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1.
Clin Cancer Res ; 26(21): 5609-5620, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32847933

RESUMO

PURPOSE: Tumor-associated macrophages correlate with increased invasiveness, growth, and immunosuppression. Activation of the colony-stimulating factor-1 receptor (CSF-1R) results in proliferation, differentiation, and migration of monocytes/macrophages. This phase I study evaluated the immunologic and clinical activity, and safety profile of CSF-1R inhibition with the mAb LY3022855. PATIENTS AND METHODS: Patients with advanced refractory metastatic breast cancer (MBC) or metastatic castration-resistant prostate cancer (mCRPC) were treated with LY3022855 intravenously in 6-week cycles in cohorts: (A) 1.25 mg/kg every 2 weeks (Q2W); (B) 1.0 mg/kg on weeks 1, 2, 4, and 5; (C) 100 mg once weekly; (D)100 mg Q2W. mCRPC patients were enrolled in cohorts A and B; patients with MBC were enrolled in all cohorts. Efficacy was assessed by RECIST and Prostate Cancer Clinical Trials Working Group 2 criteria. RESULTS: Thirty-four patients (22 MBC; 12 mCRPC) received ≥1 dose of LY3022855. At day 8, circulating CSF-1 levels increased and proinflammatory monocytes CD14DIMCD16BRIGHT decreased. Best RECIST response was stable disease in five patients with MBC (23%; duration, 82-302 days) and three patients with mCRPC (25%; duration, 50-124 days). Two patients with MBC (cohort A) had durable stable disease >9 months and a third patient with MBC had palpable reduction in a nontarget neck mass. Immune-related gene activation in tumor biopsies posttreatment was observed. Common any grade treatment-related adverse events were fatigue, decreased appetite, nausea, asymptomatic increased lipase, and creatine phosphokinase. CONCLUSIONS: LY3022855 was well tolerated and showed evidence of immune modulation. Clinically meaningful stable disease >9 months was observed in two patients with MBC.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/efeitos adversos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/efeitos adversos , Receptores de Lipopolissacarídeos/genética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptores de IgG/genética
2.
Oncogene ; 39(5): 987-1003, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31591478

RESUMO

Despite intense research and clinical efforts, patients affected by advanced colorectal cancer (CRC) have still a poor prognosis. The discovery of colorectal (CR) cancer stem cell (CSC) as the cell compartment responsible for tumor initiation and propagation may provide new opportunities for the development of new therapeutic strategies. Given the reduced sensitivity of CR-CSCs to chemotherapy and the ability of bone morphogenetic proteins (BMP) to promote colonic stem cell differentiation, we aimed to investigate whether an enhanced variant of BMP7 (BMP7v) could sensitize to chemotherapy-resistant CRC cells and tumors. Thirty-five primary human cultures enriched in CR-CSCs, including four from chemoresistant metastatic lesions, were used for in vitro studies and to generate CR-CSC-based mouse avatars to evaluate tumor growth and progression upon treatment with BMP7v alone or in combination with standard therapy or PI3K inhibitors. BMP7v treatment promotes CR-CSC differentiation and recapitulates the cell differentiation-related gene expression profile by suppressing Wnt pathway activity and reducing mesenchymal traits and survival of CR-CSCs. Moreover, in CR-CSC-based mouse avatars, BMP7v exerts an antiangiogenic effect and sensitizes tumor cells to standard chemotherapy regardless of the mutational, MSI, and CMS profiles. Of note, tumor harboring PIK3CA mutations were affected to a lower extent by the combination of BMP7v and chemotherapy. However, the addition of a PI3K inhibitor to the BMP7v-based combination potentiates PIK3CA-mutant tumor drug response and reduces the metastatic lesion size. These data suggest that BMP7v treatment may represent a useful antiangiogenic and prodifferentiation agent, which renders CSCs sensitive to both standard and targeted therapies.


Assuntos
Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/farmacologia , Neoplasias Colorretais/patologia , Mutação , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS One ; 10(4): e0125697, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25919028

RESUMO

Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.


