RESUMEN

Eradicating leukemia requires a deep understanding of the interaction between leukemic cells and their protective microenvironment. The CXCL12/CXCR4 axis has been postulated as a critical pathway dictating leukemia stem cell (LSC) chemoresistance in AML due to its role in controlling cellular egress from the marrow. Nevertheless, the cellular source of CXCL12 in the acute myeloid leukemia (AML) microenvironment and the mechanism by which CXCL12 exerts its protective role in vivo remain unresolved. Here, we show that CXCL12 produced by Prx1+ mesenchymal cells but not by mature osteolineage cells provide the necessary cues for the maintenance of LSCs in the marrow of an MLL::AF9-induced AML model. Prx1+ cells promote survival of LSCs by modulating energy metabolism and the REDOX balance in LSCs. Deletion of Cxcl12 leads to the accumulation of reactive oxygen species and DNA damage in LSCs, impairing their ability to perpetuate leukemia in transplantation experiments, a defect that can be attenuated by antioxidant therapy. Importantly, our data suggest that this phenomenon appears to be conserved in human patients. Hence, we have identified Prx1+ mesenchymal cells as an integral part of the complex niche-AML metabolic intertwining, pointing towards CXCL12/CXCR4 as a target to eradicate parenchymal LSCs in AML.

Asunto(s)

Médula Ósea , Leucemia Mieloide Aguda , Médula Ósea/metabolismo , Metabolismo Energético , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Oxidación-Reducción , Microambiente Tumoral

RESUMEN

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Asunto(s)

Aldehído Oxidorreductasas/fisiología , Carbolinas/farmacología , Ciclohexilaminas/farmacología , Ferroptosis/efectos de los fármacos , Hematopoyesis/fisiología , Leucemia Mieloide Aguda/enzimología , Proteínas de Neoplasias/fisiología , Fenilendiaminas/farmacología , Aldehído Oxidorreductasas/genética , Aldehídos/farmacología , Animales , Línea Celular Tumoral , Citarabina/administración & dosificación , Doxorrubicina/administración & dosificación , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Peroxidación de Lípido , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Ácido Oléico/farmacología , Proteínas de Fusión Oncogénica/fisiología , Oxidación-Reducción , Estrés Oxidativo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidores , Fosfolípido Hidroperóxido Glutatión Peroxidasa/fisiología

RESUMEN

Aprior phase I/II trial of bortezomib/tacrolimus/methotrexate prophylaxis after human leukocyte antigen (HLA)-mismatched reduced intensity conditioning allogeneic hematopoietic stem cell transplantation documented low acute graft-versus-host disease incidence, with promising overall and progression-free survival. We performed an open-label three-arm 1:1:1 phase II randomized controlled trial comparing grade II-IV acute graft-versus-host disease between conventional tacrolimus/methotrexate (A) versus bortezomib/tacrolimus/methotrexate (B), and versus bortezomib/sirolimus/tacrolimus (C), in reduced intensity conditioning allogeneic transplantation recipients lacking HLA-matched related donors. The primary endpoint was grade II-IV acute graft-versus-host disease incidence rate by day +180. One hundred and thirty-eight patients (A 46, B 45, C 47) with a median age of 64 years (range: 24-75), varying malignant diagnoses and disease risk (low 14, intermediate 96, high/very high 28) received 7-8/8 HLA-mismatched (40) or matched unrelated donor (98) grafts. Median follow up in survivors was 30 months (range: 14-46). Despite early immune reconstitution differences, day +180 grade II-IV acute graft-versus-host disease rates were similar (A 32.6%, B 31.1%, C 21%; P=0.53 for A vs B, P=0.16 for A vs C). The 2-year non-relapse mortality incidence was similar (A 14%, B 16%, C 6.4%; P=0.62), as were relapse (A 32%, B 32%, C 38%; P=0.74), chronic graft-versus-host disease (A 59%, B 60% C 55%; P=0.66), progression-free survival (A 54%, B 52%, C 55%; P=0.95), and overall survival (A 61%, B 62%, C 62%; P=0.98). Overall, the bortezomib-based regimens evaluated did not improve outcomes compared with tacrolimus/methotrexate therapy. clinicaltrials.gov Identifier: 01754389.

