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1.
Microbiome ; 11(1): 208, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37735685

RESUMO

BACKGROUND: The gut microbiota contributes to macrophage-mediated inflammation in adipose tissue with consumption of an obesogenic diet, thus driving the development of metabolic syndrome. There is a need to identify and develop interventions that abrogate this condition. The hops-derived prenylated flavonoid xanthohumol (XN) and its semi-synthetic derivative tetrahydroxanthohumol (TXN) attenuate high-fat diet-induced obesity, hepatosteatosis, and metabolic syndrome in C57Bl/6J mice. This coincides with a decrease in pro-inflammatory gene expression in the gut and adipose tissue, together with alterations in the gut microbiota and bile acid composition. RESULTS: In this study, we integrated and interrogated multi-omics data from different organs with fecal 16S rRNA sequences and systemic metabolic phenotypic data using a Transkingdom Network Analysis. By incorporating cell type information from single-cell RNA-seq data, we discovered TXN attenuates macrophage inflammatory processes in adipose tissue. TXN treatment also reduced levels of inflammation-inducing microbes, such as Oscillibacter valericigenes, that lead to adverse metabolic phenotypes. Furthermore, in vitro validation in macrophage cell lines and in vivo mouse supplementation showed addition of O. valericigenes supernatant induced the expression of metabolic macrophage signature genes that are downregulated by TXN in vivo. CONCLUSIONS: Our findings establish an important mechanism by which TXN mitigates adverse phenotypic outcomes of diet-induced obesity and metabolic syndrome. TXN primarily reduces the abundance of pro-inflammatory gut microbes that can otherwise promote macrophage-associated inflammation in white adipose tissue. Video Abstract.


Assuntos
Microbioma Gastrointestinal , Síndrome Metabólica , Animais , Camundongos , Síndrome Metabólica/tratamento farmacológico , RNA Ribossômico 16S/genética , Tecido Adiposo , Obesidade , Inflamação
3.
Cell Mol Life Sci ; 62(2): 214-26, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15666093

RESUMO

Expression profiles of mRNAs and proteins for various transcription factors were determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos, PU.1, C/EBPalpha, and C/EBPepsilon than monocytes and granulocytes, but higher levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2, Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos, these proteins were absent or expressed only slightly in monocytes and granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPalpha and C/EBPepsilon was below the detection limit in sMCs, but detectable in other myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem cells. The absence of C/EBPvarepsilon in sMCs, but strong expression in HMC-1, suggests it may impair MC maturation. In summary, mature human MCs can be characterized as C/EBPalpha(low), C/EBPvarepsilon-, PU.1(low), GATA-1(low), GATA-2+, SCL+, MITF(high).


Assuntos
Granulócitos/metabolismo , Mastócitos/metabolismo , Monócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA2 , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Quinases/metabolismo , Ratos , Transdução de Sinais , Fator de Transcrição Sp3 , Fatores de Transcrição/genética
4.
Blood ; 98(6): 1949-54, 2001 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-11535534

RESUMO

Complementary and genomic DNA for the murine transferrin receptor 2 (TfR2) were cloned and mapped to chromosome 5. Northern blot analysis showed that high levels of expression of murine TfR2 occurred in the liver, whereas expression of TfR1 in the liver was relatively low. During liver development, TfR2 was up-regulated and TfR1 was down-regulated. During erythrocytic differentiation of murine erythroleukemia (MEL) cells induced by dimethylsulfoxide, expression of TfR1 increased, whereas TfR2 decreased. In MEL cells, expression of TfR1 was induced by desferrioxamine, an iron chelator, and it was reduced by ferric nitrate. In contrast, levels of TfR2 were not affected by the cellular iron status. Reporter assay showed that GATA-1, an erythroid-specific transcription factor essential for erythrocytic differentiation at relatively early stages, enhanced TfR2 promoter activity. Interestingly, FOG-1, a cofactor of GATA-1 required for erythrocyte maturation, repressed the enhancement of the activity by GATA-1. Also, CCAAT-enhancer binding protein, which is abundant in liver, enhanced the promoter activity. Thus, tissue distribution of TfR2 was consistent with the reporter assays. Expression profiles of TfR2 were different from those of TfR1, suggesting unique functions for TfR2, which may be involved in iron metabolism, hepatocyte function, and erythrocytic differentiation.


