RESUMEN
Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrP(C)) into a protease resistant infectious form (PrP(res)). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrP(C) and PrP(res). In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.
Asunto(s)
Proteínas PrPSc/uso terapéutico , Scrapie/tratamiento farmacológico , Animales , Cricetinae , Estimación de Kaplan-Meier , Ratones , Proteínas PrPSc/química , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Scrapie/mortalidadRESUMEN
Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans.
Asunto(s)
Proteínas PrPC/uso terapéutico , Enfermedades por Prión/terapia , Proteínas Recombinantes/uso terapéutico , Scrapie/terapia , Animales , Células Cultivadas , Cricetinae , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Gliosis/fisiopatología , Gliosis/terapia , Ratones , Ratones Endogámicos C57BL , Proteínas PrPC/química , Enfermedades por Prión/genética , Proteínas Recombinantes/química , Resultado del TratamientoRESUMEN
Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that serves an essential role in B cell signaling and development. We examined the BTK expression profile of primary leukemic cells from infants with newly diagnosed acute lymphoblastic leukemia (ALL) (N = 14) and from pediatric patients with newly diagnosed (N = 10) or relapsed (N = 5) B-lineage ALL. Analysis of BTK protein and mRNA expression in the infant patient cells (N = 14) showed variable levels of BTK expression with the majority of samples having reduced to absent BTK expression. Sequence analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products of Btk mRNA from infant leukemia cells revealed the presence of aberrant transcripts. These Btk transcripts were characterized by either deletion of exon 16 (delta16) alone or deletion of both exons 15 and 16 (delta15 and 16). These deletions involve exact exon skipping and encode BTK proteins with either a deleted (delta16), or truncated (delta15 and 16) kinase domain. Extension of these Btk transcript sequencing studies to 15 pediatric B-lineage ALL patients revealed expression of exon 16 deleted Btk transcripts in several pediatric patients, however, none of these pediatric patients expressed transcripts with the exon 15 and 16 deletion. Both reduced expression of Btk message and expression of aberrant deleted Btk transcripts would contribute to reduced BTK protein expression and function in B-lineage leukemia cells. Since BTK is required for radiation induced apoptosis, reduced to absent expression of functional BTK in infant ALL cells could contribute to their radiation resistance.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Quinasas/genética , Adolescente , Agammaglobulinemia Tirosina Quinasa , Secuencia de Bases , Niño , Preescolar , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 4 , Cartilla de ADN , ADN de Neoplasias/genética , Exones , Femenino , Humanos , Lactante , Masculino , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Conformación Proteica , Proteínas Tirosina Quinasas/química , ARN Mensajero/genética , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transcripción Genética , Translocación GenéticaRESUMEN
Sequence analysis of the noncoding first exon (exon 1) of the Syk gene demonstrated the presence of a previously cloned CpG island (GenBank #Z 65706). Transient transfection analysis in Daudi cells demonstrated promoter activity (18-fold increase over parental luciferase plasmid) for a 348 bp BstXI-BsrBI fragment containing this island. This region exhibits a high GC content (approximately 75%), contains several SP1 binding sites and a potential initiator sequence, but lacks a strong TATA consensus. Bisulfite sequencing and methylation-specific PCR (MSP) of this region demonstrated that the Syk promoter CpG island was largely unmethylated in B-lineage leukemia cell lines, control peripheral blood cells, human thymocytes and CD3(+) T lymphocytes. However, dense methylation was seen in four T-lineage leukemia cell lines, Jurkat, H9, Molt 3 and HUT 78. MSP screening of leukemia cells from six T-lineage acute lymphoblastic leukemia (ALL) patients demonstrated methylation of the Syk promoter CpG island in one T-lineage ALL patient. Promoter methylation was correlated with reduced to absent expression of Syk mRNA and SYK protein in the T-lineage leukemia cell lines. Treatment of the leukemia lines Ha and Molt 3, with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) resulted in increased Syk mRNA expression. The presence of a methylated promoter sequence in these T-lineage leukemia cell lines and in one T-lineage patient suggests a potential role for SYK as a tumor suppressor in T-ALL.
