RESUMO
Sickle cell disease (SCD) results from a sequence defect in the ß-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human ß-globin and sickle hß-globin, as well as lentiviral-encoded therapeutic hßAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA-HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hßAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hßAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.
Assuntos
Anemia Falciforme , Lentivirus , Adulto , Camundongos , Animais , Humanos , Lentivirus/genética , Vetores Genéticos/genética , Hemoglobinas/genética , Hemoglobinas/metabolismo , Anemia Falciforme/diagnóstico , Anemia Falciforme/genética , Anemia Falciforme/terapia , Globinas beta/genética , Células Cultivadas , Espectrometria de MassasRESUMO
ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.
Assuntos
Hemoglobina Fetal/genética , Vetores Genéticos/genética , Hemoglobinopatias/terapia , Lentivirus/genética , Linhagem Celular , Células Cultivadas , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Hemoglobinopatias/genética , Humanos , Transdução Genética , Regulação para Cima , gama-Globinas/genéticaRESUMO
Lymphatic metastasis is a high-impact prognostic factor for mortality of breast cancer (BC) patients, and it directly depends on tumor-associated lymphatic vessels. We previously reported that lipopolysaccharide-induced inflammatory lymphangiogenesis is strongly promoted by myeloid-derived lymphatic endothelial cell progenitors (M-LECPs) derived from the bone marrow (BM). As BC recruits massive numbers of provascular myeloid cells, we hypothesized that M-LECPs, within this recruited population, are specifically programmed to promote tumor lymphatics that increase lymph node metastasis. In support of this hypothesis, high levels of M-LECPs were found in peripheral blood and tumor tissues of BC patients. Moreover, the density of M-LECPs and lymphatic vessels positive for myeloid marker proteins strongly correlated with patient node status. It was also established that tumor M-LECPs coexpress lymphatic-specific, stem/progenitor and M2-type macrophage markers that indicate their BM hematopoietic-myeloid origin and distinguish them from mature lymphatic endothelial cells, tumor-infiltrating lymphoid cells, and tissue-resident macrophages. Using four orthotopic BC models, we show that mouse M-LECPs are similarly recruited to tumors and integrate into preexisting lymphatics. Finally, we demonstrate that adoptive transfer of in vitro differentiated M-LECPs, but not naïve or nondifferentiated BM cells, significantly increased metastatic burden in ipsilateral lymph nodes. These data support a causative role of BC-induced lymphatic progenitors in tumor lymphangiogenesis and suggest molecular targets for their inhibition.
Assuntos
Neoplasias da Mama/patologia , Células Progenitoras Endoteliais/fisiologia , Endotélio Linfático/patologia , Células Mieloides/fisiologia , Animais , Células da Medula Óssea/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Linfangiogênese/fisiologia , Metástase Linfática , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCIDRESUMO
Various malignancies are reproducibly cured in mouse models, but most cancer immunotherapies show objective responses in a fraction of treated patients. One reason for this disconnect may be the use of young, lean mice lacking immune-altering comorbidities present in cancer patients. Although many cancer patients are overweight or obese, the effect of obesity on antitumor immunity is understudied in preclinical tumor models. We examined the effect of obesity on two immunotherapeutic models: systemic anti-CTLA-4 mAb and intratumoral delivery of a TRAIL-encoding adenovirus plus CpG. Both therapies were effective in lean mice, but neither provided a survival benefit to diet-induced obese BALB/c mice. Interestingly, tumor-bearing leptin-deficient (ob/ob) obese BALB/c mice did respond to treatment. Moreover, reducing systemic leptin with soluble leptin receptor:Fc restored the antitumor response in diet-induced obese mice. These data demonstrate the potential of targeting leptin to improve tumor immunotherapy when immune-modulating comorbidities are present.