Assuntos
Eritrócitos/fisiologia , Receptores da Transferrina/genética , Células 3T3 , Processamento Alternativo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Diferenciação Celular , Mapeamento Cromossômico , Proteínas de Ligação a DNA/fisiologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Ferro/metabolismo , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores da Transferrina/biossíntese , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Fatores de Transcrição/fisiologia , Ativação Transcricional , Células Tumorais Cultivadas
5.
Blood ; 97(9): 2561-7, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11313242

RESUMO

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. The neutrophils of individuals with SGD display atypical bi-lobed nuclei, lack expression of all secondary and tertiary granule proteins, and possess defects in chemotaxis, disaggregation, receptor up-regulation, and bactericidal activity, resulting in frequent and severe bacterial infections. Previously, a homozygous mutation in the CCAAT/enhancer binding protein-epsilon (C/EBPepsilon) gene was reported for one case of SGD. To substantiate the role of C/EBPepsilon in the development of SGD and elucidate its mechanism of inheritance, the mutational status of the gene was determined in a second individual. An A-nucleotide insertion in the coding region of the C/EBPepsilon gene was detected. This mutation completely abolished the predicted translation of all C/EBPepsilon isoforms. Microsatellite and nucleotide sequence analyses of the C/EBPepsilon locus in the parents of the proband indicated that the disorder may have resulted from homozygous recessive inheritance of the mutant allele from an ancestor shared by both parents. The mutant C/EBPepsilon(32) protein localized in the cytoplasm rather than the nucleus and was unable to activate transcription. Consistent with this, a significant decrease in the levels of the messenger RNAs (mRNAs) encoding the secondary granule protein human 18-kd cationic antimicrobial protein (hCAP-18)/LL-37 and the primary granule protein bactericidal/permeability-increasing protein were observed in the patient. The hCAP-18 mRNA was induced by overexpression of C/EBPepsilon(32) in the human myeloid leukemia cell line, U937, supporting the hypothesis that C/EBPepsilon is a key regulator of granule gene synthesis. This study strongly implicates mutation of the C/EBPepsilon gene as the primary genetic defect involved in the development of neutrophil SGD and defines its mechanism of inheritance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Mutação da Fase de Leitura , Neutropenia/genética , Adulto , Grânulos Citoplasmáticos , Feminino , Genes Recessivos , Homozigoto , Humanos , Neutropenia/congênito , Neutropenia/etiologia , Neutropenia/patologia , Neutrófilos/patologia
6.
Steroids ; 66(3-5): 433-40, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11179752

RESUMO

Induction of growth arrest and differentiation of some cancer cells by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], and its potent analogs, is well characterized. However, aggressive cancer cell lines are often either insensitive to the antiproliferative effects of 1alpha,25(OH)(2)D(3) or require toxic concentrations to recapitulate them which has, to-date, precluded its use in anticancer therapy. Therefore we are interested in mechanisms by which 1alpha,25(OH)(2)D(3) signaling has become deregulated in malignant cells in order to identify novel therapeutic targets. We observed previously that 1alpha,25(OH)(2)D(3) and its metabolites, generated via the C-24 oxidation pathway, drive simultaneous differentiation and hyper-proliferation within the same cell population. Thus we have proposed that metabolism of 1alpha,25(OH)(2)D(3) via the C-24 oxidation pathway represents a novel-signaling pathway, which integrates proliferation with differentiation. In the current study we examined further the role of this pathway and demonstrated that these effects are not restricted to leukemic cells but are observed also in both normal myeloid progenitors and breast cancer cell lines. Intriguingly, stable transfection of MCF-7 breast cancer cells with antisense vitamin D(3) receptor (VDR) reduced antiproliferative sensitivity to 1alpha,25(OH)(2)D(3) but significantly enhanced growth stimulation, which, in turn, was blocked by inhibiting metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway with ketoconazole. Taken together, these studies indicate that metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway gives rise to ligands with different biologic effects. We propose that this mechanism may allow the co-ordination of population expansion and cell maturation during differentiation. Cancer cells appear to corrupt this process during malignant transformation, by only responding to the pro-proliferative signals, thereby deriving a clonal advantage.


