RESUMEN
Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85+/-6% and 86+/-4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19(+) target cells resulting in > or =80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer.
Asunto(s)
Antígenos CD19/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Antígenos/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Electroporación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Receptores de Antígenos/genética , TransfecciónRESUMEN
The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas Inmediatas-Precoces , Proteínas de Neoplasias , Factores de Crecimiento Nervioso/farmacología , Proteínas Represoras/farmacología , Proteínas Represoras/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , División Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Células PC12 , Ratas , Factores de Transcripción/fisiología , Transfección/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study we have identified a factor, CDF-1, which interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro , in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B- myb promoter. Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes that are up-regulated early (around mid G1), such as B- myb , CDE-CHR-controlled genes, such as cdc25C , cdc2 and cyclin A , become derepressed later. Finally, the fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteínas Portadoras , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Ciclina A/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/genética , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Proteínas Represoras/fisiología , Células 3T3 , Animales , Proteína Quinasa CDC2/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Ciclina A/biosíntesis , Huella de ADN , Factores de Transcripción E2F , Sustancias Macromoleculares , Ratones , Fosfoproteínas Fosfatasas/biosíntesis , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transfección , Fosfatasas cdc25RESUMEN
The TATA- and Inr-less promoter of the human cdc25C gene is regulated during the cell cycle through binding of a repressor to two contiguous promoter-proximal elements, the CDE and CHR. In this study we have characterized in detail the region of the cdc25C promoter immediately downstream of these elements. Several lines of evidence suggest that this region of approximately 60 bp acts as the core promoter. This sequence: (i) harbors most of the transcription initiation sites; (ii) possesses basal promoter activity in vivo ; (iii) shows no stable protein binding in vivo as indicated by genomic dimethyl sulfate and phenanthroline copper footprinting; (iv) contains single-stranded regions in vivo as shown by potassium permanganate footprinting; (v) is hypersensitive to DNase I cleavage in permeabilized cells. Mutational analysis of the core promoter revealed the presence of three sites which play a role in transcription. Two of these sites were found to represent low affinity binding sites for transcription factors of the Sp1 family. Mutation of these sites led to decreased levels of transcription, while their alteration to canonical Sp1 sites impaired cell cycle regulation. Thus the transient interaction of Sp1 with the core promoter appears to be necessary for maximal transcription without perturbing cell cycle regulation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Regulación de la Expresión Génica/genética , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Transcripción Genética/genética , Sitios de Unión , Proteínas de Ciclo Celular/biosíntesis , Línea Celular , Huella de ADN , Fibroblastos , Humanos , Fosfoproteínas Fosfatasas/biosíntesis , Factores de Transcripción/metabolismo , Fosfatasas cdc25RESUMEN
The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element'; CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. We now show that CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation of all three genes. In addition, the cdc25C, cyclin A and cdc2 genes share an identical 5 bp region ('cell cycle genes homology region'; CHR) starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting showed the periodic occupation of the cyclin A CDE in the major groove, and of the CHR in the minor groove. Finally, all three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Regulación de la Expresión Génica , Genes cdc , Transcripción Genética , Fosfatasas cdc25 , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Secuencia de Consenso , Ciclinas/genética , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes , Análisis de Secuencia de ADNRESUMEN
Elongation factor-1 alpha (EF-1 alpha) is a highly conserved protein functioning in peptide elongation during translation. A cDNA, S1, was isolated; its deduced amino acid sequence shares high similarity with mammalian EF-1 alpha s (92%). While EF-1 alpha is present in all tissues, S1 mRNA can only be detected in brain, heart, and muscle. We report here that the retropseudogene phenomenon is attributable to EF-1 alpha and not S1, the latter being represented by a single copy in the rat genome. The S1 steady-state mRNA levels are consistently higher than EF-1 alpha in S1-positive tissues. S1 mRNA can only be detected late during brain, heart, and muscle development in vivo and increases to a plateau in early postnatal life. In a cultured muscle system, S1 expression is dependent upon the formation of myotubes, although the accumulation of S1 mRNA is significantly lower than that observed in adult skeletal muscle. EF-1 alpha mRNA levels are down-regulated during brain, heart, and muscle development, but stay relatively steady in liver. We show here that EF-1 alpha and S1 are differentially expressed during rat development and that the activation of S1 gene expression is subsequent to the terminal differentiation process in brain, heart, and muscle.
Asunto(s)
Regulación de la Expresión Génica , Factores de Elongación de Péptidos/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , ADN Complementario , Desarrollo Embrionario y Fetal/genética , Femenino , Datos de Secuencia Molecular , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Factor 1 de Elongación Peptídica , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The nucleotide sequence of a full-length cDNA, the deduced amino acid sequence, and the regulation of expression of a cold acclimation-specific gene, cas18, in cell suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago falcata cv Anik) have been determined. The deduced polypeptide, CAS18, is relatively small (17.6 kD), is highly hydrophilic, is rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/dehydrin family of proteins, which accumulate in response to abscisic acid (ABA) or water stress. It is intriguing that cas18 is induced by neither ABA nor water stress. The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4, and 1.0 kb, and the cDNA characterized here corresponds to the 1.0-kb transcript. The expression of this gene is about 30-fold greater in cold-acclimated cells than in nonacclimated cells. Although the accumulation of transcripts during cold acclimation is relatively slow, their disappearance during deacclimation is dramatically rapid, becoming undetectable in less than 5 h. Studies of nuclear run-on transcription show that cold acclimation enhances the transcription of this gene nearly 9-fold. The stability of cas18-detectable transcripts during deacclimation is considerably increased if transcription is inhibited with cordycepin. It therefore appears that low temperature regulates the expression of cas18 at both the transcriptional and posttranscriptional levels.