Assuntos
Adenocarcinoma/metabolismo , Envelhecimento/fisiologia , Anticorpos Monoclonais/uso terapêutico , Imunoterapia/métodos , Neoplasias Renais/metabolismo , Leptina/metabolismo , Obesidade/metabolismo , Adenocarcinoma/terapia , Adenoviridae/genética , Animais , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Dieta , Modelos Animais de Doenças , Feminino , Humanos , Imunidade , Neoplasias Renais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Obesidade/terapia , Oligodesoxirribonucleotídeos/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
BACKGROUND: Alterations in nuclear pore complex (NPC) genes have been previously associated with response to chemotherapy. Using agnostic exome sequencing, we envisioned that new alleles in NPC genes, predictive of sensitivity to platinum treatment, could be discovered. METHODS: Twenty-two platinum-sensitive and six platinum-resistant ovarian cancer patients were tested. Platinum sensitivity was defined as disease-free survival greater than 6 months. Next-generation sequencing of exomes was used to compare platinum-sensitive and platinum-resistant patients. Single nucleotide variants (SNVs) associated with platinum sensitivity in NPC genes (n=30 genes) were identified. RESULTS: SNVs in three NPC genes were associated with response to platinum on univariate analysis. SNV rs79419059 (10T>C) in Nucleoporin 107 (Nup107) was associated with platinum resistance (P=0.0061), whereas rs2302811 (3662-4A>G) in Nucleoporin 188 (Nup188) and rs77246077 (3420-67T>A) in Nucleoporin 214 (Nup214) were associated with platinum sensitivity (P=0.0483 and 0.0091, respectively). Controlling for other confounders, multivariate age-adjusted Cox proportional hazard analysis showed rs79419059 to be significantly associated with platinum resistance (odds ratio: 4.519, 95% confidence interval: 1.317-15.501, P=0.0457). CONCLUSION: We identified a variant in the 3'-UTR region Nup107 unique to sensitivity to platinum in ovarian cancer. With validation of this variant, it is possible that a new marker predictive of patient response may be identified.
Assuntos
Antineoplásicos/uso terapêutico , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-IdadeRESUMO
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the ß-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the ß-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.
Assuntos
Anemia Falciforme/genética , Anemia Falciforme/terapia , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Mutação , Globinas beta/genética , Anemia Falciforme/patologia , Animais , Antígenos CD34/análise , Sequência de Bases , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células Cultivadas , Endodesoxirribonucleases/metabolismo , Sangue Fetal/transplante , Loci Gênicos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Dedos de ZincoRESUMO
Long noncoding RNAs (lncRNAs) interact with other RNAs, DNA and/or proteins to regulate gene expression during development. Erythropoiesis is one developmental process that is tightly controlled throughout life to ensure accurate red blood cell production and oxygen transport to tissues. Thus, homeostasis is critical and maintained by competitive outcomes of pro- and anti-apoptotic pathways. LncRNAs are expressed during blood development; however, specific functions are largely undefined. Here, a culture model of human erythropoiesis revealed that lncRNA Fas-antisense 1 (Fas-AS1 or Saf) was induced during differentiation through the activity of essential erythroid transcription factors GATA-1 and KLF1. Saf was also negatively regulated by NF-κB, where decreasing NF-κB activity levels tracked with increasing transcription of Saf. Furthermore, Saf over-expression in erythroblasts derived from CD34(+) hematopoietic stem/progenitor cells of healthy donors reduced surface levels of Fas and conferred protection against Fas-mediated cell death signals. These studies reveal a novel lncRNA-regulated mechanism that modulates a critical cell death program during human erythropoiesis.
Assuntos
Apoptose , Eritroblastos/citologia , Eritrócitos/citologia , Eritropoese , RNA Longo não Codificante/genética , Receptor fas/genética , Linhagem Celular Tumoral , Eritroblastos/metabolismo , Eritrócitos/metabolismo , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Receptor fas/metabolismoRESUMO
Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.
Assuntos
Interleucina-10 , Neoplasias , Células-Tronco Neoplásicas , Microambiente Tumoral , Animais , Humanos , Camundongos , Células da Medula Óssea/patologia , Diferenciação Celular , Células Cultivadas , Interleucina-10/metabolismo , Fator Estimulador de Colônias de Macrófagos , Neoplasias/patologia , Fenótipo , Receptor 4 Toll-Like , Células-Tronco Multipotentes/metabolismo , Linfangiogênese , Células Mieloides/metabolismo , Células Mieloides/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and ß-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders that would benefit from increased fetal hemoglobin levels.
Assuntos
Hemoglobina Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transcrição Gênica/fisiologia , HumanosRESUMO
ß-Thalassemia major results from severely reduced or absent expression of the ß-chain of adult hemoglobin (α2ß2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of ß-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with ß-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34(+) cells demonstrated levels of HbF up to 21% per vector copy. For ß-thalassemic CD34(+) cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with ß-globin deficiency.
Assuntos
Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Técnicas de Transferência de Genes , Terapia Genética , Talassemia beta/terapia , gama-Globinas/genética , Antígenos CD34/metabolismo , Southern Blotting , Western Blotting , Separação Celular , Eritropoese/fisiologia , Hemoglobina Fetal/genética , Citometria de Fluxo , Vetores Genéticos , Humanos , Lentivirus/genética , Reação em Cadeia da PolimeraseRESUMO
AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50 ng/ml) and insulin (10 nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.