Assuntos
Neoplasias da Mama/patologia , Calcitriol/farmacologia , Neoplasias da Mama/metabolismo , Calcitriol/metabolismo , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Oxirredução , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos
7.
Oncogene ; 19(44): 5091-7, 2000 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-11042697

RESUMO

The anti-proliferative action of the seco-steroid hormone 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] extends to some, but not all breast and prostate cancer cell lines. By elucidating the molecular mechanisms mediating the sensitivity of these cells, we can identify critical target genes regulated directly or indirectly by 1alpha,25(OH)2D3 and pathways potentially disrupted during transformation. In this study, we demonstrated the induction of expression of BRCA1 mRNA and protein as well as transcriptional activation from the BRCA1-promoter by 1alpha,25(OH)2D3 in the sensitive breast cancer cell line MCF-7. This was not observed in the 1alpha,25(OH)2D3-resistant breast cancer cell line MDA-MB-436. The induction of BRCA1 mRNA was blocked by cyclohexamide. This indicated that transcriptional activation was mediated indirectly by the vitamin D receptor (VDR). Inhibition of VDR protein levels by stable transformation of the anti-sense VDR in MCF-7 reduced the sensitivity of MCF-7 to 1alpha,25(OH)2D3 by 50-fold. In addition, the induction of BRCA1 protein and transcriptional activation of a BRCA1 promoter-luciferase reporter construct was abrogated in the stable transformant with the greatest reduction of VDR levels. Examination of other breast and prostate cancer cell lines revealed that sensitivity to the anti-proliferative effects of 1alpha, 25(OH)2D3 was strongly associated with an ability to modulate BRCA1 protein. Furthermore, the expression of the estrogen receptor in these cell lines strongly correlated with their sensitivity to 1alpha,25(OH)2D3 and their ability to modulate BRCA1 expression. Taken together, our data support a model whereby the anti-proliferative effects of 1alpha,25(OH)2D3 are mediated, in part, by the induction of BRCA1 gene expression via transcriptional activation by factors induced by the VDR and that this pathway is disrupted during the development of prostate and breast cancers.


Assuntos
Neoplasias da Mama/genética , Calcitriol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes BRCA1/efeitos dos fármacos , Neoplasias da Próstata/genética , Animais , Proteína BRCA1/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células COS/metabolismo , Divisão Celular/efeitos dos fármacos , Feminino , Genes BRCA1/genética , Humanos , Masculino , Oligonucleotídeos Antissenso/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Calcitriol/biossíntese , Receptores de Calcitriol/genética , Receptores de Calcitriol/fisiologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
8.
Blood ; 95(4): 1451-5, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10666224

RESUMO

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by recurrent, self-limited attacks of fever and serositis and by infiltration of affected tissues by large numbers of neutrophils. A candidate gene for FMF was identified by positional cloning and named "MEFV." The corresponding protein was named "pyrin." To elucidate the currently unknown function of pyrin, we characterized its tissue distribution, regulation of expression during hematopoietic differentiation, and subcellular localization. Reverse transcription-polymerase chain reaction analysis, followed by hybridization with an internal oligonucleotide, demonstrated expression of MEFV in different populations of peripheral blood cells. Among hematopoietic cell lines, MEFV was almost exclusively expressed in cells of the myeloid lineage. Furthermore, MEFV messenger RNA was strongly expressed within 24 hours of dimethyl sulfoxide-induced granulocytic differentiation of HL-60 cells. Analysis of complementary DNA from human solid tumor-derived cell lines revealed expression of MEFV in several cell lines derived from colon and prostate cancers. Expression of MEFV fused to enhanced green fluorescent protein showed that pyrin localized in distinct patches in the cytoplasm, forming a perinuclear cap. Taken together, MEFV is predominantly expressed in myeloid cells and upregulated during myeloid differentiation, and the corresponding protein, pyrin, is expressed in the cytoplasm. (Blood. 2000;95:1451-1455)