Assuntos
Proteína Morfogenética Óssea 7/uso terapêutico , Células Endoteliais/metabolismo , Glioblastoma/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Tecido Adiposo/citologia , Animais , Proteína Morfogenética Óssea 7/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Laminina/farmacologia , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteoglicanas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Forensic Sci ; 60(1): 157-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25284026

RESUMO

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.


Assuntos
Impressões Digitais de DNA/instrumentação , DNA/análise , RNA Mensageiro/análise , Manejo de Espécimes/instrumentação , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Saliva/química
5.
PLoS One ; 9(4): e96036, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24759702

RESUMO

Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was significantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.


Assuntos
Adipócitos/citologia , Técnicas de Cocultura/métodos , Neovascularização Patológica/metabolismo , RNA Interferente Pequeno/farmacologia , Fator de Transcrição Sp1/genética , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura/economia , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Indóis/administração & dosagem , Lentivirus/genética , Camundongos Nus , Transplante de Neoplasias , Pirróis/administração & dosagem , RNA Interferente Pequeno/genética , Células-Tronco/metabolismo , Sunitinibe
6.
J Biol Chem ; 288(9): 6743-53, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23335506

RESUMO

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38ß mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.


Assuntos
Endotélio Vascular/enzimologia , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neoplasias/enzimologia , Neovascularização Patológica/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Citocinas/metabolismo , Endotélio Vascular/patologia , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
J Forensic Sci ; 57(4): 1051-8, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22309221

RESUMO

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.


Assuntos
Perfilação da Expressão Gênica , RNA Mensageiro/metabolismo , Actinas/genética , Adulto , Análise de Variância , Análise Química do Sangue , Muco do Colo Uterino/química , Ciclofilina A/genética , Feminino , Genética Forense , Expressão Gênica , Gliceraldeído 3-Fosfato/genética , Humanos , Masculino , Menstruação , Pessoa de Meia-Idade , Fosfoglicerato Quinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Saliva/química , Sêmen/química , Microglobulina beta-2/genética
8.
Forensic Sci Int Genet ; 6(2): 185-90, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21570374

RESUMO

Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework.


Assuntos
Impressões Digitais de DNA/métodos , Genoma Humano , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA/análise , Degradação Necrótica do DNA , DNA Ligases , Genótipo , Humanos , Repetições de Microssatélites , Moldes Genéticos
9.
FEBS J ; 277(1): 210-23, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19951360

RESUMO

CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a component of the euchromatic Setd1A histone H3K4 methyltransferase complex, and is a critical regulator of histone methylation, cytosine methylation, cellular differentiation, and vertebrate development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but show increased levels of global histone H3K4 methylation, suggesting that Cfp1 functions to inhibit or restrict the activity of the Setd1A histone H3K4 methyltransferase complex. The studies reported here reveal that ES cells lacking Cfp1 contain decreased levels of Setd1A and show subnuclear mislocalization of both Setd1A and trimethylation of histone H3K4 with regions of heterochromatin. Remarkably, structure-function studies reveal that expression of either the N-terminal fragment of Cfp1 (amino acids 1-367) or the C-terminal fragment of Cfp1 (amino acids 361-656) is sufficient to restore appropriate levels of Setd1A in CXXC1(-/-) ES cells. Furthermore, functional analysis of various Cfp1 point mutations reveals that retention of either Cfp1 DNA-binding activity or association with the Setd1 histone H3K4 methyltransferase complex is required to restore normal Setd1A levels. In contrast, expression of full-length Cfp1 in CXXC1(-/-) ES cells is required to restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicating that both Cfp1 DNA-binding activity and interaction with the Setd1A complex are required for appropriate genomic targeting of the Setd1A complex. These studies illustrate the complexity of Cfp1 function, and identify Cfp1 as a regulator of Setd1A genomic targeting.


