did not inhibit p-S6 and p-CAD in this cell line. Similarly, ASNase failed to increase FAO in cells with active mTORC1. These results suggest that the effect of ASNase on protein translation, de novo pyrimidine synthesis and FAO is mediated through RagB-mTORC1 pathway. By contrast, c-Myc expression was decreased in both RagB wild type and mutant cells, indicating that ASNase inhibits glycolysis in RagB-mTORC1 independent manner. The activation of FAO has been suggested to have a pro-survival function in leukemic cells under nutrient stress conditions. We tested whether the increase of FAO in ALL cells treated with ASNase also serves to cope with the metabolic stress. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, cells with the inability to increase FAO (RagB mutant) were more sensitive to ASNase compared to RagB wild type cells. Our results show that the inhibitory effect of ASNase on mTORC1 leads not only to apoptosis but also to metabolic reprogramming. We propose that inhibition of protein translation and pyrimidine synthesis are part of apoptotic processes whereas increased FAO and cell respiration represent pro-survival pathway. Altogether, our study suggests that targeting of FAO in leukemic cells resistant to ASNase is a promising new therapeutic strategy. IGA-NT1249, GAUK-632513 Disclosures No relevant conflicts of interest to declare.

scholarly journals PKM2 Inhibition Enhances the Sensitivity of Doxorubicin in ABC Diffuse Large B-Cell Lymphoma Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4336-4336
Author(s):  
Minglang Zhan ◽  
Xiaolei Wei ◽  
Weimin Huang ◽  
Yongqiang Wei ◽  
Ru Feng
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Aerobic Glycolysis ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Metabolic Reprogramming ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: Pyruvate kinase muscle isoenzyme 2 (PKM2) is a key enzyme in aerobic glycolysis and thought to contribute to cancer cell metabolic reprogramming and regulating the reactive oxygen species (ROS). Doxorubicin has been showed to induced activated-B cell types diffuse large B-cell lymphoma (ABC-DLBCL) cells death by ROS accumulation. Our purpose was to evaluate whether PKM2 inhibition could enhance the sensitivity of doxorubicin in ABC-DLBCL. Methods: MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with PKM2 inhibitor, PKM2 shRNA and doxorubicin. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green. Western Blot was used to evaluated the expression of PARP, Mcl1, Bcl2, Bax, Bim, p38 and JNK in ABC-DLBCL cells treated with PKM2 inhibition, PKM2 shRNA and doxorubicin. Results: PKM2 expression was found in both U2932 and SuDHL2 cell lines. Both PKM2 inhibitor and doxorubicin could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. PKM2 inhibitor could enhance the doxorubicin-induced apoptosis. ShRNA was used to knock down the PKM2 expression in ABC-DLBCL cell lines and PKM2 KD cell lines were more sensitive to doxorubicin. PKM2 inhibition could increase the expression of cleaved PARP, Bax, Bim, p38 and JNK as well as decrease Mcl1 and Bcl2 expression Conclusions: PKM2 inhibition could sensitize ABC-DLBCL cell lines to the cytotoxic effects of doxorubicin. Key words: PKM2, Doxorubicin, Diffuse large B cell lymphoma Disclosures No relevant conflicts of interest to declare.

MCL-1 and Mir-181c in GATA2 Mutation Associated Monomac and Familial Myelodysplastic Syndrome

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3807-3807
Author(s):  
Weixin Wang ◽  
Meghan Corrigan-Cummins ◽  
Donald C Vinh ◽  
Amy P Hsu ◽  
Dennis D. Hickstein ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Control Cell ◽  
Leukemia Cell ◽  
Differentially Expressed ◽  
Hematopoietic Stem ◽  
Functional Studies ◽  
Mrna Targets ◽  
Gata2 Deficiency

