RESUMO
Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease with heightened disease severity in children. The incomplete understanding of the precise cellular and molecular events that drive disease activity pose a significant hurdle to the development of targeted therapeutic agents. Here, we performed single-cell phenotypic and functional characterization of pediatric SLE patients and healthy controls blood via mass cytometry. We identified a distinct CD14hi monocyte cytokine signature, with increased levels of monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1ß (Mip1ß), and interleukin-1 receptor antagonist (IL-1RA). This signature was shared by every clinically heterogeneous patient, and reproduced in healthy donors' blood upon ex-vivo exposure to plasma from clinically active patients only. This SLE-plasma induced signature was abrogated by JAK1/JAK2 selective inhibition. This study demonstrates the utility of mass cytometry to evaluate immune dysregulation in pediatric autoimmunity, by identification of a multi-parametric immune signature that can be further dissected to delineate the events that drive disease pathogenesis.
RESUMO
We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.
Assuntos
Genes MDR/genética , Paclitaxel/farmacologia , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Motivos de Aminoácidos , Sequência de Aminoácidos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Dominantes/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
In autoimmune Type 1 diabetes (T1D), immune cells progressively infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, it is unclear how this process, called 'insulitis', develops and progresses within this organ. Here, using highly multiplexed CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples from pre-T1D, T1D, and non-T1D donors, we examine pseudotemporal-spatial patterns of insulitis and exocrine inflammation within large pancreatic tissue sections. We identify four sub-states of insulitis characterized by CD8 + T cells at different stages of activation. We further find that exocrine compartments of pancreatic lobules affected by insulitis have distinct cellularity, suggesting that extra-islet factors may make particular lobules permissive to disease. Finally, we identify "staging areas" - immature tertiary lymphoid structures away from islets where CD8 + T cells appear to assemble before they navigate to islets. Together, these data implicate the extra-islet pancreas in autoimmune insulitis, greatly expanding the boundaries of T1D pathogenesis.
RESUMO
Two models have been proposed for the molecular mechanism by which the Tal1 oncogene causes T cell acute lymphoblastic leukemia (T-ALL). The activation model suggests that Tal1 as heterodimers with the E2A transcription factor activates the expression of oncogenes. The inhibition model postulates that Tal1 interferes with the tumor-suppressing function of E2A. In the Jurkat T cell line, originally derived from a patient with T-ALL, Tal1 is complexed with E2A proteins and the transcriptional activity of E2A is very low. When E2A activity was restored by expressing an E2A-Tal1 fusion protein, E-T/2, the Jurkat cells underwent growth arrest and subsequently apoptosis, thus supporting the inhibition model and suggesting that E2A loss may contribute to leukemic progression.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Inibidores de Caspase , Divisão Celular , Meios de Cultura Livres de Soro , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/genética , Humanos , Células Jurkat , Proteínas Recombinantes/biossíntese , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genéticaRESUMO
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Interleucina-4/administração & dosagem , Retroviridae/genética , Animais , Terapia Genética , Vetores Genéticos , Imunização Passiva , Imunoterapia , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Camundongos , Proteína Básica da Mielina/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Transdução GenéticaRESUMO
Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.
Assuntos
Separação Celular/métodos , Drosophila melanogaster/embriologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Dissacarídeos , Escherichia coli/genética , Citometria de Fluxo , Corantes Fluorescentes , Galactosídeos/análise , Óperon Lac , Neurônios/citologia , Células-Tronco/citologiaRESUMO
The gene families encoding the proteins NF-kappa B, c-Rel and Dorsal, in conjunction with their respective inhibitors l kappa B, pp40, and Cactus, achieve specificity in gene regulation by means of common principles. The related activities of NF-kappa B and Dorsal are mediated by heterodimeric or homodimeric complexes of proteins containing the conserved dimerization and DNA-binding domain termed Rel. The l kappa Bs and Cactus, which share a core series of structural repeats termed ankyrin, inhibit cognate activators through differential interactions with the Rel-homology domain. Together, the inhibitory ankyrin proteins and their cognate Rel dimers probably define specific signalling pathways able to activate specific gene expression. Both gene families include proto-oncogenes, thus broadly implicating Rel/l kappa B in the control of both normal gene expression and the aberrant gene expression that makes cells cancerous.
Assuntos
Proteínas Sanguíneas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/fisiologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Anquirinas , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , NF-kappa B/química , NF-kappa B/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-rel , Fator de Transcrição RelBRESUMO
The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.
