RESUMEN

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen.

Asunto(s)

Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/administración & dosificación , Proteínas de Unión al ADN/genética , Terapia Genética/métodos , Inmunoterapia/métodos , Vacunas de ADN/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Interferón gamma/inmunología , Interleucina-12/genética , Interleucina-18/genética , Interleucina-4/inmunología , Ratones , Mutación , Trasplante de Neoplasias , Saponinas de Quillaja , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Saponinas/administración & dosificación , Células TH1/inmunología , Resultado del Tratamiento , Vacunas de ADN/genética

RESUMEN

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

Asunto(s)

Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Telomerasa/genética , Transcripción Genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Decitabina , Regulación hacia Abajo , Exones , Humanos , Regiones Promotoras Genéticas

RESUMEN

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Inhibidores de Crecimiento/fisiología , Proteínas Represoras , Factores de Transcripción/fisiología , Dedos de Zinc/fisiología , Células 3T3 , Animales , Factor de Unión a CCCTC , División Celular/genética , División Celular/fisiología , Línea Celular , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Genes myc , Proteínas Fluorescentes Verdes , Inhibidores de Crecimiento/genética , Células HeLa , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/genética , Transfección , Dedos de Zinc/genética

RESUMEN

Study of the folding status of the fibrinogen gamma-module (residues gamma 148-411) revealed that its COOH-terminal beta-strand (residues gamma 381-390), that is normally inserted into its central domain, can be removed without destroying its compact structure. Based on this and other observations we propose a "pull out" hypothesis that suggests a mechanism for the formation of transverse gamma-gamma crosslinks in fibrin.

Asunto(s)

Fibrinógeno/química , Fibrinógeno/metabolismo , Humanos , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Factor de Unión a CCCTC , Quinasa de la Caseína II , División Celular/genética , Línea Celular , Pollos , Proteínas de Unión al ADN/genética , Genes myc , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/genética

RESUMEN

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Asunto(s)

Fibrinógeno/química , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Simulación por Computador , Productos de Degradación de Fibrina-Fibrinógeno/genética , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Eliminación de Secuencia , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Termodinámica

RESUMEN

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Asunto(s)

Antígenos de Superficie , Neoplasias de la Próstata/terapia , Vacunas de ADN , Adenoviridae , Anciano , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígeno B7-2 , Carboxipeptidasas/genética , Terapia Combinada , Glutamato Carboxipeptidasa II , Humanos , Inmunización/métodos , Masculino , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Plásmidos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología

RESUMEN

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/genética , ARN no Traducido , Proteínas Represoras , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Factor de Unión a CCCTC , ADN/química , ADN/genética , ADN/metabolismo , Metilación de ADN , Elementos de Facilitación Genéticos , Femenino , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Datos de Secuencia Molecular , Unión Proteica , ARN Largo no Codificante , Dedos de Zinc

RESUMEN

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.

Asunto(s)

Ácidos/farmacología , Receptores de LDL/química , Receptores de LDL/metabolismo , Regulación Alostérica , Animales , Células CHO , Células COS , Cricetinae , Cartilla de ADN , ADN Complementario , Endocitosis/fisiología , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/análisis , Receptores ErbB/química , Receptores ErbB/genética , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Humanos , Radioisótopos de Yodo , Ligandos , Mutagénesis/fisiología , Fragmentos de Péptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Ensayo de Unión Radioligante , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Transferrina/análisis , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacología

RESUMEN

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Asunto(s)

Fibrinógeno/metabolismo , Antígeno de Macrófago-1/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Adhesión Celular , Cartilla de ADN , Fibrinógeno/química , Fibrinógeno/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

OBJECTIVES: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. METHODS: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears. Western blotting. RESULTS: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. CONCLUSION: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

Asunto(s)

Leucocitos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/sangre , Receptor fas/biosíntesis , Receptor fas/sangre , Separación Celular , Proteína Ligando Fas , Citometría de Flujo , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Líquido Intracelular/metabolismo , Leucocitos/inmunología , Proteínas de la Membrana/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Factores de Tiempo