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1.
Nat Cancer ; 3(4): 418-436, 2022 04.
Article in English | MEDLINE | ID: mdl-35469014

ABSTRACT

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.


Subject(s)
Antibodies, Bispecific , Neoplasms, Glandular and Epithelial , Antibodies, Bispecific/pharmacology , ErbB Receptors/metabolism , Humans , Imidazoles , Neoplasms, Glandular and Epithelial/metabolism , Neoplastic Stem Cells/metabolism , Organoids , Pyrazines , Receptors, G-Protein-Coupled/metabolism
2.
Cell Rep Med ; 1(5): 100074, 2020 08 25.
Article in English | MEDLINE | ID: mdl-33205068

ABSTRACT

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.


Subject(s)
Cell Transformation, Neoplastic/genetics , Congenital Bone Marrow Failure Syndromes/genetics , Congenital Bone Marrow Failure Syndromes/pathology , DNA-Binding Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mutation/genetics , Neutropenia/congenital , Transcription Factors/genetics , Animals , Cell Line , Cell Line, Tumor , Cell Transformation, Neoplastic/pathology , Core Binding Factor Alpha 2 Subunit/genetics , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/pathology , K562 Cells , Mice , Neutropenia/genetics , Neutropenia/pathology , Signal Transduction/genetics
3.
Gut ; 66(6): 1106-1115, 2017 06.
Article in English | MEDLINE | ID: mdl-27670374

ABSTRACT

BACKGROUND AND AIM: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. METHODS: Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. RESULTS: KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. CONCLUSIONS: RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.


Subject(s)
Adenocarcinoma/genetics , Colorectal Neoplasms/genetics , Proto-Oncogene Proteins c-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-raf/genetics , Adenocarcinoma/drug therapy , Animals , Cell Differentiation/drug effects , Cell Polarity/drug effects , Cell Polarity/genetics , Colorectal Neoplasms/drug therapy , Gene Expression , Gene Knockdown Techniques , Humans , Indoles/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Mice , Neoplasm Transplantation , Phenols/pharmacology , Phosphorylation , Primary Cell Culture , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/genetics , Tight Junctions , Tumor Cells, Cultured
4.
Gastroenterology ; 149(3): 692-704, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25962936

ABSTRACT

BACKGROUND & AIMS: Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS: We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS: Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS: Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.


Subject(s)
Biomarkers, Tumor/metabolism , Cell Differentiation , Colonic Neoplasms/enzymology , Neoplastic Stem Cells/enzymology , Paracrine Communication , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Factor/metabolism , Animals , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/genetics , Cell Hypoxia , Cell Proliferation , Coculture Techniques , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Humans , Mice, Nude , Neoplastic Stem Cells/pathology , Paracrine Communication/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/genetics , Signal Transduction , Spheroids, Cellular , Stem Cell Factor/antagonists & inhibitors , Stem Cell Factor/genetics , Time Factors , Tumor Burden , Tumor Cells, Cultured
5.
Clin Cancer Res ; 21(12): 2870-9, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25779952

ABSTRACT

PURPOSE: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. EXPERIMENTAL DESIGN: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy. RESULTS: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy. CONCLUSIONS: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable.


Subject(s)
Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Drug Resistance, Neoplasm , Oxidative Phosphorylation , Sirtuin 1/genetics , Transcription Factors/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Energy Metabolism/drug effects , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Liver Neoplasms/secondary , Mitochondria/genetics , Mitochondria/metabolism , Oxidation-Reduction , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Sirtuin 1/metabolism , Transcription Factors/metabolism
6.
Cancer Res ; 74(22): 6717-30, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25261240

ABSTRACT

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity.


Subject(s)
Colorectal Neoplasms/pathology , Glutathione Peroxidase/physiology , Hydrogen Peroxide/metabolism , Animals , Cell Differentiation , Colorectal Neoplasms/metabolism , Female , Humans , Mice , Mice, SCID , Neoplasm Metastasis , Reactive Oxygen Species/metabolism , Stress, Physiological , Thioredoxin Reductase 1/physiology
7.
Ann Surg ; 259(4): 750-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24253142

