RESUMO
A critical function of the telomere is to disguise chromosome ends from cellular recognition as double strand breaks, thereby preventing aberrant chromosome fusion events. Such chromosome end-to-end fusions are known to initiate genomic instability via breakage-fusion-bridge cycles. Telomere dysfunction and other forms of genomic assault likely result in misregulation of genes involved in growth control, cell death, and senescence pathways, lowering the threshold to malignancy and likely drive disease progression. Shortened telomeres and anaphase bridges have been reported in a wide variety of early precursor and malignant cancer lesions including those of the prostate. These findings are being extended using methods for the analysis of telomere fusions (decisive genetic markers for telomere dysfunction) specifically within human tissue DNA. Here we report that benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) prostate lesions all contain similarly high frequencies of telomere fusions and anaphase bridges. Tumor-adjacent, histologically normal prostate tissue generally did not contain telomere fusions or anaphase bridges as compared to matched PCa tissues. However, we found relatively high levels of telomerase activity in this histologically normal tumor-adjacent tissue that was reduced but closely correlated with telomerase levels in corresponding PCa samples. Thus, we present evidence of high levels of telomere dysfunction in BPH, an established early precursor (PIN) and prostate cancer lesions but not generally in tumor adjacent normal tissue. Our results suggest that telomere dysfunction may be a common gateway event leading to genomic instability in prostate tumorigenesis. .
Assuntos
Instabilidade Cromossômica , Hiperplasia Prostática/genética , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Telômero/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Quinazolinas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Análise de Sobrevida , Fatores de Tempo , TranscriptomaRESUMO
Human adipose stromal cells (ASCs) reside within the stromal-vascular fraction (SVF) in fat tissue, can be readily isolated, and include stem-like cells that may be useful for therapy. An important consideration for clinical application and functional studies of stem/progenitor cells is their capacity to maintain chromosome stability in culture. In this study, cultured ASC populations and ASC clones were evaluated at intervals for maintenance of chromosome stability. Uncultured SVF (uSVF) cells were included for comparison. G-banded chromosome analysis demonstrated that ASCs are diploid and have a normal karyotype. However since only approximately 20 cells are examined, low levels of chromosome instability would not be detected. To increase detection sensitivity, fluorescence in situ hybridization was employed, to permit chromosome enumeration in larger numbers of interphase cells. Seven cultured ASC populations, two ASC clones and four uSVF samples were examined. Chromosome X and 17 probes identified diploid, tetraploid, and aneuploid interphase cells. Both cultured ASC populations [up to approximately 35 Population Doublings (PDs)] and uSVF cells exhibited a similar level of diploidy (97.8% n = 6,355 and 98.83% n = 1,197, respectively) and numerical abnormalities, suggesting that cultured ASCs are genomically stable and supporting their suitability for transplantation applications. In comparison, cultured primary human chorionic villus cells exhibited marked genomic instability resulting in an 11.6% tetraploidy rate after 8-10 PD. Thus effects of culture on genomic stability may be cell type dependent and should be tested by appropriately scaled interphase fluorescence in situ hybridization analysis in any ex vivo expanded cell population destined for transplantation.
Assuntos
Tecido Adiposo/citologia , Instabilidade Genômica , Hibridização in Situ Fluorescente , Interfase , Células Estromais/citologia , Células Estromais/metabolismo , Adulto , Vasos Sanguíneos/citologia , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Cromossomos Humanos/genética , Células Clonais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diagnóstico Pré-Natal , Fatores de TempoRESUMO
PURPOSE: To elucidate the perspectives of patients on the conceptual framework for a new undergraduate medical curriculum organized around the healer and professional roles of the physician (their physicianship), and to illustrate how these perspectives can affect program development. METHOD: In 2006, using an adapted interpretive description design and semistructured interviews, the authors collected data from a sample of 58 patients receiving care in a major academic medical center. RESULTS: Three findings were particularly salient. (1) The concepts of the physician as healer and professional, although central to the curriculum, did not resonate strongly with patients. The words professionalism and healer occasionally accrued negative connotations. There was little concordance between the lexicon patients use to describe ideal physician behaviors and a faculty-defined list of core physicianship attributes. (2) The listening skills of physicians were highly valued and seen as an "essentia" of ideal doctoring. (3) Being treated as a number by physicians represented a threat to patients' personal identity. CONCLUSIONS: This study found important differences between patients' and physicians' perspectives on key curricular concepts. Understanding these differences represents an important resource for program design and development. The findings also suggest avenues for future research on highly topical issues such as professionalism.
Assuntos
Currículo , Assistência Centrada no Paciente , Relações Médico-Paciente , Competência Profissional , Centros Médicos Acadêmicos , Adolescente , Adulto , Idoso , Estudos Transversais , Educação de Graduação em Medicina , Feminino , Humanos , Pacientes Internados , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Assistência ao PacienteRESUMO
Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-gamma stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-gamma-induced expression of some genes in VACV-infected cells, IFN-gamma was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses.
Assuntos
Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/imunologia , Interferon gama/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Fator de Transcrição STAT1/imunologia , Vaccinia virus/imunologia , Animais , Apresentação de Antígeno , Técnicas de Cultura de Células , Citocinas/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Fator de Transcrição STAT1/genética , Transdução de Sinais , Linfócitos T/imunologia , Vírus da Varíola/imunologiaRESUMO
Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric alpha -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic alpha -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in >or=90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency.
