RESUMO
Astroblastomas (ABs) are rare brain tumors of unknown origin. We performed an integrative genetic and epigenetic analysis of AB-like tumors. Here, we show that tumors traceable to neural stem/progenitor cells (radial glia) that emerge during early to later brain development occur in children and young adults, respectively. Tumors with MN1-BEND2 fusion appear to present exclusively in females and exhibit overexpression of genes expressed prior to 25 post-conception weeks (pcw), including genes enriched in early ventricular zone radial glia and ependymal tumors. Other, histologically classic ABs overexpress or harbor mutations of mitogen-activated protein kinase pathway genes, outer and truncated radial glia genes, and genes expressed after 25 pcw, including neuronal and astrocyte markers. Findings support that AB-like tumors arise in the context of epigenetic and genetic changes in neural progenitors. Selective gene fusion, variable imprinting and/or chromosome X-inactivation escape resulting in biallelic overexpression may contribute to female predominance of AB molecular subtypes.
Assuntos
Neoplasias Neuroepiteliomatosas , Células-Tronco Neurais , Linhagem da Célula/genética , Criança , Células Ependimogliais , Feminino , Humanos , Masculino , Neuroglia , Inativação do Cromossomo X/genética , Adulto JovemRESUMO
Background: Multiplex tissue analysis has revolutionized our understanding of the tumor microenvironment (TME) with implications for biomarker development and diagnostic testing. Multiplex labeling is used for specific clinical situations, but there remain barriers to expanded use in anatomic pathology practice. Methods: We review immunohistochemistry (IHC) and related assays used to localize molecules in tissues, with reference to United States regulatory and practice landscapes. We review multiplex methods and strategies used in clinical diagnosis and in research, particularly in immuno-oncology. Within the framework of assay design and testing phases, we examine the suitability of multiplex immunofluorescence (mIF) for clinical diagnostic workflows, considering its advantages and challenges to implementation. Results: Multiplex labeling is poised to radically transform pathologic diagnosis because it can answer questions about tissue-level biology and single-cell phenotypes that cannot be addressed with traditional IHC biomarker panels. Widespread implementation will require improved detection chemistry, illustrated by InSituPlex technology (Ultivue, Inc., Cambridge, MA) that allows coregistration of hematoxylin and eosin (H&E) and mIF images, greater standardization and interoperability of workflow and data pipelines to facilitate consistent interpretation by pathologists, and integration of multichannel images into digital pathology whole slide imaging (WSI) systems, including interpretation aided by artificial intelligence (AI). Adoption will also be facilitated by evidence that justifies incorporation into clinical practice, an ability to navigate regulatory pathways, and adequate health care budgets and reimbursement. We expand the brightfield WSI system "pixel pathway" concept to multiplex workflows, suggesting that adoption might be accelerated by data standardization centered on cell phenotypes defined by coexpression of multiple molecules. Conclusion: Multiplex labeling has the potential to complement next generation sequencing in cancer diagnosis by allowing pathologists to visualize and understand every cell in a tissue biopsy slide. Until mIF reagents, digital pathology systems including fluorescence scanners, and data pipelines are standardized, we propose that diagnostic labs will play a crucial role in driving adoption of multiplex tissue diagnostics by using retrospective data from tissue collections as a foundation for laboratory-developed test (LDT) implementation and use in prospective trials as companion diagnostics (CDx).
RESUMO
We analyzed micro-RNAs (miRs) as possible diagnostic biomarkers for relevant comorbidities prior to and prognostic biomarkers for mortality following hematopoietic cell transplantation (HCT). A randomly selected group of patients (n = 36) were divided into low-risk (HCT-comorbidity index [HCT-CI] score of 0 and survived HCT) and high-risk (HCT-CI scores ≥ 4 and deceased after HCT) groups. There were 654 miRs tested and 19 met the pre-specified significance level of p < 0.1. In subsequent models, only eight miRs maintained statistical significance in regression models after adjusting for baseline demographic factors; miRs-374b and -454 were underexpressed, whereas miRs-142-3p, -191, -424, -590-3p, -29c, and -15b were overexpressed among high-risk patients relative to low-risk patients. Areas under the curve for these eight miRs ranged between 0.74 and 0.81, suggesting strong predictive capacity. Consideration of miRs may improve risk assessment of mortality and should be further explored in larger future prospective studies.
Assuntos
Regulação Leucêmica da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , MicroRNAs/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras , Período Pré-Operatório , RNA Neoplásico/biossíntese , Adulto , Idoso , Aloenxertos , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Valor Preditivo dos Testes , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an "ultra-high risk" microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson's correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event-free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.
