RESUMO
Advances in the field of tumor biology have identified that tumor cells co-opt developmental signaling pathways of embryonic stem cells and thus gain the ability to proliferate, differentiate and alter cell-cell interactions. One such pathway is the Wnt/beta-catenin signaling pathway. High levels of EMMPRIN expression have been shown to correlate with poor prognosis and metastasis in a broad range of tumors. Although a variety of functions are attributed to EMMPRIN in tumorigenesis, the specific mechanism(s) through which it can exert its effects have not been elucidated, until now. In this study, we identify EMMPRIN as a novel regulator of the canonical Wnt/beta-catenin signaling pathway in lung cancer. Increasing EMMPRIN expression levels in lung cancer epithelial cells upregulated the beta-catenin signaling pathway and silencing EMMPRIN inhibited beta-catenin signaling, cell migration, proliferation, anchorage-independent growth and tumor growth in a mouse tumor xenograft model. These results provide a compelling rationale for targeting EMMPRIN for anticancer therapies. Understanding the molecular mechanisms driving EMMPRIN-induced lung tumorigenesis will provide enormous benefits in developing new therapeutic treatments for this and other forms of cancer.
Assuntos
Basigina/fisiologia , Neoplasias Pulmonares/etiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/patologia , Metaloproteinases da Matriz/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , RNA Interferente Pequeno/genéticaRESUMO
The impact of different levels of agitation speed, carbondioxide and dissolved oxygen concentration on the key parameters and production of rhG-CSF in Escherichia coli BL21(DE3)PLysS were studied. Lower carbondioxide concentrations as well as higher agitation speeds and dissolved oxygen concentrations led to reduction in the acetate concentrations, and enhanced the cell growth, but inhibited plasmid stability and rhG-CSF expression. Similarly, higher carbondioxide concentrations and lower agitation speeds as well as dissolved oxygen concentrations led to enhanced acetate concentrations, but inhibited the cell growth and protein expression. To address the bottlenecks, a two-stage agitation control strategy (strategy-1) and two-stage dissolved oxygen control strategy (strategy-2) were employed to establish the physiological and metabolic conditions, so as to improve the expression of rhG-CSF. By adopting strategy-1 the yields were improved 1.4-fold over constant speed of 550 rpm, 1.1-fold over constant dissolved oxygen of 45%, respectively. Similarly, using strategy-2 the yields were improved 1.6-fold over constant speed of 550 rpm, 1.3-fold over constant dissolved oxygen of 45%, respectively.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/genética , Microbiologia Industrial , Oxigênio/farmacologia , Acetatos/metabolismo , Técnicas Bacteriológicas , Reatores Biológicos , Dióxido de Carbono/farmacologia , Fermentação/efeitos dos fármacos , Humanos , Oxigênio/metabolismo , Proteínas Recombinantes , Tempo , ViscosidadeRESUMO
Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl beta-D: -1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD(600) value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 microg/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.
Assuntos
Códon de Iniciação/genética , Códon/genética , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos/genética , Composição de Bases , Escherichia coli/crescimento & desenvolvimento , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Modelos Genéticos , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Recombinantes , Rifampina/farmacologiaRESUMO
PURPOSE: We hypothesized that alterations in Y chromosome gene expression may be associated with prostate cancer. To test this hypothesis we analyzed the expression of 19 Y chromosome genes in benign and malignant prostate tissue. MATERIALS AND METHODS: To study the expression of Y chromosome genes RNA was extracted from prostate cancer and benign prostatic hyperplasia (BPH) tissue as well as from prostate cancer cell lines. RNA was reverse transcribed and polymerase chain reaction amplified using specific primers. These primers were designed for each gene sequence obtained from the gene data bank. We analyzed 19 Y chromosome genes using 6 cell lines, 7 BPH and 7 prostate cancer tissues. Normal testis RNA served as a positive control. RESULTS: Of the 19 genes analyzed in cell lines BPH-1 cells expressed the RPS4Y, USP9Y, TMSB4Y and DBY genes; DUPro expressed RPS4Y, USP9Y, TMSB4Y, DBY and UTY; DU145 expressed DAZ, RPS4Y, USP9Y, TMSB4Y, DBY, EIAFIY, PRKY and SMCY; LNCaP expressed TSPY, SRY, BPY1, PRY, DAZ, RBMIH, RPS4Y, DBY, EIAFIY, PRKY and SMCY; ND1 expressed DAZ, RPS4Y, USP9Y, TMSB4Y, DBY, EIAFIY, PRKY and SMCY; and PC3 expressed RPS4Y, USP9Y and DBY. BPH tissue expressed the SRY, PRY, DBY, PRKY, RPS4Y, TMSB4Y, USP9Y and ZFY genes. Prostate cancer tissue expressed the PRY, TSPY, USP9Y, UTY, DBY, SMCY, ZFY, EIAFIY, TMSB4Y and RPS4Y genes. CONCLUSIONS: The differential expression of Y chromosome genes in prostate cancer, BPH tissue and prostate cancer cell lines indicates that they may have a role in prostate cancer.
