RESUMO
Recombinant interleukin-2 (IL-2) (aldesleukin, Proleukin, Chiron, Emeryville, CA) is approved for treatment of cancer patients and under investigation in HIV-infected individuals. However, treatment with aldesleukin is associated with toxicity, which may be due to its elicitation of inflammatory mediators from cells that express the intermediate-affinity IL-2 receptor. BAY 50-4798, a novel IL-2 analog, is a selective agonist for the high-affinity receptor. It induces the proliferation of activated T cells with a potency similar to that of aldesleukin but has reduced activity on cells expressing the intermediate-affinity receptor. In the current study, we compared cytokine responses elicited in peripheral blood mononuclear cell (PBMC) cultures stimulated with BAY 50-4798 or aldesleukin. BAY 50-4798 induced approximately 5-fold lower mean levels of endogenous IL-2 than aldesleukin, and at least 50% lower levels of proinflammatory cytokines, such as tumor necrosis fctor-alpha (TNF-alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma). Furthermore, statistically significant reductions in the levels of IL-5, IL-8, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed in response to BAY 50-4798. These findings increase our understanding of the biologic action of BAY 50-4798 and suggest a mechanism by which it may exhibit better safety than aldesleukin in humans.
Assuntos
Citocinas/biossíntese , Interleucina-2/análogos & derivados , Receptores de Interleucina-2/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Química Farmacêutica , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interleucina-2/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Cinética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Mitógenos/farmacologia , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
We examined the brains of 266 patients with prion disease (PrionD) and found that 46 patients (17%) had Alzheimer disease (AD)-like changes. To explore potential mechanistic links between PrionD and AD, we exposed human brain aggregates (BrnAggs) to a brain homogenate from a patient with sporadic Creutzfeldt-Jakob disease and found that neurons in human BrnAggs produced many ß-amyloid (Aß; Aß42) inclusions, whereas uninfected control-exposed human BrnAggs did not. Western blot analysis of 20 pooled Creutzfeldt-Jakob disease-infected BrnAggs verified Aß42 levels higher than those in controls. We next examined the CA1 region of the hippocampus from 14 patients with PrionD and found that 5 patients had low levels of scrapie-associated prion protein (PrP), many Aß42 intraneuronal inclusions, low apolipoprotein E-4 (APOE-4), and no significant nerve cell loss. Seven patients had high levels of PrP, low Aß42, high APOE-4, and 40% nerve cell loss, suggesting that APOE-4 and PrP together cause neuron loss in PrionD. There were also increased levels of hyperphosphorylated tau protein (Hτ) and Hτ-positive neuropil threads and neuron bodies in both PrionD and AD groups. The brains of 6 age-matched control patients without dementia did not contain Aß42 deposits; however, there were rare Hτ-positive threads in 5 controls, and 2 controls had few Hτ-positive nerve cell bodies. We conclude that PrionD may trigger biochemical changes similar to those triggered by AD and suggest that PrionD is a disease involving PrP, Aß42, APOE-4, and abnormal tau.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Doenças Priônicas/patologia , Idoso , Doença de Alzheimer/genética , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Doenças Priônicas/genéticaRESUMO
Prion disease is caused by a single pathogenic protein (PrPSc), an abnormal conformer of the normal cellular prion protein PrPC. Depletion of PrPC in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrPC expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrPC levels were reduced by â¼ 70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrPSc formation in the thalamic infusion site by â¼ 75%, it did not suppress PrPSc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrPC in the brain is required for successful therapy of prion diseases.