Assuntos
Febre Familiar do Mediterrâneo/genética , Hematopoese/genética , Leucócitos/metabolismo , Proteínas/genética , Células Cultivadas , Clonagem Molecular , Neoplasias do Colo , Proteínas do Citoesqueleto , Células HL-60 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Neutrófilos/metabolismo , Proteínas/análise , Pirina , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas
9.
J Biol Chem ; 274(30): 20826-32, 1999 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-10409623

RESUMO

Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: alpha (approximately 2.9 kilobase pairs), and beta (approximately 2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-alpha protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-beta protein lacked the amino-terminal portion of the TfR2-alpha protein including the putative transmembrane domain. Northern blot analysis showed that the alpha transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-alpha. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound (55)Fe uptake. Taken together, TfR2-alpha may be a second transferrin receptor that can mediate cellular iron transport.


Assuntos
Ferro/metabolismo , Receptores da Transferrina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , Humanos , Células K562 , Dados de Sequência Molecular , Receptores da Transferrina/metabolismo , Alinhamento de Sequência , Análise de Sequência , Transdução de Sinais
10.
Blood ; 94(3): 1113-20, 1999 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10419905

RESUMO

The p73 gene, a member of the p53 family, is a new candidate tumor suppressor gene. To investigate the possibility of genetic alteration of p73 in leukemia and lymphoma, we examined 55 cell lines and 39 patient samples together with 17 nonhematopoietic cancer cell lines. Gene expression of p73 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cell lines (5 of 7 pre B/B-acute lymphoblastic leukemia [ALL], 13 of 21 T-ALL/lymphoblastic lymphomas [LBL], 9 of 10 B-non-Hodgkin's lymphomas [B-NHL], 8 of 9 acute myelogenous leukemias [AML], 2 of 2 T-NHL, 3 of 3 multiple myeloma), and in patient samples (16 of 23 pre B-ALL, 5 of 8 T-ALL/LBL, 5 of 8 B-NHL). PCR-single-strand conformation polymorphism (SSCP) of cDNAs showed no mutation in 43 p73-expressing cell lines within the regions that corresponded to the 5 mutational hotspots of the p53 gene. Neither homologous deletion nor rearrangement of the p73 gene were found by Southern blot analysis in any of the cell lines that lack expression of p73. In contrast to prior published data, analysis of a polymorphic site showed that the p73 gene was expressed biallelically in cell lines and normal peripheral blood. Notably, the p73-negative cell lines were hypermethylated at a CpG island in the 5' untranslated region of the p73 mRNA, and treatment of these cell lines with 5-azacytidine (5-AC), a demethylation reagent, induced p73 expression. Taken together, we found that a sizable proportion (32%) of ALL/B-NHL cell lines and primary tumors had negligible or limited expression of the p73 gene associated with hypermethylation of the gene. These findings suggest that silencing of the p73 gene by hypermethylation may contribute to development and/or progression of lymphoid neoplasms.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Linfoma/genética , Proteínas Nucleares/genética , Metilação de DNA , DNA de Neoplasias/genética , Genes Supressores de Tumor , Humanos , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteínas Supressoras de Tumor
11.
J Clin Invest ; 103(10): 1399-408, 1999 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10330422

RESUMO

The CCAAT/enhancer binding protein epsilon (C/EBPepsilon) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPepsilon expression was through the retinoic acid receptor alpha (RARalpha) pathway. Reporter gene studies showed that C/EBPepsilon promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPepsilon. The RA-induced expression of C/EBPepsilon markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARalpha (PML/RARalpha), but not in those induced to express promyelocytic leukemia zinc finger/RARalpha (PLZF/RARalpha). In retinoid-resistant APL cell lines, C/EBPepsilon either is not induced or is induced only at very high concentrations of RA (>/=10(-6) M). In addition, forced expression of C/EBPepsilon in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPepsilon is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.


Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Proteínas Nucleares/genética , Retinoides/uso terapêutico , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Resistência a Medicamentos/genética , Elementos Facilitadores Genéticos , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Leucemia Promielocítica Aguda/metabolismo , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Tretinoína/farmacologia , Células Tumorais Cultivadas , Células U937
12.
Blood ; 93(10): 3327-37, 1999 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10233885

RESUMO

C/EBPepsilon is essential for granulocytic differentiation. We investigated the role of C/EBPepsilon in the transcriptional activation of various myeloid-specific genes. We found that two C/EBPepsilon isoforms, p32 and p30, possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBPepsilon p30 but not p32 was differentially upregulated in NB-4 promyelocytic leukemia cells treated with retinoids. Both isoforms bound specifically to C/EBP sites in myeloid promoters. The kd for C/EBPepsilon binding to the C/EBP site of the neutrophil elastase promoter was 4.2 nmol/L. In transfection assays using the nonhematopoietic cell line, CV-1, the p32 isoform activated promoters from the myeloid-specific mim-1, neutrophil elastase, and granulocyte colony-stimulating factor (G-CSF) receptor genes by 2.5-, 1.8-, and 1.6-fold, respectively. The p30 isoform lacked significant transcriptional activity, suggesting that other hematopoietic-specific factors were required for its function. Consistent with this prediction, transfections into the hematopoietic cell line Jurkat showed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphoblasts (Jurkat cells) and immature myeloid cells (eg, Kcl22 cells) expressed high levels of the c-myb hematopoietic transcription factor. Cotransfection of c-myb with either the p32 or p30 isoform of C/EBPepsilon in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respectively, and the neutrophil elastase promoter by 10-and 7-fold, respectively. Pulldown assays showed that each C/EBPepsilon isoform interacted directly with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBPepsilon, but not C/EBPalpha, C/EBPbeta, or C/EBPdelta. A mutation of the C/EBP site in the neutrophil elastase promoter markedly decreased the transactivation of the promoter in Kcl22 myeloblasts. These results demonstrate a role for C/EBPepsilon in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Hematopoese/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Células COS , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Humanos , Células Jurkat , Elastase de Leucócito/genética , Camundongos , Oncogenes , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes de Fusão/biossíntese , Ativação Transcricional , Transfecção , Células U937
13.
J Biol Chem ; 273(24): 14796-804, 1998 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-9614080

RESUMO

Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Proteínas Repressoras/genética , Ativação Transcricional/genética , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Elastase de Leucócito/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/química , Transfecção/genética
14.
Blood ; 90(7): 2591-600, 1997 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-9326225

RESUMO

Human C/EBP epsilon is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBP epsilon, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBP epsilon mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBP epsilon was the only C/EBP family member that was easily detected by RT-PCR. No C/EBP epsilon mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBP epsilon. Northern blot and RT-PCR analyses showed that C/EBP epsilon mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBP epsilon protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBP epsilon protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38-), purified from humans had very weak expression of C/EBP epsilon mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBP epsilon mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBP epsilon, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBP epsilon is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Granulócitos/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Monócitos/citologia , Proteínas Nucleares/biossíntese , Doença Aguda , Alitretinoína , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Tretinoína/farmacologia
15.
Leukemia ; 11(10): 1673-80, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9324288

RESUMO

Loss of the p16INK4A gene by homozygous deletions or point mutations is attributed to the development of many types of cancers including leukemia. T cell acute lymphoblastic leukemias (T-ALLs) and B-cell ALLs show a remarkable rate of 75 and 20% homozygous deletion of this gene, respectively. Restoration of p16 expression in p16-deficient solid tumor cell lines results in a dramatic reduction of growth and maligant phenotype. To test the hypothesis that p16INK4A suppresses the growth of p16-deficient leukemias, we utilized a retroviral system to restore wild-type (wt) or mutant p16 protein expression. We tested the efficacy of our system by expressing the wt or mutant p16 genes in the osteosarcoma cell line, U20S, which lacks p16 and retains functional retinoblastoma protein (pRb). The wt p16 protein formed complexes with both cyclin-dependent kinases (CDK) 4 and 6 and inhibited U20S growth by 30-fold. The p16 mutants E120K and R144C formed complexes with CDK4 and CDK6 in cells and inhibited cell growth as effectively as wt p16 (20-fold) while the mutant proteins that did not complex with detectable levels of CDK4 or CDK6 only inhibited growth 0.25- and five-fold (G101W and D141, respectively) or not at all (H83Y and DA4). The COOH-terminal 'tail' of the wt p16 protein (amino acid residues 141-156), missing in mutant D141, enhanced the growth suppressive capability of p16. The amino acid substitutions in mutants G101W and H83Y not only disrupted CDK4 and CDK6 binding, but decreased the protein half-lives by two- and three-fold, respectively, compared to wt p16. The wt, but not mutant p16 genes, effectively inhibited the growth of T cell acute lymphoblastic (CEM) and myeloid leukemia (NB-4 and K562) cell lines that lacked the p16 gene, but retained functional pRb. Growth of the T-ALL cell line, HSB-2, which lacked both p16 and pRb, was not inhibited, indicating the growth suppression involved the pRb pathway. These results define regions critical for the function of p16 and demonstrate that restoration of wt p16 expression in p16-deficient leukemias significantly reverted their transformed phenotype and inhibited their growth.