Assuntos
Aldeído Redutase/metabolismo , Carcinoma Hepatocelular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Neoplasias Hepáticas/metabolismo , Fator de Transcrição AP-1/metabolismo , Aldeído Redutase/genética , Aldo-Ceto Redutases , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Primers do DNA/genética , Feminino , Genes fos , Células Hep G2 , Humanos , Insulina/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Regulação para Cima/efeitos dos fármacosRESUMO
An internal circadian clock regulates timing of systemic energy homeostasis. The central clock in the hypothalamic suprachiasmatic nucleus (SCN) directs local clocks in peripheral tissues such as liver, muscle, and adipose tissue to synchronize metabolism with food intake and rest/activity cycles. Aryl hydrocarbon receptor (AhR) interacts with the molecular circadian clockworks. Activation of AhR dampens rhythmic expression of core clock genes, which may lead to metabolic dysfunction. Given the importance of appropriately-timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled adipose tissue activity. We hypothesized that AhR activation in adipose tissue would impair lipolysis by dampening adipose rhythms, leading to a decreased lipolysis rate during fasting, and subsequently, altered serum glucose concentrations. Levels of clock gene and lipolysis gene transcripts in mouse mesenchymal stem cells (BMSCs) differentiated into mature adipocytes were suppressed by the AhR agonist ß-napthoflavone (BNF), in an AhR dependent manner. BNF altered rhythms of core clock gene and lipolysis gene transcripts in C57bl6/J mice. BNF reduced serum free fatty acids, glycerol and liver glycogen. Chromatin immunoprecipitation indicated that BNF increased binding of AhR to E-Box elements in clock gene and lipolysis gene promoters. These data establish a link between AhR activation and impaired lipolysis, specifically by altering adipose tissue rhythmicity. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide necessary energy.
Assuntos
Relógios Circadianos , Receptores de Hidrocarboneto Arílico , Camundongos , Animais , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Ritmo Circadiano/fisiologia , Lipólise , Relógios Circadianos/genética , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Camundongos Endogâmicos C57BLRESUMO
The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-kappaB) family; however, the role of NF-kappaB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kappaB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-kappaB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kappaB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kappaB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kappaB pathway by inflammatory stimuli activates Prox1, and both NF-kappaB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Neovascularização Fisiológica , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Feminino , Proteínas de Homeodomínio/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Ratos , Fatores de Tempo , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34(+) hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source. When cytokine-mobilized peripheral blood CD34(+) cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells were readily transduced with lentiviral vectors when exposed to vector particles between 48 and 72 hours. Among the genetic elements that may enhance fetal hemoglobin production is an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal gamma-globin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of relatively weak erythroid-specific promoters induced significant amounts of HbF (up to 20%) in erythroblasts derived from adult CD34(+) cells without altering their capacity for erythroid maturation and only modestly reducing the total numbers of cells that accumulate in culture after transduction. These observations demonstrate the potential for sequence-specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia.
Assuntos
Eritroblastos/metabolismo , Hemoglobina Fetal/biossíntese , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Dedos de Zinco , gama-Globinas/genética , Adulto , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromatina/metabolismo , Citocinas/farmacologia , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Mobilização de Células-Tronco Hematopoéticas , Humanos , Lentivirus/genética , Modelos Genéticos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Ligação Proteica/efeitos dos fármacos , Transdução GenéticaRESUMO
Myeloid-lymphatic endothelial cell progenitors (M-LECP) are a subset of bone marrow (BM)-derived cells characterized by expression of M2-type macrophage markers. We previously showed significant contribution of M-LECP to tumor lymphatic formation and metastasis in human clinical breast tumors and corresponding mouse models. Since M2 type is induced in macrophages by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10, we hypothesized that these factors might promote pro-lymphatic specification of M-LECP during their differentiation from BM myeloid precursors. To test this hypothesis, we analyzed expression of Th2 cytokines and their receptors in mouse BM cells under conditions leading to M-LECP differentiation, namely, CSF-1 treatment followed by activation of TLR4. We found that under these conditions, all three Th2 receptors were strongly upregulated in >95% of the cells that also secrete endogenous IL-10, but not IL-4 or IL-13 ligands. However, addition of any of the Th2 factors to CSF-1 primed cells significantly increased generation of myeloid-lymphatic progenitors as indicated by co-induction of lymphatic-specific (eg, Lyve-1, integrin-a9, collectin-12, and stabilin-1) and M2-type markers (eg, CD163, CD204, CD206, and PD-L1). Antibody-mediated blockade of either IL-10 receptor (IL-10R) or IL-10 ligand significantly reduced both immunosuppressive and lymphatic phenotypes. Moreover, tumor-recruited Lyve-1+ lymphatic progenitors in vivo expressed all Th2 receptors as well as corresponding ligands, including IL-4 and IL-13, which were absent in BM cells. This study presents original evidence for the significant role of Th2 cytokines in co-development of immunosuppressive and lymphatic phenotypes in tumor-recruited M2-type myeloid cells. Progenitor-mediated increase in lymphatic vessels can enhance immunosuppression by physical removal of stimulatory immune cells. Thus, targeting Th2 pathways might simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that ultimately promote tumor spread.