Assuntos
Dependovirus/genética , Doenças Priônicas/terapia , Príons/antagonistas & inibidores , RNA Interferente Pequeno/metabolismo , Tálamo/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Camundongos , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Proteínas Priônicas , RNA Interferente Pequeno/genética , Tálamo/patologiaRESUMO
Brain aggregates (BrnAggs) derived from fetal mouse brains contain mature neurons and glial cells. We determined that BrnAggs are consistently infected with Rocky Mountain Laboratory scrapie strain prions and produce increasing levels of the pathogenic form of the prion protein (PrP). Their abundant dendrites undergo degeneration shortly after prion infection. Treatment of prion-infected BrnAggs with drugs, such as a γ-secretase inhibitors and quinacrine (Qa), which stop PrP formation and dendritic degeneration, mirrors the results from rodent studies. Because PrP is trafficked into lysosomes by endocytosis and autophagosomes by phagocytosis in neurons of prion strain-infected BrnAggs, we studied the effects of drugs that modulate subcellular trafficking. Rapamycin (Rap), which activates autophagy, markedly increased light-chain 3-II (LC3-II)-positive autophagosomes and cathepsin D-positive lysosomes in BrnAggs but could not eliminate the intracellular PrP within them. Adding Qa to Rap markedly reduced the number of LC3-II-positive autolysosomes. Rap + Qa created a competition between Rap increasing and Qa decreasing LC3-II. Rapamycin + Qa decreased total PrP by 56% compared with that of Qa alone, which reduced PrP by 37% relative to Rap alone. We conclude that the decrease was dominated by the ability of Qa to decrease the formation of PrP. Therefore, BrnAggs provide an efficient in vitro tool for screening drug therapies and studying the complex biology of prions.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Inibidores Enzimáticos/uso terapêutico , Proteínas PrPSc/metabolismo , Doenças Priônicas , Príons/metabolismo , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Azepinas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Catepsina D/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Dendritos/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunossupressores/farmacologia , Técnicas In Vitro , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Degeneração Neural/etiologia , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Proteínas do Tecido Nervoso/metabolismo , Proteínas PrPSc/antagonistas & inibidores , Gravidez , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Proteínas Priônicas , Príons/genética , Transporte Proteico/efeitos dos fármacos , Quinacrina/farmacologia , Sirolimo/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Frações Subcelulares/patologia , Fatores de TempoRESUMO
Prion diseases are disorders of protein conformation in which PrPC, the normal cellular conformer, is converted to an abnormal, protease-resistant conformer rPrPSc. Approximately 80% of rPrPSc accumulates in neuronal plasma membranes where it changes their physical properties and profoundly affects membrane functions. In this review we explain how rPrPSc is transported along axons to presynaptic boutons and how we envision the conversion of PrPC to rPrPSc in the postsynaptic membrane. This information is a prerequisite to the second half of this review in which we present evidence that rPrPSc accumulation in synaptic regions links Notch-1 signaling with the dendritic degeneration. The hypothesis that the Notch-1 intracellular domain, NICD, is involved in prion disease was tested by treating prion-infected mice with the gamma-secretase inhibitor (GSI) LY411575, with quinacrine (Qa), and with the combination of GSI + Qa. Surprisingly, treatment with GSI alone markedly decreased NICD but did not prevent dendritic degeneration. Qa alone produced near normal dendritic trees. The combined GSI + Qa treatment resulted in a richer dendritic tree than in controls. We speculate that treatment with GSI alone inhibited both stimulators and inhibitors of dendritic growth. With the combined GSI + Qa treatment, Qa modulated the effect of GSI perhaps by destabilizing membrane rafts. GSI + Qa decreased PrPSc in the neocortex and the hippocampus by 95%, but only by 50% in the thalamus where disease was begun by intrathalamic inoculation of prions. The results of this study indicate that GSI + Qa work synergistically to prevent dendrite degeneration and to block formation of PrPSc.
RESUMO
BACKGROUND: The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes. RESULTS: In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor. CONCLUSION: These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.
Assuntos
Carcinoma/genética , Ordem dos Genes , Genes Neoplásicos , Genoma Humano , Linhagem Celular Tumoral , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Quebras de DNA , Biblioteca Gênica , Humanos , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Análise de Sequência de DNA , Transcrição GênicaRESUMO
A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and co-amplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes anti-apoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project.