Assuntos
Adenocarcinoma/genética , Expressão Gênica , Neoplasias da Próstata/genética , Cromossomo Y/genética , Humanos , Masculino , Reação em Cadeia da Polimerase , Hiperplasia Prostática/genética , RNA Neoplásico/análise , Células Tumorais CultivadasRESUMO
Leukotriene B4 (LTB4), a product of arachidonic acid metabolism, plays an important role in inflammatory responses. We have cloned from human erythroleukemia cells, a G protein-coupled receptor, designated P2Y(7), which was later identified as the receptor for LTB4 (B-LTR). We have investigated the distribution of LTB4 receptors in various hematopoietic cells. Northern blotting and reverse transcription-coupled polymerase chain reaction (RT-PCR) analyses using radiolabeled LTB4 receptor cDNA as a probe indicated the presence of LTB4 receptor mRNA in peripheral blood leukocytes but not in platelets. Flow cytometry analysis of peripheral blood cells using specific LTB4 receptor antibodies revealed that monocytes, granulocytes, and lymphocytes, but not platelets, express LTB4 receptors. RT-PCR-Southern hybridization analysis revealed that peripheral blood leukocytes and human umbilical vein endothelial cells express the LTB4 receptor. Of the hematopoietic cell lines tested, promonocytic U937 cells, promyelocytic HL-60 cells, K562 cells, and human erythroleukemia cells express the LTB4 receptor. These results suggest a physiological role for the LTB4 receptor in the stimulation of monocytes, neutrophils, and endothelial cells.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Receptores do Leucotrieno B4/sangue , Sequência de Aminoácidos , Citometria de Fluxo , Células HL-60 , Humanos , Células K562 , Leucócitos/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/sangue , Receptores do Leucotrieno B4/genética , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2/genética , Células Tumorais Cultivadas , Células U937RESUMO
Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca(2+)) can bind FXI and can substitute for HK (and Zn(2+)) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 71 nm) and the rA1 domain (K(d) approximately 239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (125)I-rA1 (Glu(1)-Ser(90)) binding to prothrombin, (125)I-prothrombin binding to FXI, and (125)I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the A1 domain of FXI, acted synergistically to inhibit (125)I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.
Assuntos
Fator XI/química , Fator XI/metabolismo , Kringles , Protrombina/química , Protrombina/metabolismo , Sítios de Ligação , Ligação Competitiva , Fator XI/genética , Humanos , Radioisótopos do Iodo , Cinética , Cininogênio de Alto Peso Molecular/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Trombina/metabolismoRESUMO
Although previous data showed that the putative thiolase B PPRE located at -681/-669 bind the PPARalpha-RXRalpha heterodimer in vitro (Kliewer et al. (1992) Nature 358, 771-774), there is no evidence about the functional role of this element. By gel mobility-shift assay, we found an interaction of this PPRE with not only PPARalpha but also with HNF-4. By transfection of cells with the putative PPRE-driven luciferase reporter vector and PPARalpha, we found no significant activation of the luciferase gene expression, in contrast to the case with reporter expression driven by the PPRE of the peroxisomal bifunctional enzyme. On the other hand, HNF-4 activated the luciferase gene expression driven by the putative thiolase PPRE. We suggest that the thiolase B gene induction by peroxisome proliferators employs either another PPRE or this one in combination with other gene regulatory element(s) to lead to the strong gene expression observed in the presence of peroxisome proliferators.
Assuntos
Acetil-CoA C-Aciltransferase/genética , Proteínas de Ligação a DNA , Fígado/enzimologia , Proliferadores de Peroxissomos/metabolismo , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Células COS , DNA , Primers do DNA , Genes Reporter , Fator 4 Nuclear de Hepatócito , Ratos , TransfecçãoRESUMO
Recently, the World Wide Web has emerged as a platform for computer-based oral health records. Web-based patient records can make teledentistry an instant reality. Because an increasing number of dental care providers can access Web pages, traditional barriers to exchanging information are dropping. Web-based records also make cumulative, longitudinal patient records possible. Sophisticated security mechanisms can ensure the integrity and confidentiality of patient information. Because Web-based systems are simpler to install and configure, the cost of operating them may be reduced. However, their development is complex, difficult, and expensive because the Web was not developed as a programming environment. Furthermore, the technologies underlying the Web are constantly evolving, forcing developers to continuously reengineer their systems. In addition, several policy questions, such as storage of and access to computer-based patient records, have to be answered. This article describes CMSWeb, a Web-based clinical information system implemented at Temple University School of Dentistry.