Assuntos
Vasos Linfáticos , Neoplasias , Células Th2 , Animais , Medula Óssea/metabolismo , Diferenciação Celular , Citocinas , Interleucina-10 , Interleucina-13 , Interleucina-4/farmacologia , Ligantes , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Fator Estimulador de Colônias de Macrófagos , Camundongos , Neoplasias/patologiaRESUMO
Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24(-)/CD44(+)/ESA(+)) that were isolated from both ER+ and ER- breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ácido Graxo Sintases/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Associadas com Morte Celular , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Lipogênese/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Resveratrol , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary carnitine deficiency (PCD) is a rare autosomal recessive disorder caused by loss of function mutations in the solute carrier family 22 member 5 (SLC22A5) gene that encodes a high-affinity sodium-ion-dependent organic cation transporter protein (OCTN2). Reduced carnitine transport results in diminished fatty acid oxidation in heart and skeletal muscle and carnitine wasting in urine. We present a case of PCD diagnosed in an adult female after a positive newborn screen (NBS) for PCD that was not confirmed on follow-up testing. The mother was referred for evaluation of persistent fatigue and possible hypothyroidism even though all measurements of thyroid-stimulating hormone were well within the range of 0.4 to 2.5 mIU/L expected for reproductive-age women. She was found to have unequivocally low levels of both total carnitine and carnitine esters, and genetic testing revealed compound heterozygosity for 2 SLC22A5 mutations. One mutation (c.34G>A [p.Gly12Ser]) is a known missense mutation with partial OCTN2 activity, but the other mutation (c.41G>A [p.Trp14Ter]) is previously unreported and results in a premature stop codon and truncated OCTN2. This case illustrates that some maternal inborn errors of metabolism can be identified by NBS and that maternal carnitine levels should be checked after a positive NBS test for PCD.
Assuntos
Hiperamonemia , Adulto , Cardiomiopatias , Carnitina/deficiência , Feminino , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/genética , Recém-Nascido , Doenças Musculares , Mutação , Membro 5 da Família 22 de Carreadores de Soluto/genéticaRESUMO
Sickle cell disease (SCD) and ß-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2ß2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in ß-thalassemia or potently suppress expression of sickle ß-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult ß-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe ß-hemoglobin disorders by reversing the developmental γ- to ß-globin switch.
RESUMO
Natural killer (NK) cells are classically associated with immune surveillance and destruction of tumor cells. Inconsistent with this function, NK cells are found in advanced human tumors including renal cell carcinoma (RCC). NK cells with non-classical phenotypes (CD56+CD16dim/neg; termed decidua NK (dNK) cells) accumulate at the maternal-fetal interface during embryo implantation. These dNK cells are poorly cytotoxic, proangiogenic, and facilitate placenta development. As similarities between embryo implantation and tumor growth exist, we tested the hypothesis that an analogous shift in NK cell phenotype and function occurs in RCC tumors. Our results show that peripheral NK (pNK) cells of RCC patients were uniformly CD56+CD16bright, but lacked full cytotoxic ability. By comparison, RCC tumor-infiltrated NK (TiNK) cells were significantly enriched for CD56+CD16dim-neg cells, a phenotype of dNK cells. Gene expression analysis revealed that angiogenic and inflammatory genes were significantly increased for RCC TiNK versus RCC pNK populations, with enrichment of genes in the hypoxia inducible factor (HIF) 1α pathway. Consistent with this finding, NK cells cultured under hypoxia demonstrated limited cytotoxicity capacity, but augmented production of vascular endothelial growth factor (VEGF). Finally, comparison of gene expression data for RCC TiNK and dNK cells revealed a shared transcriptional signature of genes with known roles in angiogenesis and immunosuppression. These studies confirm conversion of pNK cells to a dNK-like phenotype in RCC tumors. These characteristics are conceivably beneficial for placentation, but likely exploited to support early tumor growth and promote metastasis.
RESUMO
UNLABELLED: Current techniques for the alteration of gene expression in the liver have a number of limitations, including the lack of stable somatic gene transfer and the technical challenges of germline transgenesis. Rapid and stable genetic engineering of the liver would allow systematic, in vivo testing of contributions by many genes to disease. After fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in FAH-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable Fah transposon cassette. First, net conversion of Fah(-/-) liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, coexpressed short hairpin RNAs (shRNAs) stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant allele of human alpha1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in transformation of transduced hepatocytes. CONCLUSION: Coexpression with FAH is an effective technique for lifelong expression of transgenes in adult hepatocytes with applicability to a wide variety of genetic studies in the liver.