Asunto(s)
Metilación de ADN , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Secuencia de Bases , Células de la Médula Ósea , Islas de CpG , Precursores Enzimáticos/genética , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Bazo/metabolismo , Quinasa SykRESUMEN
Fas (APO-1/CD95) is a 45-kDa membrane protein which regulates apoptosis in many lymphoid cell types. In the present study, FAS expression was examined in primary leukemic cells from infants with acute lymphoblastic leukemia (ALL). The cells were resistant to apoptosis induction by an anti-FAS antibody and expressed nearly undetectable amounts of FAS protein. Molecular analysis of FAS transcripts in these cells revealed no detectable expression of full-length Fas mRNA after a single round of reverse transcription and polymerase chain reaction (PCR) amplification (RT-PCR). However, a more sensitive nested RT-PCR analysis revealed alternatively spliced Fas transcripts in three of five infants (60%) with the remaining two infants showing no detectable Fas mRNA expression. The primary sequence variation of Fas mRNA seen in the samples was a previously described variant lacking exon 6 encoding soluble FAS. However, we also detected the presence of several novel alternatively spliced FAS transcripts in the ALL cells. In one patient, we observed a novel spliced form of soluble Fas, which not only lacked exon 6 but also contained an insertion of an alternative exon 7 (exon 7B). In another, a novel exon 4Del FAS mRNA variant was observed, which contained an additional 4-bp deletion at the exon 5/6-splice junction. These variants lack intact transmembrane domains and thus are predicted to encode soluble FAS variants. The low level of expression of functional full length FAS transcripts with corresponding low level of FAS protein expression in the ALL cells contribute to their resistance to CD95-mediated apoptosis.
Asunto(s)
Empalme Alternativo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptor fas/genética , Apoptosis , Secuencia de Bases , Estudios de Casos y Controles , Resistencia a Antineoplásicos , Femenino , Variación Genética , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Receptor fas/análisisRESUMEN
Here we show that the gene of the cytoplasmic tyrosine kinase SYK spans a region of 90kb with 13 coding exons, an alternative exon 14 and at least two 5' untranslated regions exons 1a and 1b. 5' RACE (Rapid amplification of cDNA ends) of human Syk cDNAs demonstrated a complex promoter usage and splicing pattern. We identified three common single nucleotide polymorphisms in the exon la promoter region of the Syk gene as well as a variant Syk cDNA haplotype. This haplotype was characterized by a constellation of 5 silent mutations in the Syk cDNA: 1065(C-T), 1302(G-C), 1338(G-A), 1521(C-T) and 1545(T-C). A hypervariable CATATA(n) repeat polymorphism was also localized to the intron between exons 11 and 12. These novel insights into the genomic organization, promoter structure and genetic variability of Syk will serve as a foundation for detailed molecular epidemiological investigation of its potential role in human cancer biology.
Asunto(s)
Mapeo Cromosómico , Precursores Enzimáticos/genética , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Bazo/enzimología , Regiones no Traducidas 5'/química , Animales , Secuencia de Bases , Clonación Molecular , Islas de CpG , ADN Complementario/genética , Exones , Estructuras Genéticas , Haplotipos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Quinasa SykRESUMEN
Janus tyrosine kinase 3 (JAK3) is one of several key regulatory enzymes in B-cell precursors which is highly conserved between multiple species. The gene for Jak3 has been mapped to human chromosome 19p12-13.1 and encompasses 23 exons. Constitutively high levels of JAK3 activity may contribute to drug resistance and enhanced clonogenicity of leukemic B-cell precursors from children and infants with acute lymphoblastic leukemia (ALL). As part of a systematic effort to accurately determine the genomic sequence of Jak3 gene in normal and leukemic B-cell precursors, we sequenced a relatively short region of Jak3 spanning two introns, originally termed introns 10 and 11. This genomic sequence appeared in certain RT-PCR products from our analysis of Jak3 gene expression in pediatric, as well as infant, primary ALL cells. Unexpectedly, a gap in the original Jak3 genomic sequence was found in intron 10 across the sequence matching to an Alu element. Furthermore, the sequence obtained from intron 11 did not match at all to that previously reported, and the length of the intron was much larger than expected at 1.1 kb. Homology to Alu elements (three regions, 699 bp total) and a LINE2 element (one region, 189 bp total) were seen across the entire region covering exons 10-12 (2.1 kb total). Two potential single nucleotide polymorphisms (SNPs) were observed in intron 11. No apparent genomic mutation was found across this region in leukemic B-cell precursors from any of the ALL patients examined. This newly described sequence corrects the previous published genomic sequence from this region rather than identifying an insertion or translocation specific to these ALL cases. Our results significantly extend previous efforts to determine the genomic sequence of Jak3 and analyze its expression in childhood pro-B ALL and other forms of ALL.