RESUMEN
Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.
Asunto(s)
Adenocarcinoma , Hiperplasia Prostática , Neoplasia Intraepitelial Prostática , Neoplasias de la Próstata , Masculino , Humanos , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologíaRESUMEN
In acute myeloid leukemia (AML), leukemia stem cells (LSCs) have self-renewal potential and are responsible for relapse. We previously showed that, in Mll-AF9/NRASG12V murine AML, CD69 expression marks an LSC-enriched subpopulation with enhanced in vivo self-renewal capacity. Here, we used CyTOF to define activated signaling pathways in LSC subpopulations in Mll-AF9/NRASG12V AML. Furthermore, we compared the signaling activation states of CD69High and CD36High subsets of primary human AML. The human CD69High subset expresses low levels of Ki67 and high levels of NFκB and pMAPKAPKII. Additionally, the human CD69High AML subset also has enhanced colony-forming capacity. We applied Bayesian network modeling to compare the global signaling network within the human AML subsets. We find that distinct signaling states, distinguished by NFκB and pMAPKAPKII levels, correlate with divergent functional subsets, defined by CD69 and CD36 expression, in human AML. Targeting NFκB with proteasome inhibition diminished colony formation.
Immunophenotypically-defined murine AML stem cells harbor self-renewing and non-self-renewing subsets that display unique signaling characteristics.CD69, an NFκB target gene, marks a subset of human AML with increased colony forming capacity and reduced proliferation.NFκB activation correlates with the global signaling pathway activation state in human AML.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Ratones , Animales , Teorema de Bayes , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal , Células Madre Neoplásicas/metabolismoRESUMEN
NRAS proteins are central regulators of proliferation, survival, and self-renewal in leukemia. Previous work demonstrated that the effects of oncogenic NRAS in mediating proliferation and self-renewal are mutually exclusive within leukemia subpopulations and that levels of oncogenic NRAS vary between highly proliferative and self-renewing leukemia subpopulations. These findings suggest that NRAS activity levels may be important determinants of leukemic behavior. To define how oncogenic NRAS levels affect these functions, we genetically engineered an acute myeloid leukemia (AML) cell line, THP-1, to express variable levels of NRASG12V. We replaced the endogenous NRASG12D gene with a tetracycline-inducible and dose-responsive NRASG12V transgene. Cells lacking NRASG12V oncoprotein were cell-cycle arrested. Intermediate levels of NRASG12V induced maximal proliferation; higher levels led to attenuated proliferation, increased G1 arrest, senescence markers, and maximal self-renewal capacity. Higher levels of the oncoprotein also induced self-renewal and mitochondrial genes. We used mass cytometry (CyTOF) to define the downstream signaling events that mediate these differential effects. Not surprisingly, we found that the levels of such canonical RAS-effectors as pERK and p4EBP1 correlated with NRASG12V levels. ß-Catenin, a mediator of self-renewal, also correlated with NRASG12V levels. These signaling intermediates may mediate the differential effects of NRASG12V in leukemia biology. Together, these data reveal that oncogenic NRAS levels are important determinants of leukemic behavior explaining heterogeneity in phenotypes within a clone. This system provides a new model to study RAS oncogene addiction and RAS-induced self-renewal in AML. IMPLICATIONS: Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Oncogenes , Proteínas Oncogénicas/genética , Proliferación Celular , Línea CelularRESUMEN
BACKGROUND: The painful conditions of the Upper quarter region (UQR) such as chronic Craniofacial Pain (CFP) and Cervicobrachial Pain (CBP) usually occur with a plethora of symptoms. Although biological and psychosocial factors are attributed to such conditions, the involvement of the Deep Cervical Fascia (DCF) is ambiguous and needs further exploration. OBJECTIVE: We reported a case of CFP and CBP with an intent to showcase the possible involvement of impaired DCF in such presentations and to explore the short-term effect of Fascia directed approach (Fascial Manipulation). METHODS: This is a report of a 25-year-old female college student with chronic head, temporomandibular, neck, and arm pain over the past four years with acute pain exacerbation. After identifying the densified Centre of Coordination points along the myofascial continuum of the DCF, Fascial Manipulation (FM) was performed by deep manual friction. The patient-reported outcomes such as the Numerical Pain Rating Scale (NPRS), Temporomandibular disability index (TMDI), and Patient-Specific Functional Scale (PSFS) were assessed. RESULTS: Following FM treatment, there is a reduction in pain and improved function between the baseline and follow up evaluation after one week based on all the outcomes (NPRS, PSFS, and TMDI). CONCLUSION: This case report highlights the possible role of dysfunction of the DCF and the importance of assessing myofascial chains in patients with pain in the UQR. The report has also shown that FM may be beneficial and can be considered an adjunct in the rehabilitation of chronic CFP and CBP. Nevertheless, future studies with multiple sessions and follow-ups are imperative.
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Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/genética , Terapia Molecular Dirigida , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Antineoplásicos/uso terapéutico , Células Clonales , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Humanos , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Represoras/genéticaRESUMEN
Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.
Asunto(s)
Proliferación Celular/genética , Autorrenovación de las Células/genética , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Análisis de la Célula Individual/métodos , Animales , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Madre Neoplásicas/metabolismoRESUMEN
Skeletal muscles have a tremendous capacity for repair and regeneration in response to injury. This capacity for regeneration is largely due to a myogenic stem cell population, termed satellite cells, which are resident in adult skeletal muscles. In order to decipher the mechanisms that govern myogenic stem cell quiescence, activation, differentiation, and self-renewal, a reproducible injury model is required. Therefore, we have utilized the delivery of the myonecrotic agent, cardiotoxin, to examine the molecular mechanisms of myogenic stem cells in response to injury. Here, we describe our experience using cardiotoxin as a potent myonecrotic agent to study skeletal muscle regeneration. We provide a detailed protocol to examine skeletal muscle injury and regeneration using morphological analyses.
