RESUMO
OBJECTIVE: The existence of mesenchymal stem cells (MSCs) in SF was previously reported. However, the behaviour and properties of MSCs derived from SF have not been fully elucidated. METHODS: Human SFs were obtained from 19 knee joints with anterior cruciate ligament injury around the time of reconstruction surgery, and from three healthy volunteers. SF was plated, cultured and examined for colony-forming number, in vitro differentiation, surface epitopes and gene profiles. Also, rabbit synovium-MSCs were injected into the knee joint in a rabbit partial anterior cruciate ligament defect model, and the injected cells were traced. RESULTS: SF-MSCs from IA ligament injury patients were 100 times more in number than those from healthy volunteers. Total colony number was positively correlated with post-injury period. No significant differences were observed among the cells derived from SF around the time of the surgery in relation to surface epitopes and differentiation potentials. Cluster analysis of gene profiles demonstrated that SF-MSCs were more similar to synovium MSCs than bone marrow MSCs. In rabbit experiments, the MSCs injected into the knee in which IA ligament was partially defective were observed more on the defected area than on the intact area of the ligament at 24 h. CONCLUSION: We demonstrated that SF-MSCs, similar to synovium MSCs, increased in number after IA ligament injury and surgery without marked alteration of the properties.
Assuntos
Lesões do Ligamento Cruzado Anterior , Células-Tronco Mesenquimais/patologia , Líquido Sinovial/citologia , Adolescente , Adulto , Animais , Ligamento Cruzado Anterior/patologia , Células da Medula Óssea/patologia , Adesão Celular , Diferenciação Celular , Células Cultivadas , Criança , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Epitopos/análise , Perfilação da Expressão Gênica/métodos , Humanos , Traumatismos do Joelho/patologia , Traumatismos do Joelho/terapia , Transplante de Células-Tronco Mesenquimais , Pessoa de Meia-Idade , Coelhos , Líquido Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and alpha-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.
Assuntos
Células da Medula Óssea/citologia , Linhagem Celular , Miocárdio/citologia , Células Estromais/citologia , Potenciais de Ação , Animais , Diferenciação Celular , Linhagem da Célula , Coração/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Proteínas Musculares/fisiologiaRESUMO
The differentiation of both embryonal carcinoma (EC) and embryonic stem (ES) cells can be triggered in culture by exposure to retinoic acid and results in the transcriptional induction of both the endogenous mouse keratin 18 (mK18) intermediate filament gene and an experimentally introduced human keratin 18 (K18) gene as well as a variety of other markers characteristic of extraembryonic endoderm. The induction of K18 in EC cells is limited, in part, by low levels of ETS and AP-1 transcription factor activities which bind to sites within a complex enhancer element located within the first intron of K18. RNA levels of ETS-2, c-Jun, and JunB increase upon the differentiation of ES cells and correlate with increased expression of K18. Occupancy of the ETS site, detected by in vivo footprinting methods, correlates with K18 induction in ES cells. In somatic cells, the ETS and AP-1 elements mediate induction by a variety of oncogenes associated with the ras signal transduction pathway. In EC cells, in addition to the induction by these limiting transcription factors, relief from negative regulation is mediated by three silencer elements located within the first intron of the K18 gene. These silencer elements function in F9 EC cells but not their differentiated derivatives, and their activity is correlated with proteins in F9 EC nuclei which bind to the silencers and are reduced in the nuclei of differentiated F9 cells. The induction of K18, associated with the differentiation of EC cells to extraembryonic endoderm, is due to a combination of relief from negative regulation and activation by members of the ETS and AP-1 transcription factor families.
