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1.
Nat Commun ; 15(1): 2203, 2024 Mar 11.
Article in English | MEDLINE | ID: mdl-38467616

ABSTRACT

The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a more minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cell intratumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.


Subject(s)
CD8-Positive T-Lymphocytes , Neoplasms , Humans , CD8-Positive T-Lymphocytes/metabolism , Mitochondria/metabolism , Neoplasms/pathology , Metabolic Networks and Pathways , Cell Movement
2.
Blood Adv ; 7(13): 3265-3275, 2023 07 11.
Article in English | MEDLINE | ID: mdl-36930820

ABSTRACT

Transcription factor Forkhead box P1 (FOXP1) belongs to the same protein family as the FOXOs that are well-known regulators of murine hematopoietic stem progenitor cell (HSPC) maintenance via dampening oxidative stress. FOXP1 and FOXOs can play opposite, or similar, roles depending on cell context; they can crossregulate each other's expression. In a previous study, we have shown that FOXP1 contributes to healthy human HSPC and acute myeloid leukemia (AML) cell growth. Here, we investigated the role of FOXP1 in HSPCs and AML cell oxidative stress defense in a human context. FOXP1 expression level was associated with an inferior survival outcome in patients with cytogenetically normal AML. FOXP1 knockdown enhanced superoxide anion levels of human-committed CD34+CD38+ cells but not stem cell-enriched CD34+CD38- HSPCs or AML cells in vitro. FOXP1 knockdown triggered enhanced NRF2 activity and increased cell oxidative stress. FOXP1 had no impact on FOXO1/3/4 expression in these cells; genetic and pharmacological inhibition of FOXOs did not change superoxide anion levels of human HSPCs or AML cells. Moreover, FOXP1 antioxidant activity was independent of changes in expression of superoxide dismutase 1 and 2 or catalase. Instead, FOXP1 upregulated expression of the stress sensor SIRT1 by stabilizing SIRT1 protein. FOXP1 loss sensitized AML cells to chemotherapy. Together, this study identified FOXP1 as a new safeguard against myeloid progenitor oxidative stress, which works independently of FOXOs but through SIRT1 and contributes to AML chemoresistance. It proposes FOXP1 expression/activity as a promising target to overcome drug resistance of AML HSPCs.


Subject(s)
Leukemia, Myeloid, Acute , Sirtuin 1 , Humans , Animals , Mice , Sirtuin 1/genetics , Sirtuin 1/metabolism , Superoxides/metabolism , Leukemia, Myeloid, Acute/genetics , Hematopoietic Stem Cells/metabolism , Oxidative Stress , Repressor Proteins/genetics , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism
3.
Haematologica ; 107(1): 268-283, 2022 Jan 01.
Article in English | MEDLINE | ID: mdl-33241676

ABSTRACT

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFß-dependent and mediated by SMAD7, a TGFß- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5'-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFß superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.


Subject(s)
DNA-Binding Proteins , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Smad7 Protein , Transcription Factors , Adult , Animals , Cell Cycle , Epigenesis, Genetic , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Myelodysplastic Syndromes/genetics , RNA, Messenger , Smad7 Protein/genetics
4.
Oncotarget ; 6(5): 2794-811, 2015 Feb 20.
Article in English | MEDLINE | ID: mdl-25605239

ABSTRACT

The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.


Subject(s)
Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Signal Transduction , Transcription, Genetic , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Carrier Proteins/genetics , Cell Line, Tumor , DNA-Binding Proteins , Female , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Leukemia, Myeloid, Acute/genetics , Male , Middle Aged , Phosphorylation , Primary Cell Culture , Prognosis , RNA Interference , RNA, Messenger/metabolism , Transcription Factors , Transfection , Tumor Cells, Cultured , Up-Regulation
5.
Oncotarget ; 4(9): 1438-48, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23988457

ABSTRACT

The retinoid-responsive gene CXXC5 localizes to the 5q31.2 chromosomal region and encodes a retinoid-inducible nuclear factor (RINF) that seems important during normal myelopoiesis. We investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients, and a wide variation in CXXC5/RINF mRNA levels was observed both in the immature leukemic myeloblasts and in immature acute lymphoblastic leukemia cells. Furthermore, patients with low-risk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. This association with prognosis was seen both when investigating (i) an unselected patient population as well as for patients with (ii) normal cytogenetic and (iii) core-binding factor AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells.