ABSTRACT

OBJECTIVE: To assess the contribution of hypoxia and bone marrow-derived cells to aggressive outgrowth of micrometastases after liver surgery. BACKGROUND: Liver surgery generates a microenvironment that fosters aggressive tumor recurrence. These areas are characterized by chronic hypoxia and influx of bone marrow-derived cells. METHODS: The contribution of hematopoietic cell types was studied in mice lacking specific components of the immune system and in irradiated mice lacking all bone marrow-derived cells. Tumor cells were derived from colorectal cancer patients and from a metastatic tumor cell line. Hypoxia-induced changes in stem cell and differentiation marker expression, clone-forming potential, and metastatic capacity were assessed. The effect of vascular clamping on cancer stem cell (CSC) characteristics was performed in mice bearing patient-derived liver metastases. RESULTS: Immune cells and bone marrow-derived cells were not required for aggressive outgrowth of micrometastases in livers treated with surgery. Rather, hypoxia was sufficient to promote invasion and accelerate metastatic outgrowth. This was associated with a rapid loss of differentiation markers and increased expression of CSC markers and clone-forming capacity. Likewise, metastases residing in ischemia-reperfusion-injured liver lobes acquired CSC characteristics. Despite their renowned general resistance to chemotherapy, clone-forming CSCs were readily killed by the hypoxia-activated prodrug tirapazamine. CONCLUSIONS: Surgery-generated hypoxia in the liver causes rapid dedifferentiation of tumor cells into immature CSCs with high clone- and metastasis-forming capacity. The results help explain the phenomenon of aggressive local tumor recurrence after liver surgery and offer a potential strategy to kill aggressive CSCs by hypoxia-activated prodrugs.


Subject(s)
Colorectal Neoplasms/pathology , Hepatectomy , Hypoxia/etiology , Liver Neoplasms, Experimental/secondary , Neoplasm Recurrence, Local/pathology , Neoplasm, Residual/pathology , Postoperative Complications , Animals , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/metabolism , Blotting, Western , Catheter Ablation , Cell Line, Tumor , Flow Cytometry , Hematopoietic Stem Cells/pathology , Hepatectomy/methods , Humans , Hypoxia/metabolism , Hypoxia/pathology , Immunohistochemistry , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasm Invasiveness/pathology , Neoplasm Micrometastasis/pathology , Neoplasm Recurrence, Local/metabolism , Neoplasm, Residual/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Phenotype , Postoperative Complications/metabolism , Postoperative Complications/pathology , Real-Time Polymerase Chain Reaction , Reperfusion Injury/etiology , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Tirapazamine , Triazines/therapeutic use
8.
Blood ; 117(12): 3320-30, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21263150

ABSTRACT

The transcription factor signal transducer and activator of transcription 5 (STAT5) fulfills essential roles in self-renewal in mouse and human hematopoietic stem cells (HSCs), and its persistent activation contributes to leukemic transformation, although little molecular insight into the underlying mechanisms has been obtained. In the present study, we show that STAT5 can impose long-term expansion exclusively on human HSCs, not on progenitors. This was associated with an enhanced cobblestone formation under bone marrow stromal cells of STAT5-transduced HSCs. Hypoxia-induced factor 2α (HIF2α) was identified as a STAT5 target gene in HSCs, and chromatin immunoprecipitation studies revealed STAT5 binding to a site 344 base pairs upstream of the start codon of HIF2α. Lentiviral RNA interference (RNAi)-mediated down-modulation of HIF2α impaired STAT5-induced long-term expansion and HSC frequencies, whereas differentiation was not affected. Glucose uptake was elevated in STAT5-activated HSCs, and several genes associated with glucose metabolism were up-regulated by STAT5 in an HIF2α-dependent manner. Our studies indicate that pathways normally activated under hypoxia might be used by STAT5 under higher oxygen conditions to maintain and/or impose HSC self-renewal properties.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Hematopoietic Stem Cells/metabolism , STAT5 Transcription Factor/physiology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Hypoxia/genetics , Cell Hypoxia/physiology , Cell Proliferation/drug effects , Cells, Cultured , Female , Fetal Blood/cytology , Fetal Blood/metabolism , Gene Expression Regulation/drug effects , Glucose/metabolism , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/physiology , Humans , Infant, Newborn , Models, Biological , Myeloid Progenitor Cells/metabolism , Myeloid Progenitor Cells/physiology , Oxygen/pharmacology , Pregnancy , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism
9.
J Biol Chem ; 286(8): 6061-70, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21169357

ABSTRACT

In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.