Assuntos
Registros Odontológicos , Internet , Sistemas Computadorizados de Registros Médicos , Segurança Computacional , Humanos , SoftwareRESUMO
The peroxisomal 3-oxoacyl-CoA thiolase (thiolase) is the last enzyme involved in the beta-oxidation of fatty acids. The enzyme cleaves long chain fatty acyl-CoA to generate acetyl-CoA and shortened acyl-CoA. The enzyme is nuclear encoded, synthesized in the cytoplasm and transported into peroxisomes. The thiolase B gene is inducible by the peroxisome proliferator compounds, like other genes involved in beta-oxidation of fatty acids in peroxisomes. The importance of studying thiolase is that it generates acetyl-CoA which is the precursor for the synthesis of molecules like cholesterol and fatty acids. The structural and functional analysis of thiolase at molecular level may add to the knowledge of fatty acid metabolism and further the obesity phenomenon. It is known that several genes mediate lipid homeostasis in target organs like liver, adipose tissue and are regulated by peroxisome proliferator activated receptors (PPAR alpha and PPAR gamma). To elucidate the mechanism of induction of rat liver thiolase B gene, an upstream 2.8 kb fragment containing promoter element has been subcloned and partially sequenced. The sequence analysis revealed a putative PPRE (Peroxisome Proliferator Response Element) of AGACCT T TGAACC sequence at -681 to -668 [Kliever et al. (1992) Nature 358:771-774]. By transient expression of a luciferase reporter gene in HeLa cells, we conclude that the identified PPRE could be functional in induction of thiolase B gene, but other sequences of genes might be involved.
Assuntos
Acetil-CoA C-Aciltransferase/genética , Fígado/enzimologia , Peroxissomos/metabolismo , Regiões Promotoras Genéticas , Animais , Células HeLa , Humanos , Luciferases/genética , Oxirredução , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Deleção de Sequência , TransfecçãoRESUMO
1. RT-PCR-southern hybridization analyses with radiolabelled P2Y receptor cDNAs as probes indicated that the peripheral blood leukocytes and the human umbilical vein endothelial cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 2. Of the haematopoietic cell lines tested, promonocytic U937 cells express P2Y2 and P2Y6, but not P2Y1 or P2Y4; promyelocytic HL-60 cells express the P2Y1, P2Y2 and P2Y6 receptors but not the P2Y4 receptor; K562 cells express P2Y1 but not P2Y2, P2Y4 or P2Y6; and Dami cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 3. Of the peripheral blood leukocytes tested, polymorphonuclear cells express P2Y4 and P2Y6 but not P2Y1 or P2Y2 receptors; monocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors and lymphocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 4. These results suggest a physiological role for different P2Y receptor subtypes in the extracellular nucleotide-mediated stimulation of monocytes, neutrophils, lymphocytes and endothelial cells.
Assuntos
Leucócitos/metabolismo , Receptores Purinérgicos P2/sangue , Southern Blotting , Linhagem Celular , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Receptores Purinérgicos P2/classificação , Receptores Purinérgicos P2/genéticaRESUMO
Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.
Assuntos
Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Receptores Purinérgicos P2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.
Assuntos
Leucemia Eritroblástica Aguda/metabolismo , Receptores Purinérgicos P2/análise , Difosfato de Adenosina/farmacologia , Marcadores de Afinidade/metabolismo , Animais , Cálcio/metabolismo , Humanos , Camundongos , Receptores Purinérgicos P2/classificação , Receptores Purinérgicos P2/genética , Células Tumorais CultivadasRESUMO
We mapped a human P2U purinergic receptor gene to chromosome 11q13.5-14.1. Oligonucleotide primers complementary to a part of the human P2U purinergic receptor cDNA were used to amplify a region from genomic DNAs from a panel of mouse/human somatic cell hybrid cell lines, each containing a single human chromosome. A PCR product of the expected size (378 bp) resulted from a single hybrid cell line containing human chromosome 11. The gene was further localized to a region of chromosome 11 using a sub-chromosomal hybrid panel containing different segments of chromosome 11. Based on the specific PCR product obtained and its Southern hybridization to the P2U receptor cDNA, the human P2U receptor gene was localized to chromosome 11q13.5-14.1.