Subject(s)
Carrier Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/physiology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Growth Processes/physiology , Cohort Studies , DNA-Binding Proteins , Humans , K562 Cells , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis , Transcription Factors
6.
Mol Pharmacol ; 83(5): 1057-65, 2013 May.
Article in English | MEDLINE | ID: mdl-23455313

ABSTRACT

Acute promyelocytic leukemia (APL) is characterized by granulopoietic differentiation arrest at the promyelocytic stage. In most cases, this defect can be overcome by treatment with all-trans-retinoic acid (ATRA), leading to complete clinical remission. Cyclic AMP signaling has a key role in retinoid treatment efficacy: it enhances ATRA-induced maturation in ATRA-sensitive APL cells (including NB4 cells) and restores it in some ATRA-resistant cells (including NB4-LR1 cells). We show that the two cell types express identical levels of the Cα catalytic subunit and comparable global cAMP-dependent protein kinase A (PKA) enzyme activity. However, the maturation-resistant NB4-LR1 cells have a PKA isozyme switch: compared with the NB4 cells, they have decreased content of the juxtanuclearly located PKA regulatory subunit IIα and PKA regulatory subunit IIß, and a compensatory increase of the generally cytoplasmically distributed PKA-RIα. Furthermore, the PKA regulatory subunit II exists mainly in the less cAMP-responsive nonautophosphorylated state in the NB4-LR1 cells. By the use of isozyme-specific cAMP analog pairs, we show that both PKA-I and PKA-II must be activated to achieve maturation in NB4-LR1 as well as NB4 cells. Therefore, special attention should be paid to activating not only PKA-I but also PKA-II in attempts to enhance ATRA-induced APL maturation in a clinical setting.


Subject(s)
Antineoplastic Agents/pharmacology , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/pathology , Tretinoin/pharmacology , Cell Differentiation/drug effects , Cell Line, Tumor , Cyclic AMP/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Humans , Isoenzymes/metabolism , Leukemia, Promyelocytic, Acute/enzymology
7.
Mol Cancer Ther ; 11(11): 2384-93, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22933702

ABSTRACT

Telomerase, a ribonucleoprotein complex mainly composed of the reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and the RNA component (hTR), is a key enzyme of cancer progression. That aggressive stage 4-neuroblastoma expressed high levels of telomerase activity, whereas favorable tumors had no or little telomerase expression and activity, prompted us to investigate the role of this enzyme in this tumor model of altered proliferation, neuronal differentiation, and apoptosis. A human MYCN-amplified neuroblastoma cell line (IGR-N-91) was engineered to stably express either the normal hTERT protein (WT-hTERT) or a catalytically inactive dominant-negative mutant of this protein (DN-hTERT). We showed that DN-hTERT expression inhibited the endogenous hTERT in the malignant neuroblasts without telomere shortening nor loss of in vitro proliferative capacity. Importantly, DN-hTERT expression induced major changes in cell morphology of neuroblasts that switched them from a neuronal to a substrate adherent phenotype, which was more prone to apoptosis and lost their tumorigenic properties in nude mice. These biologic effects arose from modifications in the expression of genes involved in both apoptosis and neuroblastoma biology. Taken together these results highlighted the functional relevance of noncanonical functions of hTERT in the determination of neuroblast cell fate. Therefore, our results envision new therapeutic strategies for metastatic neuroblastoma therapeutic management.


Subject(s)
Biocatalysis , Genes, Dominant/genetics , Mutant Proteins/metabolism , Neuroblastoma/enzymology , Neuroblastoma/pathology , Telomerase/metabolism , Animals , Apoptosis , Caspase 8/metabolism , Cell Line, Tumor , Cell Shape , Cell Transformation, Neoplastic/pathology , Child , Genome, Human/genetics , Humans , Male , Mice , Mice, Nude , N-Myc Proto-Oncogene Protein , Neuroblastoma/genetics , Nuclear Proteins , Oncogene Proteins , Phenotype , Telomere Homeostasis , Transduction, Genetic , Tumor Suppressor Protein p53/metabolism
8.
Blood ; 118(9): 2551-5, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21734233

ABSTRACT

TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34(+) cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34(+) cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.