Subject(s)
Antigens, CD34 , Cell Differentiation/physiology , Hematopoietic Stem Cells/metabolism , MAP Kinase Signaling System/physiology , Monocytes/metabolism , Proto-Oncogene Proteins/metabolism , ras Proteins/metabolism , Amino Acid Substitution , Angiopoietin-Like Protein 6 , Angiopoietin-like Proteins , Angiopoietins/genetics , Angiopoietins/metabolism , Cell Line , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cytokines/biosynthesis , Cytokines/genetics , Genome-Wide Association Study , Hematopoietic Stem Cells/cytology , Humans , Leukemia/genetics , Leukemia/metabolism , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Monocytes/cytology , Mutation, Missense , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Transduction, Genetic , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , ras Proteins/genetics
10.
Diabetes ; 59(10): 2390-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20622167

ABSTRACT

OBJECTIVE: The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of ß-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates ß-cell mass and function. RESEARCH DESIGN AND METHODS: The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS: Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in ß-cell mass. The lack of ß-cell mass expansion resulted from decreased G(1)-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS: This work defines the importance of S6K in regulation of ß-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces ß-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in ß-cell failure and diabetes in conditions of nutrient overload.


Subject(s)
Insulin-Secreting Cells/cytology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Animals , Apoptosis , Cell Cycle , Cell Division , Cell Size , Glucose/pharmacology , Glucose Tolerance Test , Insulin/genetics , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/enzymology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Transgenic , Multiprotein Complexes , Promoter Regions, Genetic , Proteins , Rats , Signal Transduction , TOR Serine-Threonine Kinases , Transcription Factors/metabolism
11.
Exp Hematol ; 36(10): 1254-65, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18640764

ABSTRACT

OBJECTIVE: Mucin1 is a membrane glycoprotein that is overexpressed in a variety of human cancers. Here, we analyzed the role of Mucin1 in human hematopoietic stem/progenitor cells as well as in acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: Mucin1 expression was determined within the normal stem cell and progenitor compartment, as well as in the AML CD34+ and CD34- subfractions of patient samples. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays in limiting dilution and progenitor frequencies in colony-forming cell (CFC) assays in methylcellulose, and consequences of elevated Mucin1 expression were studied using retroviral overexpression systems in cord blood (CB) CD34+ cells. RESULTS: Ten percent of CB and 5% of peripheral blood CD34+ cells expressed Mucin1. Retroviral overexpression of Mucin1 in CB CD34+ cells resulted in elevated stem cell and progenitor frequencies as determined in LTC-IC and CFC assays without affecting differentiation, which coincided with increased proliferation. Overexpression of intercellular adhesion molecule-1, a ligand for Mucin1, in MS5 stromal cells further increased LTC-IC frequencies. Mucin1 overexpression was associated with increased nuclear factor-kappaB p50 nuclear translocation, suggesting that Mucin1-induced phenotypes involve increased cell survival mechanisms. Finally, we observed increased Mucin1 expression in 70% of the AML cases (n=24), suggesting that elevated Mucin1 levels might be involved in regulating the proliferative potential of the immature leukemic compartment as well. CONCLUSIONS: Our data indicate that hematopoietic stem cells as well as CD34+ AML subfractions are enriched for Mucin1 expression, and that overexpression of Mucin1 in CB cells is sufficient to increase both progenitor and LTC-IC frequencies.


Subject(s)
Fetal Blood/physiology , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid, Acute/genetics , Mucin-1/genetics , Stem Cells/physiology , Antigens, CD/analysis , Antigens, CD34/analysis , Colony-Forming Units Assay , DNA Primers , Flow Cytometry , Humans , Infant, Newborn , Intercellular Adhesion Molecule-1/genetics , Leukemia, Myeloid, Acute/pathology , Polymerase Chain Reaction , Retroviridae/genetics , Up-Regulation
12.
Stem Cells ; 26(7): 1732-42, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18436865

ABSTRACT

Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.


Subject(s)
Antigens, CD34/biosynthesis , Down-Regulation , Erythropoiesis , Megakaryocytes/cytology , STAT5 Transcription Factor/biosynthesis , STAT5 Transcription Factor/physiology , Cell Differentiation , Cell Line, Tumor , Flow Cytometry , Humans , Megakaryocytes/metabolism , Platelet Membrane Glycoprotein IIb/biosynthesis , RNA Interference , Receptors, Thrombopoietin/metabolism , Stem Cell Factor/metabolism , Stem Cells/cytology , Thrombopoietin/metabolism
13.
Mol Cell Biol ; 27(13): 4953-67, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17485446

ABSTRACT

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.


