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1.
Int J Cancer ; 123(8): 1741-50, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18324679

RESUMEN

Extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. Extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. The present study shows that the interaction between recombinant E-selectin (rE-selectin) and colorectal cancer (CRC) cells alters the gene expression profile of the cancer cells. A DNA microarry analysis indicated that E-selectin-mediated alterations were significantly more pronounced in the metastatic CRC variants SW620 and KM12SM than in the corresponding non-metastatic local SW480 and KM12C variants. The number of genes altered by E-selectin in the metastatic variants was about 10-fold higher than the number of genes altered in the corresponding local variants. Aiming to identify genes involved in CRC metastasis, we focused, by using a DNA microarry analysis, on genes that were altered by E-selectin in a similar fashion exclusively in both metastatic variants. This analysis indicated that E-selectin down regulated (at least by 1.6-folds) the expression of 7 genes in a similar fashion, in both metastatic cells. The DNA microarry analysis was validated by real time PCR or by RT-PCR. HMGB1 was among these genes. Confocal microscopy indicated that E-selectin down regulated the cellular expression of the HMGB1 protein and enhanced the release of HMGB1 into the culture medium. The released HMGB1 in turn, activated endothelial cells to express E-selectin.


Asunto(s)
Neoplasias Colorrectales/genética , Selectina E/metabolismo , Selectina E/farmacología , Regulación Neoplásica de la Expresión Génica/fisiología , Proteína HMGB1/biosíntesis , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Selectina E/biosíntesis , Células Endoteliales/metabolismo , Células Endoteliales/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteína HMGB1/genética , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
2.
J Mol Biol ; 352(2): 245-52, 2005 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-16083908

RESUMEN

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.


Asunto(s)
Amiloide/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Cobre/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Oligopéptidos/genética , Factores de Terminación de Péptidos , Priones/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido
3.
Immunol Lett ; 104(1-2): 138-45, 2006 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-16386315

RESUMEN

The dual-specificity phosphatase Pyst2-L was found to be over expressed in leukocytes derived from AML and ALL patients as well as in certain other solid tumors and lymphoblastoid cell lines. Pyst2-L, binds and dephosphorylates both pERKs and pJNKs proteins, and thus, plays a role in regulating the MAP kinase signaling pathway. In the present study, a comparative genomic application was used and sequence analysis of multi-organisms databases were searched in order to identify genes homologous to Pyst2-L. The Xenopus laevis MAP kinase phosphatase X17c gene and the Yeast nitrogen starvation-induced protein phosphatase Yvh1p gene were revealed to be highly homologous with Pyst2-L. Both X17c and Yvh1p genes play a role in cell cycle regulation. A down regulated expression of the Yvh1p gene occurred in Saccharomyces cerevisiae that were synchronized to the G2-phase of the cell cycle by alpha-factor. In conformity with this result, a reduction in Pyst2-L expression levels was observed in G2-phase-synchronized Human K562 cells. Finally, we were able to show that cells in highly crowded cultures express high levels of the Pyst2-L phosphatase. These observations may indicate that low levels of the Pyst2-L phosphatase are essential for the G2-phase of the cell cycle and that this phosphatase might play a role in signaling cascades induced by cellular crowding.


Asunto(s)
Ciclo Celular , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular/genética , Células Cultivadas , Fosfatasa 6 de Especificidad Dual , Fosfatasas de Especificidad Dual , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas de Xenopus/genética
4.
Cancer Lett ; 242(1): 1-10, 2006 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-16413116

RESUMEN

The research area of tumor microenvironment is considered, at present, to be an important factor in tumorigenesis and especially in tumor progression. The present mini review is focused on three principles characterizing the nature of the tumor microenvironment. We first discuss the regulatory functions of the tumor microenvironment and the complexity of the combinatorial signaling pathways operating in it. We then address the aspect that the tumor microenvironment incorporates both pro and anti malignancy factors and that a balance between these factors regulates tumor progression. Thirdly we provide evidence that the non-tumor cells in the tumor microenvironment and their products may be different from those of their counterparts residing in non-tumor microenvironments. The conclusion of this mini review is that the tumor microenvironment, by exerting regulatory functions and selective pressures drives cancer cells into one of several molecular evolution pathways thereby determining and shaping their malignancy phenotype.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/patología , Transformación Celular Neoplásica , Progresión de la Enfermedad , Evolución Molecular , Humanos , Metástasis de la Neoplasia , Neovascularización Patológica , Fenotipo , Transducción de Señal
5.
Oncogene ; 22(48): 7649-60, 2003 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-14576828