Assuntos
Neuroblastoma/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise por Conglomerados , Estudos de Coortes , Perfilação da Expressão Gênica/instrumentação , Humanos , Lactente , Neuroblastoma/diagnóstico , PrognósticoRESUMO
BACKGROUND: Acute pyelonephritis (APN) versus acute rejection (AR) is a frequently encountered diagnostic and therapeutic dilemma in kidney transplants. Variable culture results, overlapping histologic features, and persistent graft dysfunction despite antibiotics are frequently encountered. Therefore, we explored the utility of intragraft microRNA profiles to distinguish between allograft APN and AR. MATERIALS AND METHODS: Between 2003 and 2011, we identified 49 patients with biopsy features of APN, within the first 2 years posttransplant. MicroRNA profiling was performed on 20 biopsies (normal kidney, n=4; unequivocal AR, n=5; features of APN, n=11). RESULTS: Only 32% (16/49) of the patients had concomitant positive urine cultures at biopsy, and in 8 of 16 patients, colony count was less than 10 CFU/mL. In 14 of 49 patients, positive urine culture did not coincide with the biopsy, and in 19 of 49 patients, urine cultures were negative. On microRNA profiling, good clustering was seen among the normal kidneys and among AR biopsies. Among the 11 biopsies with features of APN, 4 biopsies showed good clustering with a pattern distinct from AR; (these patients recovered graft function with antibiotics); 7 of 11 biopsies showed heterogeneity in microRNA profiles and variable outcomes with antibiotic treatment. We identified a panel of 25 microRNAs showing statistical difference in expression between AR and APN. MiR-99b, miR-23b let-7b-5p, miR-30a, and miR-145 were validated using qPCR. CONCLUSION: Allograft pyelonephritis can be a diagnostic and therapeutic challenge. A gestalt approach is required. In addition to histology and cultures, differential intragraft microRNA expression may prove helpful to distinguish APN from AR in renal allograft biopsies.
Assuntos
Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/epidemiologia , Transplante de Rim , MicroRNAs , Pielonefrite/diagnóstico , Pielonefrite/epidemiologia , Doença Aguda , Adulto , Idoso , Aloenxertos , Antibacterianos/uso terapêutico , Biópsia , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Rim/metabolismo , Rim/patologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Pielonefrite/tratamento farmacológico , Estudos RetrospectivosRESUMO
Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual's response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation.
Assuntos
Bioensaio/métodos , MicroRNAs/sangue , Radiometria/métodos , Soro/metabolismo , Animais , Biomarcadores/sangue , Exposição Ambiental/efeitos adversos , Exposição Ambiental/análise , Humanos , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Doses de Radiação , Radiação Ionizante , Liberação Nociva de RadioativosRESUMO
Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia , Autofagia , Proteína Beclina-1 , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/fisiologia , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timócitos/metabolismo , Timócitos/ultraestrutura , Proteínas Supressoras de Tumor/genéticaRESUMO
Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenilato Quinase/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos , Estresse Oxidativo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Decitabine (also referred to as 5-aza-2'-deoxycytidine) is a drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome (MDS). The mechanism of action is believed to be the blocking of DNA methylation and thereby reactivating silenced genes involved in harnessing MDS. When analyzing reactivation of genes involved in Burkitt lymphoma (BL), we discovered that decitabine also sensitizes tumor cells by inducing DNA damage. This sensitization is grossly augmented by the MYC oncogene, which is overexpressed in BL, and occurs in cells lacking a functional p53 tumor suppressor pathway. In p53-deficient BL cells and p53(-/-) mouse embryo fibroblasts, Myc overrides a transient G2-block exerted by decitabine via activation of Chk1. This triggers aneuploidy and cell death that correlates with, but can occur in the absence of, Epstein-Barr virus (EBV) reactivation, caspase activation, and/or expression of the BH3-only protein Puma. In vivo modeling of Myc-induced lymphoma suggests that decitabine constitutes a potential new drug against lymphoma that would selectively sensitize tumor cells but spare normal tissue.