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1.
Mol Imaging Biol ; 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37610610

RESUMO

PURPOSE: Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy. PROCEDURES: These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO2), extracellular acidity (pHe), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes. RESULTS: Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pHe × pO2)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3 mM indicated that the tumors were pre-malignant 74% of the time, and when > 3 mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pHe and pO2 and increase in GSH at later stages. CONCLUSION: Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pHe due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO2 was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.

2.
Anal Chem ; 95(2): 946-954, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36537829

RESUMO

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO2, pH, and [Pi] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Neoplasias , Oxigênio , Compostos de Tritil , Animais , Camundongos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Concentração de Íons de Hidrogênio , Hipóxia , Oxigênio/química , Microambiente Tumoral , Compostos de Tritil/química , Técnicas Biossensoriais
3.
Int J Mol Sci ; 23(23)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36499305

RESUMO

There are currently no effective biomarkers for prognosis and optimal treatment selection to improve non-small cell lung cancer (NSCLC) survival outcomes. This study further validated a seven-gene panel for diagnosis and prognosis of NSCLC using RNA sequencing and proteomic profiles of patient tumors. Within the seven-gene panel, ZNF71 expression combined with dendritic cell activities defined NSCLC patient subgroups (n = 966) with distinct survival outcomes (p = 0.04, Kaplan-Meier analysis). ZNF71 expression was significantly associated with the activities of natural killer cells (p = 0.014) and natural killer T cells (p = 0.003) in NSCLC patient tumors (n = 1016) using Chi-squared tests. Overexpression of ZNF71 resulted in decreased expression of multiple components of the intracellular intrinsic and innate immune systems, including dsRNA and dsDNA sensors. Multi-omics networks of ZNF71 and the intracellular intrinsic and innate immune systems were computed as relevant to NSCLC tumorigenesis, proliferation, and survival using patient clinical information and in-vitro CRISPR-Cas9/RNAi screening data. From these networks, pan-sensitive and pan-resistant genes to 21 NCCN-recommended drugs for treating NSCLC were selected. Based on the gene associations with patient survival and in-vitro CRISPR-Cas9, RNAi, and drug screening data, MEK1/2 inhibitors PD-198306 and U-0126, VEGFR inhibitor ZM-306416, and IGF-1R inhibitor PQ-401 were discovered as potential targeted therapy that may also induce an immune response for treating NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Multiômica , Proteômica , Carcinogênese , Proliferação de Células/genética , Biomarcadores Tumorais/genética
4.
Int J Mol Sci ; 22(7)2021 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-33916522

RESUMO

Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Docetaxel/farmacologia , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias/genética , Paclitaxel/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Taxa de Sobrevida
6.
J Immunol ; 205(8): 2301-2311, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32938724

RESUMO

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Macrófagos/imunologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/imunologia , Proteínas de Neoplasias/imunologia , Receptor TIE-2/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteínas de Neoplasias/genética , Oxigênio/imunologia , Receptor TIE-2/genética
7.
J Org Chem ; 85(16): 10388-10398, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32698583

RESUMO

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.


Assuntos
Oxigênio , Compostos de Tritil , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Interações Hidrofóbicas e Hidrofílicas , Camundongos
8.
Stem Cells Int ; 2016: 8270464, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26880992

RESUMO

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

9.
Cancer Treat Res ; 158: 25-40, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24222353

RESUMO

Deregulation of gene expression plays a pivotal role in tumorigenesis, so the ability to detect RNA alterations is of great value in cancer diagnosis and management. DNA microarrays have been used to measure changes in mRNA or microRNA level, but less often the change of RNA isoforms. Here we appraise the utilization of microarray in detecting alternatively processed RNAs, which have alternative splice forms, retained introns, or altered 3' untranslated regions. We cover the methodology and focus on cancer studies. Recent development in parallel or deep sequencing used in transcriptome analysis is also discussed.


Assuntos
Processamento Alternativo , Análise de Sequência com Séries de Oligonucleotídeos , Perfilação da Expressão Gênica , Humanos , Íntrons , Neoplasias/genética
10.
Eur J Haematol ; 90(6): 469-78, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23551534

RESUMO

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Etoposídeo/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Melfalan/farmacologia , Osteoblastos/metabolismo , Nicho de Células-Tronco/efeitos dos fármacos , Animais , Linhagem Celular , Quimiocina CXCL12/metabolismo , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Matriz Extracelular/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Osteoblastos/citologia , Osteocalcina/metabolismo , Osteopontina/metabolismo
11.
Cytokine ; 58(2): 245-52, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22356805

RESUMO

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.