RESUMEN

Northern blotting confirmed previous results indicating that the mitogen-activated protein kinase (MAPK) phosphatase Pyst2-L was highly expressed in leukocytes obtained from acute myeloid leukemia (AML) patients. High levels of Pyst2-L mRNA were expressed in bone marrow (BM) and peripheral leukocytes from nine AML and acute lymphoblastic leukemia (ALL) patients. BM from healthy individuals expressed very low levels of Pyst2-L. Whereas high levels of Pyst2-L mRNA and protein were detected in several leukemia cell lines, Pyst2-L mRNA was detected neither in 33/34 samples of normal peripheral blood mononuclear cells (PBMC) nor in leukocyte fractions enriched with CD34+ cells. Certain solid tumor and lymphoblastoid cell lines expressed high levels of Pyst2-L mRNA. In view of the association of Pyst2-L to MAPK signaling cascades, we tested if cell activation, a process involving MAPK signaling, influences Pyst2-L expression. Indeed, activation of T cells and endothelial cells increased Pyst2-L in these cells. Furthermore, TPA, a known MAPK activator, induces the expression of both Pyst2-L mRNA as well as the Pyst2-L protein in leukemia cells. This induction was partially inhibited by PD098059, an Mek1/2-specific inhibitor. Based on the results of this and previous studies, we hypothesize that the high levels of Pyst2-L detected in the active state of AML and ALL diseases and in other types of cancer reflect an altered MAPK signaling pathway in such malignant processes. This alteration may be the result of a failed attempt to counter the constitutive activation of MAPK in transformed cells or alternatively, may represent the activated state of such cells.


Asunto(s)
Leucemia Mieloide/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Línea Celular Tumoral , Fosfatasas de Especificidad Dual , Regulación Enzimológica de la Expresión Génica , Humanos , Leucemia Mieloide/patología , Leucocitos/enzimología , Leucocitos/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Especificidad por Sustrato , Regulación hacia Arriba
6.
Methods Enzymol ; 366: 103-13, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-14674243

RESUMEN

The information contained in the draft sequence of the human genome offers a solid basis to study gene function. The DNA microarray technology is one of the most important tools enabling the performance of such studies. The global expression analysis using microarrays enables the construction of a simultaneous expression pattern of thousands of genes and thus an unprecedented opportunity to obtain molecular signatures of the state of activity of diseased cells. Microarray analysis may thus provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments. Ultimately such analyses may lead to improved early diagnosis and innovative therapeutic approaches. Yet, by employing this advanced technology in order to identify new target molecules for diagnosis or therapy, one is faced with the necessity to focUs on one or several genes and to investigate them further. A huge number of differentially expressed genes are usually identified when 2 cell samples are comparatively analyzed in an individual assay. Several factors, mainly a possible genetic diversity between the samples, minimize the capability of reaching an educated conclusion as to which gene or genes to select for further studies. Gene expression arrays were employed to identify genes whose expression characterizes leukemic leukocytes. Many different genes were differentially expressed by "leukemic-phase leukocytes," derived from three untreated acute myelogenous leukemia (AML) patients and by "remission-phase leukocytes," obtained from the same patients following induction of remission. However, only two of these genes, the dual-specificity MAPK phosphatase PYST2 and the tryptophan 5-hydroxylase, were found to be more highly expressed by the leukemic-phase than by remission-phase leukocytes of all three patients. The microarray results of only the PYST2 gene could be verified by RT-PCR. By focusing on genes that had a similar expression pattern in cells from multiple donors we overcame the problem of genetic diversity and identified, out of 60 differentially expressed genes, a single candidate target gene in AML. Pooling specimens in order to overcome the problem of genetic diversity (e.g., clinical material obtained from treated patients and from untreated ones), is not recommended because it may dilute the treatment effects. Therefore, it is essential to perform each experiment using individual preparations.


Asunto(s)
Leucemia Mieloide Aguda/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Tirosina Fosfatasas/genética , Adulto , Secuencia de Bases , Cartilla de ADN , Fosfatasas de Especificidad Dual , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Fosfoproteínas Fosfatasas , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
7.
Cancer Lett ; 199(2): 185-92, 2003 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-12969791

RESUMEN

In a previous study we used gene expression arrays to identify genes that are more highly expressed by leukemic than by non-leukemic leukocytes from acute myelogenous leukemia patients. One of such genes was Phosphorylates tyrosine serine threonine 2 (PYST2), a dual-specificity Mitogen-activated protein (MAP) kinase (MAPK) phosphatase. In the present study, high levels of PYST2 mRNA were detected by RT-PCR and by Northern blotting in bone marrow (BM) leukocytes and in peripheral blood mononuclear cells from additional eight AML patients. No PYST2 mRNA was detected in nine out of twelve samples of Peripheral blood mononuclear cells (PBMC) from healthy blood bank donors and very low levels were detected by the same techniques in the other three PBMC samples from the healthy donors. Relatively high levels of PYST2 were detected in a variety of myeloid leukemia and other cancer cell lines. In view of the potential role played by PYST2 in MAPK signaling cascades we propose that an over expression of PYST2 in malignant cells may reflect a disrupted or an altered MAPK signaling pathway in malignancy processes.