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Linfoma de Burkitt/patologia , Dano ao DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Aneuploidia , Animais , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Western Blotting , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/virologia , Ciclo Celular , Metilação de DNA , DNA de Neoplasias/efeitos dos fármacos , Decitabina , Embrião de Mamíferos , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Humanos , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação/genéticaRESUMO
The histone deacetylase inhibitor SAHA enhances cell death stimulated by the proteasome inhibitor bortezomib (BZ) by disrupting BZ-induced aggresome formation. Here we report that Myc regulates the sensitivity of multiple myeloma (MM) cells to BZ + SAHA-induced cell death. In MM cells, Myc expression directly correlated with intracellular ER content, protein synthesis rates, the percentage of aggresome-positive cells, and the sensitivity to BZ + SAHA-induced cell death. Accordingly, Myc knockdown markedly reduced the sensitivity of MM cells to BZ + SAHA-mediated apoptosis. Furthermore, activation of Myc was sufficient to provoke aggresome formation and thus sensitivity to BZ + SAHA, and these responses required de novo protein synthesis. BZ + SAHA-mediated stimulation of apoptosis includes the induction of the proapoptotic BH3-only protein Noxa as well as endoplasmic reticular stress, a disruption of calcium homeostasis, and activation of capase-4. Finally, knockdown studies demonstrated that both caspase-4 and Noxa play significant roles in Myc-driven sensitivity to BZ + SAHA-induced apoptosis. Collectively, our results establish a mechanistic link between Myc activity, regulation of protein synthesis, increases in HDAC6 expression and aggresome formation, induction of Noxa, and sensitivity to BZ + SAHA-induced apoptosis. These data suggest that MM patients with elevated Myc activity may be particularly sensitive to the BZ + SAHA combination.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Estruturas do Núcleo Celular/efeitos dos fármacos , Estruturas do Núcleo Celular/metabolismo , Ácidos Hidroxâmicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazinas/farmacologia , Bortezomib , Linhagem Celular Tumoral , Estruturas do Núcleo Celular/ultraestrutura , Cicloeximida/farmacologia , Diploide , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Mieloma Múltiplo/patologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , VorinostatRESUMO
Despite great interest in cancer chemoprevention, effective agents are few. Here we show that chloroquine, a drug that activates the stress-responsive Atm-p53 tumor-suppressor pathway, preferentially enhances the death of Myc oncogene-overexpressing primary mouse B cells and mouse embryonic fibroblasts (MEFs) and impairs Myc-induced lymphomagenesis in a transgenic mouse model of human Burkitt lymphoma. Chloroquine-induced cell death in primary MEFs and human colorectal cancer cells was dependent upon p53, but not upon the p53 modulators Atm or Arf. Accordingly, chloroquine impaired spontaneous lymphoma development in Atm-deficient mice, a mouse model of ataxia telangiectasia, but not in p53-deficient mice. Chloroquine treatment enhanced markers of both macroautophagy and apoptosis in MEFs but ultimately impaired lysosomal protein degradation. Interestingly, chloroquine-induced cell death was not dependent on caspase-mediated apoptosis, as neither overexpression of the antiapoptotic protein Bcl-2 nor deletion of the proapoptotic Bax and Bak affected chloroquine-induced MEF death. However, when both apoptotic and autophagic pathways were blocked simultaneously, chloroquine-induced killing of Myc-overexpressing cells was blunted. Thus chloroquine induces lysosomal stress and provokes a p53-dependent cell death that does not require caspase-mediated apoptosis. These findings specifically demonstrate that intermittent chloroquine use effectively prevents cancer in mouse models of 2 genetically distinct human cancer syndromes, Burkitt lymphoma and ataxia telangiectasia, suggesting that agents targeting lysosome-mediated degradation may be effective in cancer prevention.