Assuntos
Proteínas de Transporte/fisiologia , Leucemia/terapia , Animais , Células COS , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Divisão Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/terapia , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/terapia , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/terapia , Mutação , Osteossarcoma/genética , Osteossarcoma/metabolismo , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Retroviridae/metabolismo , Células Tumorais Cultivadas
16.
Oncogene ; 15(13): 1605-9, 1997 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-9380413

RESUMO

Tumor necrosis factor alpha (TNFalpha) is a cytotoxic/cytostatic compound for a variety of human cancer cells. The p21WAF1 protein is a cyclin-dependent kinase inhibitor (CDKI) that binds to cyclin/cyclin-dependent kinase (CDK) complexes and inhibits their kinase activities, thereby leading to cell cycle arrest. We found that the cytostatic effect of TNFalpha on the cervical cancer cell line, ME180, was concomitant with an arrest of these cells in the G0/G1 phase of the cell-cycle. This corresponded with an increase in both p21WAF1 mRNA and protein levels which likely occurred via a p53-independent pathway since ME180 is infected with the human papilloma virus. To elucidate the role of p21WAF1 in the TNFalpha-mediated growth and cell cycle arrest, we stably transformed ME180 cells with an antisense p21WAF1 expression vector. Two clones with reduced levels of p21WAF1 both in their basal state as well as after their exposure to TNFalpha were selected. The growth of these cells was still inhibited by TNFalpha and they arrested in G0/G1 similar to wildtype or empty vector transfected cells. These results indicate that although p21WAF1 expression increases dramatically with TNFalpha treatment, it may not play a critical role in the cytostatic effect of TNFalpha on ME180 cervical cancer cells.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Humanos , RNA Mensageiro/análise , Transformação Genética , Células Tumorais Cultivadas
17.
Biochem Biophys Res Commun ; 238(1): 77-80, 1997 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-9299455

RESUMO

Allelic variations of the vitamin D receptor (VDR) gene have been associated with the risk of developing prostate cancer in men and osteoporosis in postmenopausal women. Three RFLPs (TaqI, ApaI, BsmI) define two common haplotypes: BAt and baT. None of these polymorphisms change the translated protein. Since sequence variations in the 3' UTR of VDR have been linked to the different haplotypes, investigators have proposed that the stability of VDR mRNA is influenced by allelic variations. Indirect evidence suggested that allele T is less stable than allele t. In this study, we used a RT-PCR based approach to compare the stability of the big T and small t allele in normal heterozygous lymphocytes and the heterozygous cell lines NB4 (myeloid leukemia) and PC-3 and DU 145 (prostate cancers). In all three cases, we did not find a significant difference in stability. Interestingly, we consistently observed 30% less RT-PCR product derived from the small t allele mRNA in steady state, a finding which also speaks against a higher stability of the small t allele mRNA. These results indicate a variation in transcriptional regulation rather than mRNA stability between the alleles. We hypothesize that an unknown gene or genes in linkage with the polymorphisms is (are) responsible for the relationship between risk of prostate cancer and VDR polymorphisms.