Asunto(s)

Bortezomib/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/métodos , Terapia de Inmunosupresión/métodos , Acondicionamiento Pretrasplante/métodos , Adulto , Anciano , Aloinjertos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedades Hematológicas/mortalidad , Enfermedades Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Análisis de Supervivencia , Tacrolimus/uso terapéutico , Adulto Joven

RESUMEN

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.

Asunto(s)

Epigenómica , Células Madre Hematopoyéticas/citología , Animales , Linaje de la Célula , Células Clonales/citología , Fluorescencia , Hematopoyesis , Inflamación/patología , Ratones , Transcripción Genética

RESUMEN

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Asunto(s)

Acetil-CoA Carboxilasa/metabolismo , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Leucemia Mieloide Aguda/metabolismo , Prolina/metabolismo , Acetil-CoA Carboxilasa/antagonistas & inhibidores , Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/genética , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Redes y Vías Metabólicas/genética , Ratones , Ratones Endogámicos NOD , Modelos Moleculares , Trasplante de Neoplasias , Oxidación-Reducción , Prolina/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Homología Estructural de Proteína , Análisis de Supervivencia

RESUMEN

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

RESUMEN

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.

Asunto(s)

Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Heparitina Sulfato/biosíntesis , N-Acetilglucosaminiltransferasas/metabolismo , Células del Estroma/metabolismo , Acondicionamiento Pretrasplante , Animales , Anticoagulantes/farmacología , Unión Competitiva/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Proteínas Fluorescentes Verdes/genética , Heparina/farmacología , Heparitina Sulfato/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Células del Estroma/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16(INK4a) expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury.

Asunto(s)

Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Receptores Purinérgicos P2Y/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Envejecimiento/efectos de la radiación , Animales , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Senescencia Celular/fisiología , Senescencia Celular/efectos de la radiación , Desarrollo Embrionario/fisiología , Desarrollo Embrionario/efectos de la radiación , Femenino , Hematopoyesis/fisiología , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/citología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2Y/deficiencia , Receptores Purinérgicos P2Y/genética , Transducción de Señal , Estrés Fisiológico/fisiología , Estrés Fisiológico/efectos de la radiación

RESUMEN

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Asunto(s)

Médula Ósea/metabolismo , Oxígeno/análisis , Animales , Arterias/metabolismo , Médula Ósea/irrigación sanguínea , Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Busulfano/farmacología , Hipoxia de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hipoxia/diagnóstico , Hipoxia/metabolismo , Mediciones Luminiscentes , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía , Nestina/metabolismo , Oxígeno/metabolismo , Fotones , Nicho de Células Madre/efectos de los fármacos , Nicho de Células Madre/efectos de la radiación

RESUMEN

Distinctive metabolism associated with particular cell states is increasingly being defined in normal and malignant cells. Ito et al. (2012) now show that fatty acid oxidation is associated with hematopoietic stem cells and determines whether they undergo symmetric or asymmetric cell division, driving a fundamental property of the stem cell state.

Asunto(s)

Leucemia Mieloide Aguda/metabolismo , Microambiente Tumoral , Humanos , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/etiología , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/metabolismo

RESUMEN

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.

Asunto(s)

Factores de Transcripción Forkhead/metabolismo , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Antígenos CD34/metabolismo , Apoptosis , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Proteína Forkhead Box O3 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo

RESUMEN

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

Asunto(s)

Anemia/etiología , Anemia/prevención & control , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Inflamación/complicaciones , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Proteínas Portadoras/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Hep G2 , Hepcidinas , Humanos , Interleucina-6/farmacología , Ratones , Ratones Endogámicos C57BL , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Trementina/toxicidad , Pez Cebra , Proteínas de Pez Cebra/metabolismo

RESUMEN

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Asunto(s)

Metabolismo Energético , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Autofagia , Médula Ósea/metabolismo , Médula Ósea/patología , Ciclo Celular , Proliferación Celular , Supervivencia Celular , Activación Enzimática , Femenino , Hematopoyesis , Células Madre Hematopoyéticas/patología , Homeostasis , Metabolismo de los Lípidos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Complejos Multiproteicos , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

Asunto(s)