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Cardiotoxinas/efectos adversos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/patología , Regeneración , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Factores de TiempoRESUMEN
Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Witanólidos/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/patología , Tubulina (Proteína)/genética , Witanólidos/farmacocinéticaRESUMEN
Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.
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Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/efectos de los fármacos , Isotiocianatos/farmacología , Proteínas Proto-Oncogénicas/fisiología , Antineoplásicos , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Humanos , Proteínas de la Membrana , Proteínas Proto-Oncogénicas c-bcl-2 , Verduras/químicaRESUMEN
Health benefits of garlic and other Allium vegetables (e.g., onions), such as lipid lowering and anticancer effects, are credited to metabolic byproducts, including diallyl trisulfide (DATS). Evidence for anticancer effects of garlic derives from both population-based case-control studies, and clinical and laboratory investigations using purified garlic constituents such as DATS. Studies have shown that DATS can offer protection against chemically-induced neoplasia as well as oncogene-driven spontaneous cancer development in experimental rodents. Mechanisms underlying cancer chemopreventive effects of DATS are not completely understood, but known pharmacological responses to this natural product include alteration in carcinogen-metabolizing enzymes, cell cycle arrest, induction of apoptotic cell death, suppression of oncogenic signal transduction pathways, and inhibition of neoangiogenesis. This article reviews mechanisms and targets of cancer chemoprevention by DATS.
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Compuestos Alílicos/uso terapéutico , Anticarcinógenos/uso terapéutico , Ajo/química , Neoplasias/prevención & control , Sulfuros/uso terapéutico , Compuestos Alílicos/química , Animales , Anticarcinógenos/química , Apoptosis , Puntos de Control del Ciclo Celular , Proliferación Celular , Quimioprevención , Humanos , Redes y Vías Metabólicas , Ratones , Sulfuros/químicaRESUMEN
PURPOSE: Loss of sensitivity to transforming growth factor beta (TGF-beta) signaling typically occurs in human ovarian cancer cells, but there is paucity of information regarding this in human ovarian tumors. Thus the association of inactivating mutations and/or variations in expression levels of TGF-beta signaling components with human ovarian tumors was evaluated. METHODS: Forty human ovarian tissue samples were analyzed for mutations and/or variations in the expression of transforming growth factor beta signaling components. Mutation studies were done through reverse transcription (RT) PCR, single strand conformation polymorphism analysis and automated DNA sequencing. Expression studies were carried out by semi quantitative RT PCR and western blotting. DNA binding ability of Smad complexes and expression of downstream targets were also analyzed. RESULTS: The six alanine repeat containing variant of TGF-beta RI was seen in 27% of the tumor cases studied, in addition to the 45 bp nucleotide deletions in exon 1 of the receptor in two ovarian tumor samples. A deletion in the polyadenine tract of exon 3 of TGF-beta RII was seen in 22% of the tumor samples. We also report a loss or decrease in the expression of Smad 4 protein in tumor samples with a concurrent loss or reduced DNA binding ability of the Smad complex and deregulated expression of p21 and c-Myc. CONCLUSIONS: Our results suggest that mutations and/or alterations in expression of TGF-beta receptors and loss of Smad 4 are frequent in human ovarian cancers and may potentially explain the frequent loss of TGF-beta responsiveness that typically occurs in human ovarian cancer.
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Carcinoma/genética , Mutación , Neoplasias Ováricas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteína Smad4/genética , Nucleótidos de Adenina/genética , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Carcinoma/metabolismo , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Persona de Mediana Edad , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/fisiología , Neoplasias Ováricas/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad4/metabolismoRESUMEN
Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.
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Adenosina Trifosfatasas/fisiología , Proteínas de Ciclo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica , Receptor Notch1/biosíntesis , Retina/embriología , Factor de Transcripción Brn-3B/biosíntesis , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Cromatina/metabolismo , ADN Helicasas , Modelos Biológicos , Proteínas Nucleares , Ratas , Ratas Sprague-Dawley , Retina/citología , Transducción de Señal , Células Madre/citología , Transcripción GenéticaRESUMEN
Mutations in Smads, intermediates of transforming growth factor-beta signaling, are known to contribute to the loss of sensitivity to transforming growth factor-beta, a common feature of many neoplastic cells. However, not much information is available on Smad alterations in cervical cancer and so we probed, for the first time, for alterations in Smad 2 and Smad 4 genes using human cervical cancer cell lines and human cervical tissue samples. Using PCR/reverse transcription-PCR, single-stranded conformation polymorphism analysis and DNA sequencing, we observed a deletion of 'G' in the L3 loop (crucial in Smad-receptor interaction) in C-33A cells, and an insertion of 'A' in codon 122 (loss of MH2 domain) from a cervical tumor sample, both of which caused frame shift and pretermination in Smad 2. In addition, a G/A transition at 31 bp upstream-nontranslated regions of exon 8 of Smad 4 was found in Bu 25TK cells. Smad 2 expression was less in some of the cervical tumor samples than that of nonmalignant samples and six cancer samples showed C-terminal deletions that abolish Smad 2 phosphorylation sites. The loss of expression of Smad 4 found in some cervical tumor samples was due to transcription loss rather than deletion of the gene. Our results highlight an important role for Smad 2 and Smad 4 in human cervical tumors.