Assuntos
Células da Medula Óssea/metabolismo , Interleucina-6/metabolismo , Melfalan/toxicidade , Osteoblastos/metabolismo , Células Estromais/metabolismo , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/genética , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real
12.
PLoS One ; 7(2): e30758, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22363485

RESUMO

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-ß1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-ß1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.


Assuntos
Antineoplásicos/efeitos adversos , Células da Medula Óssea/patologia , Osteoblastos/patologia , Animais , Antígenos CD34/metabolismo , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CXCL12/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Melfalan/efeitos adversos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta1/farmacologia
13.
J Biol Chem ; 286(18): 16039-51, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21398516

RESUMO

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.


Assuntos
Processamento Alternativo/fisiologia , Antígenos CD/biossíntese , Moléculas de Adesão Celular/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Éxons/fisiologia , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Íntrons/fisiologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
14.
BMC Mol Biol ; 11: 79, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-21050451

RESUMO

BACKGROUND: Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast). RESULTS: Using in vivo footprinting and chromatin immunoprecipitation experiments we show that the CEACAM1 proximal promoter in breast cells is bound in its active state by SP1, USF1/USF2, and IRF1/2. When down-regulated the CEACAM1 promoter remains accessible to USF2 and partially accessible to USF1. Interferon-γ up-regulates CEACAM1 mRNA by a mechanism involving further induction of IRF-1 and USF1 binding at the promoter. As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells. The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure. CONCLUSIONS: Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.


Assuntos
Antígenos CD/genética , Mama/citologia , Moléculas de Adesão Celular/genética , Células Epiteliais/metabolismo , Ativação Transcricional , Linhagem Celular , Cromatina/química , Regulação para Baixo , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 2 de Interferon/genética , Fator Regulador 2 de Interferon/metabolismo , Interferon gama/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismo
15.
Mol Cell Neurosci ; 27(1): 8-21, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15345239

RESUMO

The Sam68-like mammalian protein SLM-1 is a member of the STAR protein family and is related to SAM68 and SLM-2. Here, we demonstrate that rSLM-1 interacts with itself, scaffold-attachment factor B, YT521-B, SAM68, rSLM-2, SRp30c, and hnRNP G. rSLM-1 regulates splice site selection in vivo via a purine-rich enhancer. In contrast to the widely expressed SAM68 and rSLM-2 proteins, rSLM-1 is found primarily in brain and, to a much smaller degree, in testis. In the brain, rSLM-1 and rSLM-2 are predominantly expressed in different neurons. In the hippocampal formation, rSLM-1 is present only in the dentate gyrus, whereas rSLM-2 is found in the pyramidal cells of the CA1, CA3, and CA4 regions. rSLM-1, but not rSLM-2, is phosphorylated by p59(fyn). p59(fyn)-mediated phosphorylation abolishes the ability of rSLM-1 to regulate splice site selection, but has no effect on rSLM-2 activity. This suggests that rSLM-1-positive cells could respond with a change of their splicing pattern to p59(fyn) activation, whereas rSLM-2-positive cells would not be affected. Together, our data indicate that rSLM-1 is a tissue-specific splicing factor whose activity is regulated by tyrosine phosphorylation signals emanating from p59(fyn).


Assuntos
Encéfalo/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Quinases da Família src/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , DNA Complementar/análise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Hipocampo/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-fyn , Células Piramidais/metabolismo , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Ratos , Receptores de Estrogênio/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Processamento de Serina-Arginina , Quinases da Família src/genética , Quinases da Família src/isolamento & purificação
16.
DNA Cell Biol ; 21(11): 803-18, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12489991

RESUMO

Humans possess a surprisingly low number of genes and intensively use pre-mRNA splicing to achieve the high molecular complexity needed to sustain normal body functions and facilitate responses to altered conditions. Because hundreds of thousands of proteins are generated by 25,000 to 40,000 genes, pre-mRNA processing events are highly important for the regulation of human gene expression. Both inherited and acquired defects in pre-mRNA processing are increasingly recognized as causes of human diseases, and almost all pre-mRNA processing events are controlled by a combination of protein factors. This makes defects in these processes likely candidates for causes of diseases with complicated inheritance patterns that affect seemingly unrelated functions. The elucidation of genetic mechanisms regulating pre-mRNA processing, combined with the development of drugs targeted at consensus RNA sequences and/or corresponding proteins, can lead to novel diagnostic and therapeutic approaches.


Assuntos
Predisposição Genética para Doença , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Transformação Celular Neoplásica/genética , Códon sem Sentido , Predisposição Genética para Doença/etiologia , Predisposição Genética para Doença/prevenção & controle , Humanos , Oligonucleotídeos Antissenso/uso terapêutico , Interferência de RNA , RNA Catalítico/uso terapêutico , Transativadores/uso terapêutico
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