Assuntos
Alelos , Heterozigoto , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/metabolismo , Receptores de Calcitriol/genética , Linhagem Celular , DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Células-Tronco Hematopoéticas , Humanos , Linfócitos , Masculino , Neoplasias da Próstata , DNA Polimerase Dirigida por RNA , Taq Polimerase , Células Tumorais Cultivadas
18.
Leukemia ; 10(12): 1897-900, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8946928

RESUMO

Cyclin-dependent kinase inhibitors (CDKIs) can be classified into two groups based on the structure of the proteins. One group includes the p21 (CIP1, WAF1, CAP20), p27 (Kip1), and p57 (Kip2) CDKIs, which contain a homologous amino-terminal cyclin-dependent kinase (cdk) inhibitory domain. The p16 (INK4A), p15 (INK4B), and p18 (INK4C) CDKIs, which have an ankyrin repeat motifs, belong to the other group. The p16 and p15 CDKI genes are very frequently altered in a variety of cancers including hematopoietic malignancies. The p19 (INK4D) gene is a newly cloned CDKI which belongs to the latter group. To determine if p19 genetic alterations play a role in hematopoietic malignancies, we examined DNA from 45 childhood newly diagnosed acute lymphocytic leukemias (ALLs), 30 acute myeloblastic leukemias (AMLs), 10 chronic myelocytic leukemias (CMLs), 45 adult T cell leukemias (ATLs), 70 non-Hodgkin's lymphomas (NHLs), and 20 multiple myelomas (MM) as well as 14 ALL, 20 AML, two ATL, and five lymphoma cell lines. Using Southern blot analysis, one homozygous deletion of the p19 gene was detected in a human immunodeficiency virus (HIV)-related Burkitt-like lymphoma sample. No point mutations in any of the samples were found by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Our investigation suggests that alterations of p19 do not play an important role in the development of most hematopoietic malignancies.


Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina , Neoplasias Hematológicas/genética , Mutação Puntual , Sequência de Bases , Southern Blotting , Inibidor de Quinase Dependente de Ciclina p19 , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Células Tumorais Cultivadas
19.
Blood ; 87(12): 5225-31, 1996 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8652837

RESUMO

Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/genética , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/biossíntese , Proteínas Repressoras , Fatores de Transcrição , Doença Aguda , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Proteína 2 Inibidora de Diferenciação , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/patologia , Leucemia Mieloide/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Células Tumorais Cultivadas/efeitos dos fármacos
20.
J Cell Physiol ; 166(3): 568-76, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8600160

RESUMO

The p53 protein directly regulates the expression of the WAF1 (wild-type p53-activated fragment 1) protein which is a cyclin-dependent kinase inhibitor (CDK1). DNA damaging agents such as ionizing or UV radiation, and some chemical agents induce WAF1 in wild-type p53 containing cells, thereby halting cell cycle progression. WAF1 expression is also induced through a p53-independent pathway. Tumor necrosis factor alpha (TNF alpha) is a cytotoxic/cytostatic compound for some human cancer cells. We examined a series of myeloid leukemic cell lines that expressed either no p53 (HL-60, K562) or mutant inactive p53 (KG-1, KCL22,THP-1, U937). The KG-1, HL-60, K562, and KCL22 myeloid leukemic cells increased their levels of WAF1 mRNA in the presence of TNF alpha. We focused on KG-1 cells to determine how TNF alpha modulated WAF1 expression. WAF1 mRNA increased in a dose-dependent manner in the cells after exposure to increasing concentrations of TNF alpha, and this increase occurred in the absence of new protein synthesis. An increase of WAF1 protein and a concominant decrease of cyclin-dependent kinase 2 activity also was found in KG-1 cells. Flow cytometry using 5-bromo-2'-deoxyuridine showed an increase in the proportion of TNF alpha- treated KG-1 cells in the G0/G1 phase of the cell cycle. TNF alpha enhanced the rate of WAF1 transcription only 1.4 fold in TNF alpha-treated KG-1 cells as compared to untreated cells. Notably, however, the half-life (t 1/2) of WAF1 mRNA in TNF alpha-treated cells was 2.5 hours as compared to 0.5 hours in untreated cells. These results indicate that TNF alpha increases WAF1 levels at least in part via a postttranscriptional stabilization of the mRNA; and TNF alpha may mediate its cytostatic effects through WAF1 in some cell types.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclinas/genética , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Ciclo Celular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/biossíntese , Cicloeximida/farmacologia , Inibidores Enzimáticos , Humanos , Leucemia Mieloide , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , RNA Neoplásico/metabolismo , Proteínas Recombinantes/farmacologia , Transcrição Gênica , Células Tumorais Cultivadas
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