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1.
Int J Mol Sci ; 24(1)2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36614027

RESUMO

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.


Assuntos
Córtex Suprarrenal , Adenoma Adrenocortical , Humanos , Zona Reticular/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/metabolismo , Corticosteroides/metabolismo , Linhagem Celular
2.
PLoS One ; 11(2): e0148807, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26859746

RESUMO

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Gencitabina
3.
PLoS One ; 8(6): e65624, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23785438

RESUMO

BACKGROUND: Pheochromocytomas are rare tumors generally arising in the medullary region of the adrenal gland. These tumors release excessive epinephrine and norepinephrine resulting in hypertension and cardiovascular crises for which surgery is the only definitive treatment. Molecular mechanisms that control tumor development and hormone production are poorly understood, and progress has been hampered by the lack of human cellular model systems. To study pheochromocytomas, we developed a stable progenitor pheochromocytoma cell line derived from a primary human tumor. METHODS: After IRB approval and written informed consent, human pheochromocytoma tissue was excised, minced, dispersed enzymatically, and cultured in vitro. Primary pheochromocytoma cells were infected with a lentivirus vector carrying the catalytic subunit of human telomerase reverse transcriptase (hTERT). The hTERT immortalized cells (hPheo1) have been passaged >300 population doublings. The resulting cell line was characterized morphologically, biochemically and for expression of neuroendocrine properties. The expression of marker enzymes and proteins was assessed by immunofluorescence staining and immunoblotting. Telomerase activity was determined by using the telomeric repeat amplification protocol (TRAP) assay. RESULTS: We have established a human pheochromocytoma precursor cell line that expresses the neuroendocrine marker, chromogranin A, when differentiated in the presence of bone morphogenic protein 4 (BMP4), nerve growth factor (NGF), and dexamethasone. Phenylethanolamine N-methyltransferase (PNMT) expression is also detected with this differentiation regimen. CD-56 (also known as NCAM, neural cell adhesion molecule) is expressed in these cells, but CD31 (also known as PECAM-1, a marker of endothelial cells) is negative. CONCLUSIONS: We have maintained hTERT-immortalized progenitor cells derived from a pheochromocytoma (hPheo1) in culture for over 300 population doublings. This progenitor human cell line is normal diploid except for a deletion in the p16 region and has inducible neuroendocrine biomarkers. These cells should be a valuable reagent for studying mechanisms of tumor development and for testing novel therapeutic approaches.


Assuntos
Células-Tronco Neoplásicas/patologia , Feocromocitoma/patologia , Cariótipo Anormal , Adulto , Linhagem Celular Transformada , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imunofenotipagem , Células-Tronco Neoplásicas/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Interferência de RNA , Telomerase/genética , Telomerase/metabolismo , Transcriptoma , Transdução Genética
4.
Biochim Biophys Acta ; 1792(4): 248-59, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19233264

RESUMO

The terms cancer-initiating or cancer stem cells have been the subject of great interest in recent years. In this review we will use pancreatic cancer as an overall theme to draw parallels with historical findings to compare to recent reports of stem-like characteristics in pancreatic cancer. We will cover such topics as label-retaining cells (side-population), ABC transporter pumps, telomerase, quiescence, cell surface stem cell markers, and epithelial-mesenchymal transitions. Finally we will integrate the available findings into a pancreatic stem cell model that also includes metastatic disease.


Assuntos
Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Humanos , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Telomerase/metabolismo
5.
J Immunol ; 179(9): 6072-9, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17947681

RESUMO

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, including alpha- and beta-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (gamma tryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. The alpha-tryptase Gly(216)Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before the alphabeta split. However, the Arg(-3)Gln processing mutation appeared recently, affecting only human alpha. By comparison, the transmembrane gamma-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of alpha- and beta-like tryptases was acquired during primate evolution.


Assuntos
Membrana Celular/enzimologia , Evolução Molecular , Mastócitos/enzimologia , Triptases/metabolismo , Animais , Sequência de Bases , Éxons/genética , Genoma/genética , Humanos , Íntrons/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solubilidade , Fatores de Tempo , Triptases/genética , Vertebrados
6.
Am J Physiol Lung Cell Mol Physiol ; 287(5): L928-35, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15246975

RESUMO

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.


Assuntos
Mucosa Respiratória/citologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sódio/metabolismo , Aprotinina/farmacologia , Linhagem Celular , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio , Epitopos , Escherichia coli , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Glicosilfosfatidilinositóis/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Sódio/metabolismo , Solubilidade , Tripsina/farmacologia
7.
J Biol Chem ; 278(5): 3363-71, 2003 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-12441343

RESUMO

In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term "pancreasin" because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and gamma-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.


Assuntos
Pâncreas/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Complementar/genética , Feminino , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Tripsina/química , Tripsina/metabolismo
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