Subject(s)
Adipose Tissue, White/growth & development , Energy Metabolism , Glucose/metabolism , Homeostasis , Polyamines/metabolism , AMP-Activated Protein Kinases , Acetyltransferases/metabolism , Adenosine Triphosphate/metabolism , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue, White/cytology , Adipose Tissue, White/drug effects , Adipose Tissue, White/enzymology , Animals , Body Composition/drug effects , Energy Metabolism/drug effects , Feeding Behavior/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Food Deprivation , Gene Expression Regulation, Enzymologic/drug effects , Glucose Intolerance , Homeostasis/drug effects , Hydrogen Peroxide/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Organ Size/drug effects , Oxidative Phosphorylation/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors , p38 Mitogen-Activated Protein Kinases/metabolism
14.
J Cell Mol Med ; 10(4): 933-45, 2006.
Article in English | MEDLINE | ID: mdl-17125596

ABSTRACT

The N(1)-acetylation of spermidine or spermine by spermidine/spermine N(1)-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSATdeficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion. Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N(1),N(11)-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polyamine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild-type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes.


Subject(s)
Acetyltransferases/physiology , Aging , Insulin Resistance , Polyamines/metabolism , Acetyltransferases/biosynthesis , Acetyltransferases/genetics , Animals , Carbon Tetrachloride/toxicity , Disease Models, Animal , Enzyme Induction , Glucose , Homeostasis , Liver/drug effects , Liver/metabolism , Mice , Mice, Knockout , Spermidine/metabolism , Spermine/analogs & derivatives , Spermine/metabolism , Spermine/toxicity
15.
Diabetes ; 55(2): 318-25, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16443763

ABSTRACT

Proliferation is the major component for maintenance of beta-cell mass in adult animals. Activation of phosphoinositide 3-kinase/Akt-kinase pathway is a critical regulator of beta-cell mass. Pancreatic beta-cell overexpression of constitutively active Akt in mice (caAkt(Tg)) resulted in marked expansion of beta-cell mass by increase in beta-cell proliferation and size. The current studies provide new insights into the molecular mechanisms involved in beta-cell proliferation by Akt. Proliferation of beta-cells in caAkt(Tg) was associated with increased cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 (cdk4) activity. To determine the role of cdk4 in beta-cell proliferation induced by Akt, we generated caAkt(Tg) mice that were homozygous, heterozygous, or nullizygous for cdk4. The results of these studies showed that deletion of one cdk4 allele significantly reduced beta-cell expansion in caAkt(Tg) mice by decreased proliferation. CaAkt(Tg) mice deficient in cdk4 developed beta-cell failure and diabetes. These experiments suggest that Akt induces beta-cell proliferation in a cdk4-dependent manner by regulation of cyclin D1, cyclin D2, and p21 levels. These data also indicate that alteration in levels of these cell cycle components could affect the maintenance of beta-cell mass in basal states and the adaptation of beta-cells to pathological states resulting in diabetes.


Subject(s)
Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclins/metabolism , Insulin-Secreting Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Animals, Newborn/metabolism , Cell Proliferation , Cyclin D2 , Cyclin-Dependent Kinase 4/genetics , Female , Gene Deletion , Gene Expression Regulation, Enzymologic , Glucose/metabolism , Insulin-Secreting Cells/cytology , Male , Mice , Mice, Transgenic , Signal Transduction
16.
Clin Chem Lab Med ; 43(12): 1346-50, 2005.
Article in English | MEDLINE | ID: mdl-16309371