Subject(s)
5-Methylcytosine/metabolism , Cytosine/analogs & derivatives , DNA Methylation/genetics , DNA-Binding Proteins/physiology , Erythropoiesis/genetics , Hematopoietic Stem Cells/cytology , Myelopoiesis/genetics , Proto-Oncogene Proteins/physiology , RNA Interference , RNA, Small Interfering/genetics , Cell Line, Tumor , Cell Lineage , Colony-Forming Units Assay , Cytosine/biosynthesis , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Dioxygenases , Erythropoiesis/physiology , Fetal Blood/cytology , Genetic Vectors/genetics , Granulocytes/metabolism , Granulocytes/pathology , Humans , Lentivirus/genetics , Monocytes/metabolism , Monocytes/pathology , Mutation , Myelopoiesis/physiology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/administration & dosage
9.
Mol Cancer Ther ; 10(5): 711-9, 2011 May.
Article in English | MEDLINE | ID: mdl-21364010

ABSTRACT

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However, despite an overall significant hematological and cytogenetic response, imatinib therapy may favor the emergence of drug-resistant clones, ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression, either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid, a clinically used drug. Furthermore, we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore, combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Telomerase/metabolism , Apoptosis/drug effects , Benzamides , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/genetics , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , HEK293 Cells , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Piperazines/chemistry , Pyrimidines/chemistry , Telomerase/genetics , Transcription, Genetic/drug effects , Tretinoin/chemistry , Tretinoin/pharmacology
10.
Blood ; 113(14): 3172-81, 2009 Apr 02.
Article in English | MEDLINE | ID: mdl-19182210

ABSTRACT

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene.


Subject(s)
Carrier Proteins/physiology , Hematologic Neoplasms/genetics , Intracellular Signaling Peptides and Proteins/physiology , Myelopoiesis/genetics , Amino Acid Sequence , Carrier Proteins/genetics , DNA-Binding Proteins , Gene Expression Profiling , Gene Expression Regulation/drug effects , Granulocyte Precursor Cells/drug effects , Granulocyte Precursor Cells/physiology , HL-60 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , K562 Cells , Models, Biological , Molecular Sequence Data , Myelopoiesis/drug effects , Oligonucleotide Array Sequence Analysis , Sequence Homology, Amino Acid , Transcription Factors , Tretinoin/pharmacology , Tumor Cells, Cultured
11.
Mol Cancer ; 6: 31, 2007 Apr 23.
Article in English | MEDLINE | ID: mdl-17451600

ABSTRACT

BACKGROUND: Relapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML. RESULTS: About 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75. CONCLUSION: Our results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.


Subject(s)
Apoptosis , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Intercellular Signaling Peptides and Proteins/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Lymphocyte Activation/genetics , Alternative Splicing/drug effects , Alternative Splicing/genetics , Amino Acid Sequence , Apoptosis/drug effects , Base Sequence , Cell Line, Tumor , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Daunorubicin/pharmacology , Exons/genetics , Gene Expression Regulation, Neoplastic/drug effects , Genes, Neoplasm , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Molecular Sequence Data , Neoplasm Recurrence, Local , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
12.
J Cell Sci ; 119(Pt 13): 2797-806, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16772337

ABSTRACT

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.


Subject(s)
DNA-Binding Proteins/analysis , Phosphoproteins/analysis , RNA-Binding Proteins/analysis , Telomerase/analysis , Antibodies/metabolism , Antibody Specificity , Cell Differentiation , Cells, Cultured , Cross Reactions , DNA-Binding Proteins/deficiency , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Gene Expression Profiling , HeLa Cells , Humans , Immunoprecipitation , Mass Spectrometry , Peptide Mapping , RNA, Messenger/metabolism , Telomerase/deficiency , Nucleolin
13.
BMC Biochem ; 7: 13, 2006 Apr 25.
Article in English | MEDLINE | ID: mdl-16638120

ABSTRACT

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing epsilon-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal alpha-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-alpha-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-alpha-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-alpha-acetylation in human cells as compared to lower organisms which only have one ARD.