Células de la Médula Ósea/citología , Células Madre Hematopoyéticas/citología , Animales , Movimiento Celular/fisiología , Citometría de Flujo/métodos , Trasplante de Células Madre Hematopoyéticas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Coloración y Etiquetado/métodos

RESUMEN

In the search for novel genes involved in the paclitaxel resistance phenotype, prior studies of gene expression in paclitaxel-resistant cell lines and their paired drug-sensitive parental lines using high-density Affymetrix GeneChip arrays identified guanylate-binding protein 1 (GBP1) gene as an overexpressed transcript. The GBP1 gene encodes a large GTPase that is induced by interferon gamma (IFN-gamma) in a variety of eukaryotic cells. In this report we characterize GBP1 and demonstrate that GBP1 expression is consistently upregulated in 7 of 8 paclitaxel or doxorubicin-resistant human cancer cell lines as compared to its expression in the relevant drug-sensitive parental lines. Analysis of GBP1 expression using the Cancer Profiling Array showed that GBP1 is ubiquitously expressed with no significant difference in expression levels between normal and tumor tissue. Parallel analysis of the Cancer Cell Line Profiling Array determined that GBP1 expression in a majority of cell lines derived from human tumors of different tissue origin was induced to variable levels following exposure to multiple stress agents including paclitaxel and doxorubicin. Importantly, stable expression of a GBP1 transgene in the paclitaxel-sensitive ovarian cancer cell line OVCAR8 was sufficient to confer moderate paclitaxel resistance. Our data suggest that increased expression of the GBP1 gene may play an important role in the development of multi-drug resistance (MDR).

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Resistencia a Antineoplásicos/genética , Proteínas de Unión al GTP/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Paclitaxel/farmacología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Arriba

RESUMEN

PURPOSE: To identify genes involved in the paclitaxel resistance phenotype. METHODS: High-density Affymetrix HG-U95Av2 microarrays were used to quantify gene expression in the resulting cell lines, SKOV-3TR, OVCAR8TR and MCF-7TR, and their drug-sensitive parental lines, SKOV-3, OVCAR8 and MCF-7. RESULTS: Three paclitaxel-resistant human ovarian and breast cancer cell lines were established. We identified 790 (SKOV-3TR), 689 (OVCAR8TR) and 964 (MCF-7TR) transcripts that were more than twofold overexpressed relative to their expression in the corresponding parental cell line. A comparison of these transcripts identified eight genes that were significantly overexpressed in all three drug-resistant daughter cell lines. These genes included MDR1, a gene often implicated in both in vitro and in vivo resistance to multiple chemotherapeutics, including paclitaxel. The remaining seven genes have not been previously associated with resistance to paclitaxel in human cancer. Furthermore, we identified 815 (SKOV-3TR), 430 (OVCAR8TR) and 332 (MCF-7TR) transcripts that were more than twofold decreased relative to their expression in the corresponding parental cell line. Comparison of these transcripts identified three genes that were significantly underexpressed in all three drug-resistant cell lines, none of which have been previously associated with paclitaxel resistance. CONCLUSIONS: Our results confirm that the paclitaxel resistance phenotype is associated with a large number of transcriptional changes. In addition, acquired paclitaxel resistance was associated with distinct transcriptional changes in each of the cell lines studied, suggesting that paclitaxel resistance is a complex phenotype that can arise through multiple mechanisms.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Paclitaxel/farmacología , Femenino , Genes MDR , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

OBJECTIVES: Cytogenetic evaluation of product of conception (POC) is essential to determine the cause of pregnancy loss and aid the prenatal diagnosis of subsequent pregnancies. The purpose of this study is twofold. (1) To profile cytogenetic abnormalities, their relationship with maternal and gestational age and analyze sex ratios in our case series of 2052 consecutive samples of POC referred to the Baystate Medical Center, Laboratory Genetics between January 1992 and January 1999. (2) To present a comprehensive review of such data published in the last 15 years, in order to study temporal differences in the above parameters and make this information readily available for cytogeneticists and genetic counselors. MATERIALS AND METHODS: Data was entered and analyzed in Epi Info version 6.0 using the Z-test, chi-square test of significance and linear correlation coefficient. RESULTS AND CONCLUSIONS: The profile of cytogenetic abnormalities detected in POC has not changes significantly over time. The mean maternal age in our study (overall and for trisomies) was higher than that reported previously, which is consistent with the noted trend of increasing age at pregnancy in recent years. Our study provides evidence that abnormal karyotypes are aborted earlier and that tetraploidies have the least survival amongst all abnormalities. The higher mean gestational age for trisomic abortions in our study, as compared with previously reported figures, can be attributed to the increasing practice of active maternal screening with subsequent therapeutic abortions. Analysis of sex ratios may reaffirm a female specific developmental disadvantage in early stages of pregnancy.