ABSTRACT

BACKGROUND: The enzyme catalase is the main regulator of hydrogen peroxide metabolism. Deficiency of catalase may cause high concentrations of hydrogen peroxide and increase the risk of the development of pathologies for which oxidative stress is a contributing factor, for example, type 2 diabetes mellitus. Catalase deficiency has been reported to be associated with increased frequency of diabetes mellitus in a cohort of patients in Hungary. In this cohort, the majority of mutations in the catalase gene occur in exon 2. METHODS: Type 2 diabetic patients (n=308) were evaluated for mutations in intron 1 (81 bp), exon 2 (172 bp) and intron 2 (13 bp) of the catalase gene. Screening for mutations utilized PCR single-strand conformational polymorphism (SSCP) and PCR heteroduplex methods. Verification of detected mutations was by nucleotide sequence analysis. RESULTS: A total of 11 catalase gene mutations were detected in the 308 subjects (3.57%, p<0.001). Five of the 11 were at two previously reported mutation sites: exon 2 (79) G insertion and (138) GA insertion. Six of the 11 were at five previously unreported catalase mutation sites: intron 1 (60) G-->T; intron 2 (7) G-->A and (5) G-->C; exon 2 (96) T-->A; and exon 2 (135) T-->A. The novel missense mutations on exon 2 (96 and 135) are associated with 59% and 48% decreased catalase activity, respectively; the novel G-->C mutation on intron 2 (5) is associated with a 62% decrease in catalase activity. Mutations detected on intron 1 (60) and intron 2 (7) showed no change in catalase activity. The G-->C mutation on intron 2 (5) might be a splicing mutation. The two missense mutations on exon 2 (96) and (135) cause substitutions of amino acids 53 (Asp-->Glu) and 66 (Glu-->Cys) of the catalase protein. These are close to amino acids that are important for the binding of heme to catalase, 44 (Val) and 72-75 (Arg, Val, Val, His). Changes in heme binding may be responsible for the activity losses. CONCLUSION: Mutations that cause decreased catalase activity may contribute to susceptibility to inherited type 2 diabetes mellitus. Exon 2 and neighboring introns of the catalase gene may be minor hot spots for type 2 diabetes mellitus susceptibility mutations.


Subject(s)
Acatalasia/genetics , Catalase/genetics , Diabetes Mellitus, Type 2/genetics , Polymorphism, Single-Stranded Conformational , Acatalasia/blood , Aged , Amino Acid Substitution , Catalase/blood , DNA Mutational Analysis , Exons/genetics , Female , Heme/metabolism , Humans , Hungary , Introns/genetics , Male , Mass Screening/methods , Middle Aged , Mutation , Oxidative Stress , Polymerase Chain Reaction/methods
17.
Diabetes ; 54(4): 968-75, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15793234

ABSTRACT

An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet beta-cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in beta-cell survival, we sought to determine whether this pathway is involved in ER stress-induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress-induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress-induced apoptosis and reduced levels of phospho-glycogen synthase kinase 3beta (GSK3beta). In control cells, ER stress-induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3beta. To further assess the role of GSK3beta in ER stress-induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3beta expression. These cells were found to resist ER stress-induced apoptosis. These results illustrate that ER stress-induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3beta pathway and that GSK3beta represents a novel target for agents to promote beta-cell survival.


Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/physiology , Glycogen Synthase Kinase 3/metabolism , Insulin/physiology , Islets of Langerhans/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/drug effects , Brefeldin A/pharmacology , Cell Line, Tumor , Gene Expression , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Insulin-Like Growth Factor I/pharmacology , Insulinoma , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt , Signal Transduction , Thapsigargin/pharmacology , Tunicamycin/pharmacology
18.
J Clin Invest ; 114(7): 928-36, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15467831

ABSTRACT

The insulin and IGF signaling pathways are critical for development and maintenance of pancreatic beta cell mass and function. The serine-threonine kinase Akt is one of several mediators regulated by these pathways. We have studied the role of Akt in pancreatic beta cell physiology by generating transgenic mice expressing a kinase-dead mutant of this enzyme in beta cells. Reduction of Akt activity in transgenic animals resulted in impaired glucose tolerance due to defective insulin secretion. The mechanisms involved in dysregulation of secretion in these mice lie at the level of insulin exocytosis and are not the result of abnormalities in glucose signaling or function of voltage-gated Ca2+ channels. Therefore, transgenic mice showed increased susceptibility to developing glucose intolerance and diabetes following fat feeding. These observations suggest that Akt plays a novel and important role in the regulation of distal components of the secretory pathway and that this enzyme represents a therapeutic target for improvement of beta cell function in diabetes.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Insulin/metabolism , Islets of Langerhans/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/physiology , Blood Glucose/metabolism , Body Weight , Calcium/metabolism , Culture Techniques , Diabetes Mellitus, Experimental/genetics , Dietary Fats , Disease Susceptibility , Gene Expression Regulation , Insulin Resistance/physiology , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Signal Transduction/physiology
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