Subject(s)
Acetyltransferases/genetics , Gene Duplication , Acetylation , Acetyltransferases/biosynthesis , Acetyltransferases/isolation & purification , Acetyltransferases/metabolism , Acetyltransferases/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation/drug effects , Cell Line/metabolism , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Chromosomes, Human, Pair 4/genetics , Cloning, Molecular , Enzyme Induction , Evolution, Molecular , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/isolation & purification , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Macropodidae/genetics , Mice , Models, Molecular , Molecular Sequence Data , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , Phylogeny , Protein Conformation , Protein Processing, Post-Translational , RNA, Messenger/biosynthesis , Rats , Retroelements/genetics , Sequence Alignment , Sequence Homology , Species Specificity , Tretinoin/pharmacology
14.
Oncogene ; 23(45): 7469-74, 2004 Sep 30.
Article in English | MEDLINE | ID: mdl-15326479

ABSTRACT

Human telomerase has been implicated in cell immortalization and cancer. Recent works suggest that telomerase confers additional function required for tumorigenesis that does not depend on its ability to maintain telomeres. This new action may influence tumor therapy outcomes by yet unraveled mechanisms. Here, we show that overexpression of the catalytic subunit of telomerase (hTERT) protects a maturation-resistant acute promyelocytic leukemia (APL) cell line from apoptosis induced by the tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL) and not from apoptosis induced by chemotherapeutic drugs such as etoposide or cisplatin. Conversely, in these cells, TRAIL-induced cell death is magnified by all-trans retinoic acid (ATRA) treatment, independently of telomerase activity on telomeres. Of note, this response is subordinated neither to maturation nor to telomere shortening. This work underlines that retinoids and death receptor signaling cross-talks offer new perspectives for antitumor therapy.


Subject(s)
Apoptosis/physiology , Membrane Glycoproteins/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction , Telomerase/metabolism , Telomerase/physiology , Telomere , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Apoptosis Regulatory Proteins , DNA-Binding Proteins , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Membrane Glycoproteins/physiology , Receptors, Tumor Necrosis Factor/metabolism , TNF-Related Apoptosis-Inducing Ligand , Tretinoin/pharmacology , Tumor Necrosis Factor-alpha/physiology
15.
Oncogene ; 22(57): 9142-50, 2003 Dec 11.
Article in English | MEDLINE | ID: mdl-14668795

ABSTRACT

Retinoids modulate growth and differentiation of cancer cells through activation of gene transcription via the nuclear retinoic-acid receptors (RAR) and retinoid-X receptors (RXR). Their use in differentiation therapy of acute promyelocytic leukemia (APL) represents a model concept for reprogramming cancer cells. However, they also regulate antiproliferative genes whose functions do not mechanistically concur to this program. Recently, we have shown that, independently of maturation, a long-term all-trans retinoic acid (ATRA) treatment of the maturation-resistant APL cell line (NB4-LR1) represses telomerase (hTERT), leading to telomere shortening and death. Using retinoid-receptor-specific agonists, we demonstrate herein that cross-talk between RARalpha and RXR dual-liganded to their respective agonists resulted in strong synergistic downregulation of hTERT and subsequent cell death. Importantly, unlike ATRA, this synergy was obtained at very low agonist concentrations and occurred in other ATRA maturation-resistant APL cells. These findings provide the first demonstration that dual-liganded RXR and RARalpha signaling should allow efficient targeting of telomerase in differentiation-resistant tumor cells. Such a combination therapy might hold promise in clinic to avoid side effects of ATRA whose administration can indiscriminately activate all RARs. Given the tissue-specific expression of RARs, a tissue-selective therapy targeting telomerase in tumor cells by synthetic agonists can be envisioned.


Subject(s)
Cell Death/drug effects , Receptors, Retinoic Acid/agonists , Telomerase/antagonists & inhibitors , Transcription Factors/agonists , Tretinoin/pharmacology , Cell Division/drug effects , DNA-Binding Proteins , Drug Synergism , Genetic Vectors , Humans , Kinetics , Leukemia, Promyelocytic, Acute , Plasmids , Receptors, Retinoic Acid/genetics , Recombinant Fusion Proteins , Retinoic Acid Receptor alpha , Retinoid X Receptors , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/genetics , Telomerase/metabolism , Telomere/ultrastructure , Transcription Factors/genetics , Transfection , Tumor Cells, Cultured
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