Abstract Abstract 3807 Background: Somatic and germline mutations in GATA2 were recently identified in patients diagnosed with MonoMAC, the hallmarks of which include mono cytopenia, B-cell and NK-cell lymphopenia, susceptibility to opportunistic infections (e.g. MAC), and a strong propensity to develop hypocellular MDS/AML or CMML. GATA2 mutations were also recently identified in other related disorders: Emberger syndrome (primary lymphedema with myelodysplasia), Familial MDS/AML, and DCML (Dendritic Cell, Monocytes, Lymphoid Deficiency). Family members with GATA2 mutations show variable penetrance and expressivity indicating that other factors may be required for development of disease and phenotype. GATA2 mutations are thought to result in loss of function or haploinsufficiency, but the precise mechanism for the development of cytopenias, immunodeficiency, and susceptibility to MDS remains to be elucidated. MicroRNA (miR) represents a unique mechanism of post-transcriptional gene regulation. In this study we generated microRNA profiles of patient derived MonoMAC cell lines followed by functional studies to identify aberrant miRs and their targets, which could potentially cooperate with GATA2 deficiency in generating hematologic disease. Inducible deletion of Myeloid Leukemia Cell 1 (Mcl1), a member of the Bcl2 family, in mice results in the loss of hematopoietic stem cells (HSCs) and progenitors, and in development of cytopenias. Design: RNA was isolated from EBV-immortalized B cells of 10 healthy controls and 13 MonoMAC patients with MDS and defined mutations in GATA2. microRNA expression profiles were generated using the Agilent high density human microRNA array. Array data were normalized to the data point of 75th percentile signal strength and to a set of spike-in and control probes. The differences between the means of experimental groups were analyzed by Mann-Whitney rank sum test. The miRs with significant p values (p≤ 0.05) and fold change (≥ 2-fold) in both normalization methods were selected for further analysis. TargetScan was utilized to predict the mRNA targets of aberrantly expressed miRs. miR targets were validated by functional studies in the Ly8 cell line. Results: Eight miRs were significantly differentially expressed (≥ 2-fold; p ≤ 0.05) as determined by microRNA microarray profiles. Six miRs showed increased expression in monoMAC cell lines compared to controls (miR-9, −181a-2–3p, −181c, −181c-3p, −486–3p, −582–5p) while two miRs showed significantly decreased expression (miR-223, −424–3p). Among the differentially expressed miRs that were validated by quantitative RT-PCR was miR-181c, which demonstrated a 2.2 fold increase in expression in MonoMAC cell lines (p = 0.013). Among the target transcripts potentially regulated by miR-181c, MCL1 expression was significantly decreased (2 fold; p = 0.018) in monoMAC cell lines in comparison to control cell lines. Transient transfection of miR-181c in Ly8 cells resulted in 40% decrease of MCL1 mRNA level, suggesting that miR-181c negatively regulates MCL1 in MonoMAC. Conclusions: These findings indicate that MonoMAC/GATA2 deficiency is associated with significantly decreased expression of MCL1 possibly through negative regulation involving miR-181c. Deletion of Mcl1 is known to cause apoptosis, loss of HSCs, and cytopenias in murine studies. Thus, down-regulation of MCL1 seen in MonoMAC/GATA2 deficiency may similarly favor unregulated apoptosis and the depletion of hematopoietic progenitors resulting in cytopenias, immunodeficiency, and risk of MDS/AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Metabolic Anti-Cancer Effects of Melatonin: Clinically Relevant Prospects

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3018
Author(s):  
Marek Samec ◽  
Alena Liskova ◽  
Lenka Koklesova ◽  
Kevin Zhai ◽  
Elizabeth Varghese ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Glucose Transporter ◽  
Aerobic Glycolysis ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Effective Prevention ◽  
Anti Cancer ◽  
Glucose 6 Phosphate Dehydrogenase

Metabolic reprogramming characterized by alterations in nutrient uptake and critical molecular pathways associated with cancer cell metabolism represents a fundamental process of malignant transformation. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone secreted by the pineal gland. Melatonin primarily regulates circadian rhythms but also exerts anti-inflammatory, anti-depressant, antioxidant and anti-tumor activities. Concerning cancer metabolism, melatonin displays significant anticancer effects via the regulation of key components of aerobic glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP) and lipid metabolism. Melatonin treatment affects glucose transporter (GLUT) expression, glucose-6-phosphate dehydrogenase (G6PDH) activity, lactate production and other metabolic contributors. Moreover, melatonin modulates critical players in cancer development, such as HIF-1 and p53. Taken together, melatonin has notable anti-cancer effects at malignancy initiation, progression and metastasing. Further investigations of melatonin impacts relevant for cancer metabolism are expected to create innovative approaches supportive for the effective prevention and targeted therapy of cancers.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Koichiro Suemori ◽  
Taichi Azuma ◽  
Yoshihiro Yakushijin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cellular Immunotherapy ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Leukemia Cell Lines

Abstract Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

scholarly journals The Key Role of the WNT/β-Catenin Pathway in Metabolic Reprogramming in Cancers under Normoxic Conditions

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5557
Author(s):  
Alexandre Vallée ◽  
Yves Lecarpentier ◽  
Jean-Noël Vallée
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Warburg Effect ◽  
Target Genes ◽  
Glucose Transporter ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Metabolic Reprogramming ◽  
Pyruvate Dehydrogenase Kinase ◽  
The Warburg Effect