Asunto(s)
Leucemia Mieloide/genética , Proteínas Tirosina Fosfatasas/genética , Regulación hacia Arriba/fisiología , Enfermedad Aguda , Adulto , Northern Blotting , Médula Ósea/patología , Células Cultivadas , Cartilla de ADN , Fosfatasa 1 de Especificidad Dual , Fosfatasas de Especificidad Dual , Femenino , Citometría de Flujo , Humanos , Leucemia Mieloide/metabolismo , Masculino , Persona de Mediana Edad , Fosfoproteínas Fosfatasas , Reacción en Cadena de la Polimerasa , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/análisis , ARN Neoplásico/genética , ARN Neoplásico/metabolismo
8.
Int J Med Microbiol ; 295(4): 227-36, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16128397

RESUMEN

Extraintestinal pathogenic Escherichia coli (ExPEC) strains have been shown to code for several virulence factors involved in adherence to host tissues. Here we show the existence of an additional adherence gene cluster, coding for long polar fimbriae--LPF--in several strains of serotype O78 from septicemia and newborn meningitis. The complete gene cluster was sequenced in strain 789 (lpf789), where it is located between the genes glmS and pstS, and contains four ORFs, lpfA to lpfD. The lpf operon is expressed and is important for adherence to epithelial cells. The lpf operon was found only in four of the ExPEC strains tested and is likely to have been acquired by horizontal gene transfer.


Asunto(s)
Adhesión Bacteriana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Adhesión Bacteriana/fisiología , Línea Celular , Escherichia coli/clasificación , Escherichia coli/patogenicidad , Escherichia coli/fisiología , Proteínas de Escherichia coli/fisiología , Operón , Virulencia
9.
Cytokine ; 29(3): 105-17, 2005 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-15613278

RESUMEN

Bone marrow stroma cells secrete the chemokine CXCL12 that may support bone marrow metastasis formation by neuroblastoma cells. The present study demonstrates that bone marrow stroma cell lines also secrete CXCL10, a chemokine that was shown in the past to have anti-malignancy functions. A receptor recognized by antibodies against CXCR3 was shown to be expressed by six neuroblastoma cell lines. Further detailed analysis was performed on the NUB6 and SK-NMC neuroblastoma cells, showing that CXCL10 induced potent Erk phosphorylation in a G(alpha)i-dependent manner. The role of a CXCR3-like receptor in Erk phosphorylation was substantiated by the ability of CXCL11, another potent CXCR3 ligand, to induce Erk phosphorylation in the NUB6 and SK-NMC cells. Further characterization of CXCL10 activities indicated that CXCL10 partly inhibited the growth of the NUB6 and SK-NMC cells. Both NUB6 and SK-NMC cells did not migrate to CXCL10, although their migratory machinery was intact, as evidenced by their migration to bone marrow constituents. Altogether, these results suggest that CXCL10 interacts with a CXCR3-like receptor in neuroblastoma cell lines, raising the possibility that following the homing of the tumor cells to the bone marrow (through a CXCL10-independent mechanism), CXCL10 may partly inhibit neuroblastoma cell growth at this site.


Asunto(s)
Quimiocinas CXC/metabolismo , Neuroblastoma/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Anticuerpos/inmunología , Quimiocina CXCL10 , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunohistoquímica , Ratones , Fosforilación , Receptores CXCR3 , Receptores de Quimiocina/inmunología
10.
Genes Chromosomes Cancer ; 39(1): 37-47, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14603440

RESUMEN

PYST2 is a member of a structurally homologous subfamily of MAP kinase phosphatases. A computer-based analysis of the PYST2 locus revealed that it harbors two alternative open reading frames promoted by two conserved promoter regions. Using Northern blot analysis and reverse transcription-polymerase chain reaction followed by sequencing and alignment of the products, we confirmed the existence of two mRNAs that were transcribed from this genomic region. Western blot analysis indicated that these transcripts were translated. Functional bioinformatic analysis of both transcripts revealed that exon 2 exists in only one of the PYST2 transcripts, designated PYST2-L, and has the consensus elements of the phosphatase catalytic domain (PCD). We found that the translation from the PYST2-L transcript starts 46 codons upstream from the (already-known) PYST2 5' sequence. Furthermore, the existence of three PYST2-L transcripts was indicated. These transcripts differ only in their 5' untranslated regions (5'UTRs). Unlike PYST2-L, the other mRNA (PYST2-S) is devoid of any known PCD. Analysis of the predicted Pyst2-S protein revealed the presence of the vertebrate metallothionein signature I, the mammalian defensin, and the zinc-containing alcohol dehydrogenase motifs. These motifs might confer on this protein the ability to sense changes in the cellular environment. From these and previous results, we speculate that Pyst2-S may function as a negative regulator of Pyst2-L.


Asunto(s)
Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Línea Celular Tumoral , Fosfatasas de Especificidad Dual , Humanos , Leucemia Mieloide/enzimología , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas , Especificidad por Sustrato
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