Assuntos
Antirreumáticos/farmacologia , Apoptose/efeitos dos fármacos , Ataxia Telangiectasia/prevenção & controle , Autofagia/efeitos dos fármacos , Linfoma de Burkitt/prevenção & controle , Transformação Celular Neoplásica/metabolismo , Cloroquina/farmacologia , Lisossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antirreumáticos/uso terapêutico , Apoptose/genética , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Autofagia/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Caspases/genética , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Cloroquina/uso terapêutico , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lisossomos/patologia , Masculino , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The use of agents to prevent the onset of and/or the progression to breast cancer has the potential to lower breast cancer risk. We have previously shown that the tumor-suppressor gene p53 is a potential mediator of hormone (estrogen/progesterone)-induced protection against chemical carcinogen-induced mammary carcinogenesis in animal models. Here, we show for the first time a breast cancer-protective effect of chloroquine in an animal model. Chloroquine significantly reduced the incidence of N-methyl-N-nitrosourea-induced mammary tumors in our animal model similar to estrogen/progesterone treatment. No protection was seen in our BALB/c p53-null mammary epithelium model, indicating a p53 dependency for the chloroquine effect. Using a human nontumorigenic mammary gland epithelial cell line, MCF10A, we confirm that in the absence of detectable DNA damage, chloroquine activates the tumor-suppressor p53 and the p53 downstream target gene p21, resulting in G(1) cell cycle arrest. p53 activation occurs at a posttranslational level via chloroquine-dependent phosphorylation of the checkpoint protein kinase, ataxia telangiectasia-mutated (ATM), leading to ATM-dependent phosphorylation of p53. In primary mammary gland epithelial cells isolated from p53-null mice, chloroquine does not induce G(1) cell cycle arrest compared with cells isolated from wild-type mice, also indicating a p53 dependency. Our results indicate that a short prior exposure to chloroquine may have a preventative application for mammary carcinogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Cloroquina/farmacologia , Proteínas de Ligação a DNA/genética , Genes p53 , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/prevenção & controle , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Mama/citologia , Mama/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Myc oncoproteins are commonly activated in malignancies and are sufficient to provoke many types of cancer. However, the critical mechanisms by which Myc contributes to malignant transformation are not clear. DNA damage seems to be an important initiating event in tumorigenesis. Here, we show that although Myc does not directly induce double-stranded DNA breaks, it does augment activation of the Atm/p53 DNA damage response pathway, suggesting that Atm may function as a guardian against Myc-induced transformation. Indeed, we show that Atm loss augments Myc-induced lymphomagenesis and impairs Myc-induced apoptosis, which normally harnesses Myc-driven tumorigenesis. Surprisingly, Atm loss also augments the proliferative response induced by Myc, and this augmentation is associated with enhanced suppression of the expression of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, regulation of cell proliferation and p27(Kip1) seems to be a contributing mechanism by which Atm holds tumor formation in check.
Assuntos
Apoptose , Proteínas de Ligação a DNA/deficiência , Linfoma/metabolismo , Linfoma/patologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Supressoras de Tumor/deficiência , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Proliferação de Células/efeitos da radiação , Dano ao DNA , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Camundongos , Fosforilação/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismoRESUMO
Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.
Assuntos
Linfoma de Burkitt/etiologia , Proteína Quinase CDC2/genética , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/antagonistas & inibidores , Linfoma de Células B/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Células da Medula Óssea/citologia , Proteína Quinase CDC2/metabolismo , Cruzamentos Genéticos , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Retroviridae/genética , Células Tumorais CultivadasRESUMO
Metabolic syndrome is associated with insulin resistance and atherosclerosis. Here, we show that deficiency of one or two alleles of ATM, the protein mutated in the cancer-prone disease ataxia telangiectasia, worsens features of the metabolic syndrome, increases insulin resistance, and accelerates atherosclerosis in apoE-/- mice. Transplantation with ATM-/- as compared to ATM+/+ bone marrow increased vascular disease. Jun N-terminal kinase (JNK) activity was increased in ATM-deficient cells. Treatment of ATM+/+apoE-/- mice with low-dose chloroquine, an ATM activator, decreased atherosclerosis. In an ATM-dependent manner, chloroquine decreased macrophage JNK activity, decreased macrophage lipoprotein lipase activity (a proatherogenic consequence of JNK activation), decreased blood pressure, and improved glucose tolerance. Chloroquine also improved metabolic abnormalities in ob/ob and db/db mice. These results suggest that ATM-dependent stress pathways mediate susceptibility to the metabolic syndrome and that chloroquine or related agents promoting ATM activity could modulate insulin resistance and decrease vascular disease.
Assuntos
Cloroquina/uso terapêutico , Proteínas de Ligação a DNA/deficiência , Doenças Metabólicas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/deficiência , Transdução de Sinais , Estresse Fisiológico/metabolismo , Proteínas Supressoras de Tumor/deficiência , Animais , Apolipoproteínas E/genética , Proteínas Mutadas de Ataxia Telangiectasia , Aterosclerose/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Macrófagos/efeitos dos fármacos , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Knockout , Mutação , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Apoptosis of murine and human macrophages induced by group B Streptococcus agalactiae (GBS) is likely an important virulence mechanism that is used by the bacteria to suppress the host immune response and to persist at sites of infection. The mechanisms by which GBS induces apoptosis are, however, largely unknown. In this study, we report that in murine macrophages GBS induces unique changes in the regulation and localization of the apoptotic regulators Bad, 14-3-3, and Omi/high-temperature requirement A2 and leads to the release of cytochrome c and the activation of caspase-9 and caspase-3. Furthermore, inhibition of caspase-3 impaired GBS-induced apoptosis of macrophages. The ability to modulate the activity of effector caspases may therefore represent an unexploited avenue for therapeutic intervention in GBS infections.