Asunto(s)

Aberraciones Cromosómicas , Cigoto/metabolismo , Cigoto/patología , Adulto , Análisis Citogenético , Pérdida del Embrión/epidemiología , Pérdida del Embrión/patología , Femenino , Edad Gestacional , Humanos , Masculino , Edad Materna , Persona de Mediana Edad , Embarazo , Razón de Masculinidad , Técnicas de Cultivo de Tejidos , Trisomía/genética , Trisomía/patología

RESUMEN

Previous studies directed at identifying paclitaxel resistance genes in a paclitaxel-resistant subclone of the human ovarian cancer cell line SKOV-3 identified a novel cancer testis antigen, Taxol resistance-associated gene 3 (TRAG-3). Because investigation suggested that TRAG-3, located on chromosome Xq28, does not directly participate in the paclitaxel-resistant phenotype, it was hypothesized that TRAG-3 might be linked to a neighboring gene that is directly involved in the drug-resistant phenotype, or alternatively, overexpression of TRAG-3 might be attributable to coregulation with other cancer testis antigens. To distinguish between these two hypotheses, expression of the genes that flank TRAG-3 was evaluated, namely the Centrin 2 gene and several members of the MAGE gene cluster. Northern analysis demonstrates overexpression of MAGE2 but not Centrin 2. Extension of this analysis to other neighboring and non-neighboring representative cancer testis antigens reveals overexpression of MAGE3, MAGE6, MAGE11, and MAGE12, as well as GAGE-2, GAGE-4, GAGE-5, GAGE-6, and GAGE-7 (clustered on Xp11) in SKOV-3(TR), as compared with SKOV-3. In addition, Affymetrix-based analysis of gene expression in SKOV-3 subclones with variable paclitaxel resistance demonstrates MAGE gene overexpression occurs early in the development of the paclitaxel-resistant phenotype, whereas GAGE gene overexpression occurs somewhat later. Evaluation of additional breast and ovarian cancer cell lines reveals MAGE/GAGE overexpression in both paclitaxel- and doxorubicin-resistant cell lines, whereas gemcitabine-resistant subclones of several ovarian cancer cell lines, including SKOV-3(GR), reveals no change in MAGE/GAGE expression. To determine whether MAGE gene overexpression contributes directly to the drug-resistant phenotype, MAGE2 or MAGE6, cDNA was introduced into the paclitaxel-sensitive human ovarian cancer cell line OVCAR8. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity analysis of both MAGE2 and MAGE6 transfectants demonstrates a 4-fold increase in resistance to paclitaxel and 2-fold increase in resistance to doxorubicin but not to other drugs, such as topotecan and cisplatin, through a nonmultidrug resistance-1 mechanism. MAGE2 or MAGE6 overexpression also induces a growth advantage in OVCAR8-transfected cells. These studies suggest that the in vitro acquisition of paclitaxel and doxorubicin resistance can be associated with increased expression of a variety of both neighboring and non-neighboring cancer testis antigens genes. This does not appear to be a consequence of random genetic instability or genomic amplification of the X chromosome. These antigens, because of limited expression in normal tissues, may be suitable targets for immunotherapy and novel therapeutic strategies in the treatment of chemotherapy-resistant epithelial tumors.

Asunto(s)

Antígenos de Neoplasias/biosíntesis , Cromosomas Humanos X , Desoxicitidina/análogos & derivados , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Proteínas de Neoplasias/biosíntesis , Paclitaxel/farmacología , Northern Blotting , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular/biosíntesis , Línea Celular Tumoral , Análisis por Conglomerados , Cartilla de ADN , ADN Complementario/metabolismo , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Testículo/metabolismo , Factores de Tiempo , Transfección , Gemcitabina