Assuntos
Apoptose , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Proteína Ligante Fas , Glicoproteínas de Membrana/farmacologia , Ligação Proteica , Proteínas/metabolismo , Fator 2 Associado a Receptor de TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The REL/NF-kappaB/IkappaB superfamily of signal transducers and transcription factors are paradigmatic of molecular mechanisms by which rapid responses in the immune system can be achieved. NF-kappaB proteins have been implicated in diverse processes such as the ontogeny of the immune system, immune responses to pathogens and, importantly, in contributions to the multistage processes of oncogenesis, as described in this review. NF-kappaB and its regulators, the IkappaBs, are linked to pro- and anti-apoptotic events as well as signaling systems contributing to cellular transformation. How are these disparate events controlled to effect normal and abnormal processes in cells? Here we explore a few of the many events in which NF-kappaB appears to participate and processes that integrate signals to control important stages of oncogenesis.
Assuntos
Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Apoptose , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Genéticos , Inibidor de NF-kappaB alfa , Oncogenes , Proteínas Proto-Oncogênicas c-rel , Transdução de SinaisRESUMO
The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Animais , Proteína 3 do Linfoma de Células B , Baculoviridae/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Mariposas , NF-kappa B/antagonistas & inibidores , Oligodesoxirribonucleotídeos , Sondas de Oligonucleotídeos , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Mapeamento por Restrição , Fatores de Transcrição , TransfecçãoRESUMO
We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.
Assuntos
Diversidade de Anticorpos , DNA Nucleotidiltransferases/análise , Genes de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfócitos/imunologia , Animais , Linfócitos B/imunologia , Linhagem Celular , Células Clonais , Citometria de Fluxo/métodos , Rearranjo Gênico , VDJ RecombinasesRESUMO
Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.
Assuntos
Fibroblastos/fisiologia , Fase G1 , Genoma Humano , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sobrevivência Celular , Senescência Celular/genética , Senescência Celular/fisiologia , DNA , Dano ao DNA , Reparo do DNA , Humanos , Hibridização in Situ Fluorescente , Transdução de Sinais , TelômeroRESUMO
We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- gamma (IFN-gamma)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-gamma, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the beta-chain of the IFN-gamma receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.
Assuntos
Biblioteca Gênica , Fosfoproteínas/análise , Retroviridae/genética , Transdução de Sinais , DNA Complementar/análise , Proteínas de Ligação a DNA/metabolismo , Fixadores , Citometria de Fluxo , Formaldeído/química , Humanos , Interferon gama/farmacologia , Metanol/química , Fosforilação , Reação em Cadeia da Polimerase , Receptores de Interferon/genética , Fator de Transcrição STAT1 , Transativadores/metabolismo , Células U937RESUMO
Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single-cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting gene set enrichment analysis (GSEA) of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.
Assuntos
Leucemia Mieloide Aguda/metabolismo , NF-kappa B/metabolismo , Mielofibrose Primária/metabolismo , Transdução de Sinais , Antígenos CD34 , Medula Óssea , Linhagem Celular , Citometria de Fluxo/métodos , Humanos , Janus Quinase 2/genética , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/patologia , Mielofibrose Primária/patologiaRESUMO
Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.
Assuntos
Carbamatos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Citocinas/genética , Adolescente , Animais , Linhagem Celular Tumoral , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Nitrilas , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirazóis/farmacologia , Pirimidinas , Fator de Transcrição STAT5/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.
Assuntos
DNA de Cadeia Simples/genética , Biblioteca Gênica , Oligonucleotídeos/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Clonagem Molecular/métodos , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Pirrolidinonas/farmacologia , Reprodutibilidade dos Testes , Retroviridae/genética , Moldes Genéticos , TransfecçãoRESUMO
We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Herpesvirus Humano 4/genética , Proteínas Luminescentes/metabolismo , Retroviridae/genética , Células 3T3 , Animais , Humanos , Proteínas Luminescentes/genética , Camundongos , Células Tumorais CultivadasRESUMO
To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.
Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas Genéticas , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Anfirregulina , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Células Cultivadas/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Meios de Cultura Livres de Soro , DNA Complementar/genética , Família de Proteínas EGF , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/fisiologia , Feminino , Biblioteca Gênica , Genes do Tumor de Wilms , Teste de Complementação Genética , Vetores Genéticos/genética , Glicoproteínas/genética , Substâncias de Crescimento/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Retroviridae/genética , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
BACKGROUND: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo. RESULTS: A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here 'fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat pIII protein of the filamentous bacteriophage M13. The 'best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red. Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution. In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye. CONCLUSIONS: Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection. Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes. Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.
Assuntos
Proteínas de Transporte/síntese química , Corantes Fluorescentes/metabolismo , Peptídeos/síntese química , Sequência de Aminoácidos , Bacteriófagos , Sequência de Bases , Proteínas de Transporte/metabolismo , Escherichia coli , Fluoresceína/química , Fluoresceína/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Rodaminas/metabolismo , Xantenos/metabolismoRESUMO
The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.