Assuntos
Eucromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Histona-Lisina N-Metiltransferase/química , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transativadores/química , Transativadores/deficiência , Transativadores/genética , Transfecção
10.
DNA Repair (Amst) ; 8(12): 1411-23, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19836314

RESUMO

Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1(-/-) ES cells accumulate more DNA damage and exhibit decreased protein expression and endonuclease activity of AP endonuclease (Ape1/Ref-1), an enzyme involved in DNA base excision repair. Expression in CXXC1(-/-) ES cells of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) restores normal Ape1/Ref-1 protein expression and rescues the hypersensitivity to DNA-damaging agents, demonstrating that Cfp1 contains redundant functional domains. Furthermore, retention of either the DNA-binding activity of Cfp1 or interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes is required to restore normal sensitivity of CXXC1(-/-) ES cells to DNA-damaging agents. These results implicate Cfp1 as a regulator of DNA repair processes.


Assuntos
Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonuclease I/metabolismo , Células-Tronco Embrionárias/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Cisplatino/farmacologia , Metilação de DNA , Camundongos , Ligação Proteica/efeitos dos fármacos , Transativadores/deficiência
11.
Mol Cell Biol ; 29(14): 3817-31, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19433449

RESUMO

CXXC finger protein 1 (Cfp1) is a regulator of both cytosine methylation and histone methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a decreased plating efficiency, decreased cytosine methylation, elevated global levels of histone H3-Lys4 trimethylation, and a failure to differentiate in vitro. Remarkably, transfection studies reveal that expression of either the amino half of Cfp1 (amino acids 1 to 367 [Cfp1(1-367)]) or the carboxyl half of Cfp1 (Cfp1(361-656)) is sufficient to correct all of the defects observed with ES cells that lack Cfp1. However, a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the Cfp1(1-367) fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1 histone H3-Lys4 methyltransferase complexes ablates the rescue activity of the Cfp1(361-656) fragment. Introduction of both the C169A and C375A point mutations ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either the Cfp1 DNA-binding domain or Setd1 interaction domain is required for Cfp1 rescue activity, and they illustrate the functional complexity of this critical epigenetic regulator. A model is presented for how epigenetic cross talk may explain the finding of redundant functional domains within Cfp1.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Citosina/metabolismo , Metilação de DNA , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transativadores/deficiência , Transativadores/genética , Transfecção
12.
DNA Cell Biol ; 28(5): 223-31, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19388845

RESUMO

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias/metabolismo , Biossíntese de Proteínas , Transativadores/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Regulação para Baixo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Meia-Vida , Humanos , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Knockout , Polirribossomos/metabolismo
13.
J Biol Chem ; 282(18): 13419-28, 2007 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-17355966

RESUMO

We previously identified a mammalian Set1A complex analogous to the yeast Set1/COMPASS histone H3-Lys4 methyltransferase complex (Lee, J.-H., and Skalnik, D. G. (2005) J. Biol. Chem. 280, 41725-41731). Data base analysis indicates that human Set1A protein shares 39% identity with an uncharacterized SET domain protein, KIAA1076, hereafter denoted Set1B. Immunoprecipitation and mass spectrometry reveal that Set1B associates with a approximately 450 kDa complex that contains all five non-catalytic components of the Set1A complex, including CFP1, Rbbp5, Ash2, Wdr5, and Wdr82. These data reveal two human protein complexes that differ only in the identity of the catalytic histone methyltransferase. In vitro assays demonstrate that the Set1B complex is a histone methyltransferase that produces trimethylated histone H3 at Lys(4). Both Set1A and Set1B are widely expressed. Inducible expression of the carboxyl terminus of either Set1A or Set1B decreases steady-state levels of both endogenous Set1A and Set1B protein, but does not alter the expression of the non-catalytic components of the Set1 complexes. A 123-amino acid fragment upstream of the Set1A SET domain is necessary for interaction with CFP1, Ash2, Rbbp5, and Wdr5. This protein domain is also required to mediate feedback inhibition of Set1A and Set1B expression, which is a consequence of reduced Set1A and Set1B stability when not associated with the methyltransferase complex. Confocal microscopy reveals that Set1A and Set1B each localize to a largely non-overlapping set of euchromatic nuclear speckles, suggesting that Set1A and Set1B each bind to a unique set of target genes and thus make non-redundant contributions to the epigenetic control of chromatin structure and gene expression.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Epigênese Genética/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Proteínas de Ligação a DNA , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Células K562 , Camundongos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína/fisiologia
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