Assuntos
Apoptose/imunologia , Macrófagos/citologia , Macrófagos/microbiologia , Streptococcus agalactiae/imunologia , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/fisiologia , Animais , Caspase 3 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Ativação Enzimática , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Macrófagos/enzimologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Proteínas Mitocondriais , Serina Endopeptidases/metabolismo , Serina Endopeptidases/fisiologia , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína de Morte Celular Associada a bcl/fisiologiaRESUMO
Rel/NF-kappaB transcription factors are critical arbiters of immune responses, cell survival, and transformation, and are frequently deregulated in cancer. The p50 NF-kappaB 1 component of Rel/NF-kappaB DNA-binding dimers regulates genes involved in both cell cycle traverse and apoptosis. Nfkb 1 loss accelerates B cell growth and leads to increased B cell turnover in vivo, phenotypes akin to those manifested in B cells of Emu-Myc transgenic mice, a model of human Burkitt lymphoma. Interestingly, Emu-Myc B cells express reduced levels of cytoplasmic and nuclear NF-kappaB 1 and have reduced Rel/NF-kappaB DNA-binding activity, suggesting that Myc-mediated repression of NF-kappaB 1 might mediate its proliferative and apoptotic effects on B cells. Furthermore, Nfkb 1 expression was reduced in the majority of Emu-Myc lymphomas and was also suppressed in human Burkitt lymphoma. Nonetheless, loss of Nfkb 1 did not appreciably affect Myc's proliferative or apoptotic responses in B cells and had no effect on lymphoma development in Emu-Myc mice. Therefore, Nfkb 1 is dispensable for Myc-induced lymphomagenesis..
Assuntos
Subunidade p50 de NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Sequência de Bases , Linfoma de Burkitt/genética , DNA/metabolismo , Primers do DNA , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subunidade p50 de NF-kappa B/genética , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Myc oncoproteins are overexpressed in most cancers and are sufficient to accelerate cell proliferation and provoke transformation. However, in normal cells Myc also triggers apoptosis. All of the effects of Myc require its function as a transcription factor that dimerizes with Max. This complex induces genes containing CACGTG E-boxes, such as Ornithine decarboxylase (Odc), which harbors two of these elements. Here we report that in quiescent cells the Odc E-boxes are occupied by Max and Mnt, a putative Myc antagonist, and that this complex is displaced by Myc-Max complexes in proliferating cells. Knockdown of Mnt expression by stable retroviral RNA interference triggers many targets typical of the "Myc" response and provokes accelerated proliferation and apoptosis. Strikingly, these effects of Mnt knockdown are even manifest in cells lacking c-myc. Moreover, Mnt knockdown is sufficient to transform primary fibroblasts in conjunction with Ras. Therefore, Mnt behaves as a tumor suppressor. These findings support a model where Mnt represses Myc target genes and Myc functions as an oncogene by relieving Mnt-mediated repression.
Assuntos
Apoptose , Transformação Celular Neoplásica , Proteínas Nucleares/deficiência , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras , Transcrição Gênica/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Divisão Celular , Células Cultivadas , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Genes Supressores , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ornitina Descarboxilase/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Alterations in MYC and p53 are hallmarks of cancer. p53 coordinates the response to gamma irradiation (gamma-IR) by either triggering apoptosis or cell cycle arrest. c-Myc activates the p53 apoptotic checkpoint, and thus tumors overexpressing MYC often harbor p53 mutations. Nonetheless, many of these cancers are responsive to therapy, suggesting that Myc may sensitize cells to gamma-IR independent of p53. In mouse embryo fibroblasts (MEFs) and in E micro -myc transgenic B cells in vivo, c-Myc acts in synergy with gamma-IR to trigger apoptosis, but alone, when cultured in growth medium, it does not induce a DNA damage response. Surprisingly, c-Myc also sensitizes p53-deficient MEFs to gamma-IR-induced apoptosis. In normal cells, and in precancerous B cells of E micro -myc transgenic mice, this apoptotic response is associated with the suppression of the antiapoptotic regulators Bcl-2 and Bcl-X(L) and with the concomitant induction of Puma, a proapoptotic BH3-only protein. However, in p53-null MEFs only Bcl-X(L) expression was suppressed, suggesting levels of Bcl-X(L) regulate the response to gamma-IR. Indeed, Bcl-X(L) overexpression blocked this apoptotic response, whereas bcl-X-deficient MEFs were inherently and selectively sensitive to gamma-IR-induced apoptosis. Therefore, MYC may sensitize tumor cells to DNA damage by suppressing Bcl-X.