The canonical WNT/β-catenin pathway is upregulated in cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Nuclear β-catenin accumulation is associated with cancer. Hypoxic mechanisms lead to the activation of the hypoxia-inducible factor (HIF)-1α, promoting glycolytic and energetic metabolism and angiogenesis. However, HIF-1α is degraded by the HIF prolyl hydroxylase under normoxia, conditions under which the WNT/β-catenin pathway can activate HIF-1α. This review is therefore focused on the interaction between the upregulated WNT/β-catenin pathway and the metabolic processes underlying cancer mechanisms under normoxic conditions. The WNT pathway stimulates the PI3K/Akt pathway, the STAT3 pathway and the transduction of WNT/β-catenin target genes (such as c-Myc) to activate HIF-1α activity in a hypoxia-independent manner. In cancers, stimulation of the WNT/β-catenin pathway induces many glycolytic enzymes, which in turn induce metabolic reprogramming, known as the Warburg effect or aerobic glycolysis, leading to lactate overproduction. The activation of the Wnt/β-catenin pathway induces gene transactivation via WNT target genes, c-Myc and cyclin D1, or via HIF-1α. This in turn encodes aerobic glycolysis enzymes, including glucose transporter, hexokinase 2, pyruvate kinase M2, pyruvate dehydrogenase kinase 1 and lactate dehydrogenase-A, leading to lactate production. The increase in lactate production is associated with modifications to the tumor microenvironment and tumor growth under normoxic conditions. Moreover, increased lactate production is associated with overexpression of VEGF, a key inducer of angiogenesis. Thus, under normoxic conditions, overstimulation of the WNT/β-catenin pathway leads to modifications of the tumor microenvironment and activation of the Warburg effect, autophagy and glutaminolysis, which in turn participate in tumor growth.

scholarly journals A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2591-2600 ◽  
Author(s):  
Roberta Morosetti ◽  
Dorothy J. Park ◽  
Alexey M. Chumakov ◽  
Isabelle Grillier ◽  
Masaaki Shiohara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Nb4 Cells

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

scholarly journals Mitochondrial genome and its regulator TFAM modulates head and neck tumourigenesis through intracellular metabolic reprogramming and activation of oncogenic effectors

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Yi-Ta Hsieh ◽  
Hsi-Feng Tu ◽  
Muh-Hwa Yang ◽  
Yi-Fen Chen ◽  
Xiang-Yun Lan ◽  
...  
Keyword(s):  
Head And Neck ◽  
Tongue Cancer ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Cellular Level ◽  
Metabolic Reprogramming ◽  
Transport Chain ◽  
Genetically Manipulated ◽  
The Impact

AbstractMitochondrial transcriptional factor A (TFAM) acts as a key regulatory to control mitochondrial DNA (mtDNA); the impact of TFAM and mtDNA in modulating carcinogenesis is controversial. Current study aims to define TFAM mediated regulations in head and neck cancer (HNC). Multifaceted analyses in HNC cells genetically manipulated for TFAM were performed. Clinical associations of TFAM and mtDNA encoded Electron Transport Chain (ETC) genes in regulating HNC tumourigenesis were also examined in HNC specimens. At cellular level, TFAM silencing led to an enhanced cell growth, motility and chemoresistance whereas enforced TFAM expression significantly reversed these phenotypic changes. These TFAM mediated cellular changes resulted from (1) metabolic reprogramming by directing metabolism towards aerobic glycolysis, based on the detection of less respiratory capacity in accompany with greater lactate production; and/or (2) enhanced ERK1/2-Akt-mTORC-S6 signalling activity in response to TFAM induced mtDNA perturbance. Clinical impacts of TFAM and mtDNA were further defined in carcinogen-induced mouse tongue cancer and clinical human HNC tissues; as the results showed that TFAM and mtDNA expression were significantly dropped in tumour compared with their normal counterparts and negatively correlated with disease progression. Collectively, our data uncovered a tumour-suppressing role of TFAM and mtDNA in determining HNC oncogenicity and potentially paved the way for development of TFAM/mtDNA based scheme for HNC diagnosis.

scholarly journals Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation

2018 ◽  
Author(s):  
Zhizhou Ye ◽  
Donald E. Ayer
Keyword(s):  
Protein Synthesis ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Aerobic Glycolysis ◽  
Protein Translation ◽  
Underlying Mechanism ◽  
Metabolic Reprogramming ◽  
Coding Region ◽  
Interacting Protein ◽  
Thioredoxin Interacting Protein

ABSTRACTOncogenic Ras upregulates aerobic glycolysis to meet the bioenergetic and biosynthetic demands of rapidly growing cells. In contrast, Thioredoxin interacting protein (TXNIP) is a potent inhibitor of glucose uptake and is frequently downregulated in human cancers. Our lab previously discovered that Ras activation suppresses TXNIP transcription and translation. In this report, we developed a system to study how Ras affects TXNIP translation in the absence of transcriptional affects. We show that whereas Ras drives a global increase in protein translation, it suppresses TXNIP protein synthesis by reducing the rate at which ribosomes transit the coding region of TXNIP mRNA. To investigate the underlying mechanism(s), we randomized or optimized the codons in the TXNIP message without altering the TXNIP primary amino acid sequence. Translation from these mRNA variants is still repressed by Ras, intimating that mRNA secondary structure, miRNAs, RNA binding proteins, or codon usage do not contribute to the blockade of TXNIP synthesis. Rather, we show that the N-terminus of the growing TXNIP polypeptide is the target for Ras-dependent translational repression. Our work demonstrates how Ras suppresses TXNIP translation elongation in the face of a global upregulation of protein synthesis and provides new insight into Ras-dependent metabolic reprogramming.

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