Assuntos
Cromossomos Artificiais Humanos/genética , Fator IX/genética , Animais , Northern Blotting , Southern Blotting , Células Cultivadas , Cromossomos Artificiais Bacterianos/genética , Cricetinae , Cricetulus , Imunofluorescência , Humanos , Hibridização in Situ Fluorescente , Camundongos , Modelos Genéticos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Transfecção/métodos , Transgenes/genéticaRESUMO
Viral infections produce severe respiratory morbidity in children with cystic fibrosis (CF). CF cells are more susceptible to virus in part because of impaired airway epithelial activation of signal transducer and activator of transcription 1 (Stat1). As Stat1 is a fundamental regulator of antiviral defenses, we hypothesized that there may be multiple alterations in the antiviral defense of CF epithelium compared with normal (NL). To obtain a comprehensive view of mucosal host responses to influenza and characterize the difference between CF and NL responses to influenza, gene expression profiles of primary human airway epithelial cells (HAEC) were evaluated using an interferon (IFN)-stimulated genes/AU/double-stranded RNA (dsRNA) microarray or quantitative real-time polymerase chain reaction (PCR) following influenza A infection. Gene expression was significantly modified by influenza in NL (228 genes) and CF (101 genes), with a similar pattern of gene response but with overall less numbers of responsive genes in CF (p < 0.05). Moreover, CF cells had less IFN-related antiviral gene induction at 24 h but greater inflammatory cytokine gene induction at 1 h after infection. Taken together, the lesser antiviral and greater early inflammatory response likely contribute to the severe respiratory illness of CF patients with viral infections.
Assuntos
Fibrose Cística/patologia , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Vírus da Influenza A/metabolismo , Influenza Humana , Mucosa Respiratória/citologia , Células Cultivadas , Criança , Fibrose Cística/fisiopatologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Ativação TranscricionalRESUMO
Primary T helper 2 cells are heterogeneous, expressing subsets of cytokines at varying levels. Mechanisms controlling this spectrum of phenotypes are still unclear. The ETS family transcription factor PU.1 is expressed in Th2 but not Th1 cells. Th2 cytokine production is decreased in cultures transduced with a PU.1-expressing retrovirus and increased in Th2 cells following RNAi that decreases PU.1 expression. In primary cultures, PU.1 expression is restricted to a subpopulation of Th2 cells that express CCL22 and a subset of Th2 cytokines. PU.1 regulates the Th2 phenotype by interfering with GATA-3 DNA binding without altering GATA-3 protein levels. Thus, the expression of PU.1 in subsets of Th2 cells establishes a defined cytokine profile and contributes towards establishing the spectrum of cytokine production observed in Th2 populations.
Assuntos
Citocinas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Células Th2/imunologia , Transativadores/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Citometria de Fluxo , Fator de Transcrição GATA3 , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Proteínas Proto-Oncogênicas/imunologia , Interferência de RNA , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Transativadores/imunologiaRESUMO
Phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)- and growth factor-inducible, calcium-binding protein that either inserts into the plasma membrane or binds DNA in the nucleus depending on its state of palmyitoylation. In certain hematopoietic cells, PLSCR1 is required for normal maturation and terminal differentiation from progenitor cells as regulated by select growth factors, where it promotes recruitment and activation of Src kinases. PLSCR1 is a substrate of Src (and Abl) kinases, and transcription of the PLSCR1 gene is regulated by the same growth factor receptor pathways in which PLSCR1 potentiates afferent signaling. The marked transcriptional upregulation of PLSCR1 by IFNs led us to explore whether PLSCR1 plays an analogous role in cellular responses to IFN, with specific focus on antiviral activities. Accordingly, human cells in which PLSCR1 expression was decreased with short interfering RNA were rendered relatively insensitive to the antiviral activity of IFNs, resulting in higher titers of vesicular stomatitis virus (VSV) and encephalomyocarditis virus. Similarly, VSV replicated to higher titers in mouse PLSCR1(-/-) embryonic fibroblasts than in identical cells transduced to express PLSCR1. PLSCR1 inhibited accumulation of primary VSV transcripts, similar to the effects of IFN against VSV. The antiviral effect of PLSCR1 correlated with increased expression of a subset of IFN-stimulated genes (ISGs), including ISG15, ISG54, p56, and guanylate binding proteins. Our results suggest that PLSCR1, which is itself an ISG-encoded protein, provides a mechanism for amplifying and enhancing the IFN response through increased expression of a select subset of potent antiviral genes.
Assuntos
Antivirais/farmacologia , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos , Proteínas/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Vírus da Encefalomiocardite/fisiologia , Fibroblastos/virologia , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Fator de Transcrição STAT1 , Transativadores/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Replicação Viral/fisiologia , eIF-2 Quinase/metabolismoRESUMO
Viral infection is the primary cause of respiratory morbidity in cystic fibrosis (CF) infants. Here, we identify that host factors allow increased virus replication and cytokine production, providing a mechanism for understanding the severity of virus disease in CF. Increased virus is due to lack of nitric oxide synthase 2 (NOS2) and 2', 5' oligoadenylate synthetase (OAS) 1 induction in response to virus or IFNgamma. This can be attributed to impairment of activation of signal transducer and activator of transcription (STAT)1, a fundamental component to antiviral defense. NO donor or NOS2 overexpression provides protection from virus infection in CF, suggesting that NO is sufficient for antiviral host defense in the human airway and is one strategy for antiviral therapy in CF children.