Assuntos
Queratinas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores , Fator de Transcrição AP-1/genética , Fatores de Transcrição , Tretinoína/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Sequência Consenso , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas In Vitro , Íntrons , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-2 , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Transcrição Gênica/efeitos dos fármacos , Ativação TranscricionalRESUMO
Several lines of evidence indicate that DNA methylation plays a role in the transcriptional regulation of the murine alpha 1(I) collagen gene. To study the molecular mechanisms involved, a reporter gene construct containing the alpha 1(I) promoter and part of the first exon linked to the luciferase gene (Col3luc) was methylated in vitro and transfected into murine fibroblasts and embryonal carcinoma cells. Methylation resulted in repression of the alpha 1(I) promoter in both cell types, although it was less pronounced in embryonal carcinoma cells than in fibroblasts. The extent of repression depended on the density of methylation. DNase footprint and mobility shift assays indicated that the trans-acting factors binding to the alpha 1(I) promoter and first exon are ubiquitous factors and that their DNA binding is not inhibited by methylation. Transfection of Col3luc into Drosophila SL2 cells together with expression vectors for the transcription factors Sp1 and NF-1 showed that DNA methylation also inhibits the alpha 1(I) promoter in nonvertebrate cells, although to a much lesser extent than in murine cells. However, Sp1 and NF-1 transactivated the unmethylated and methylated reporter gene in SL2 cells equally well, confirming that these factors can bind and transactivate methylated DNA and indicating that DNA methylation represses the alpha 1(I) promoter by an indirect mechanism. This was further confirmed by cotransfection experiments with unspecific methylated competitor DNA which partially restored the activity of the methylated alpha 1(I) promoter. Our results suggest that DNA methylation can inhibit promoter activity by an indirect mechanism independent of methyl-C-binding proteins and that in vertebrate cells, chromatin structure and methyl-C-binding proteins cooperatively mediate the transcriptional inhibitory effect of DNA methylation.
Assuntos
Colágeno/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Cromatina/ultraestrutura , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Metilação , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Ligação Proteica , RNA Mensageiro/genética , Fatores de Transcrição/metabolismoRESUMO
We isolated clonal sublines of the established mouse marrow stromal cell line, H-1. These clonal sublines underwent differentiation into adipocytes in various degrees. One subline, H-1/A, underwent adipocyte differentiation after confluence, while another subline, H-1/D, did not differentiate. In H-1/A cells, the 4.5- and 2.5-kb major mRNA species of colony-stimulating factor 1 (CSF-1) were expressed before differentiation and were down-regulated at a posttranscriptional level during the differentiation of H-1/A cells. The down-regulation of the CSF-1 gene was not a result of arrested cellular growth, because no down-regulation was detected in the nondifferentiating sister line, H-1/D. This down-regulation appeared to be an early event in differentiation. Cachectin/tumor necrosis factor transiently induced the expression of CSF-1 and inhibited the differentiation of H-1/A cells into adipocytes. This induced expression of CSF-1 was due to an increased rate of transcription.
Assuntos
Tecido Adiposo/citologia , Fator Estimulador de Colônias de Macrófagos/genética , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Dactinomicina/farmacologia , Glucosefosfato Desidrogenase/genética , Células-Tronco Hematopoéticas/citologia , Cinética , Camundongos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The activities of ETS transcription factors are modulated by posttranscriptional modifications and cooperation with other proteins. Another factor which could alter the regulation of genes by ETS transcription factors is DNA methylation of their cognate binding sites. The optimal activity of the keratin 18 (K18) gene is dependent upon an ETS binding site within an enhancer region located in the first intron. The methylation of the ETS binding site was correlated with the repression of the K18 gene in normal human tissues and in K18 transgenic mouse tissues. Neither recombinant ETS2 nor endogenous spleen ETS binding activities bound the methylated site effectively. Increased expression of the K18 gene in spleens of transgenic mice by use of an alternative, cryptic promoter 700 bp upstream of the enhancer resulted in modestly decreased methylation of the K18 ETS site and increased RNA expression. Expression in transgenic mice of a mutant K18 gene, which was still capable of activation by ETS factors but was no longer a substrate for DNA methylation of the ETS site, was fivefold higher in spleen and heart. However, expression in other organs such as liver and intestine was similar to that of the wild-type gene. This result suggests that DNA methylation of the K18 ETS site may be functionally important in the tissue-specific repression of the K18 gene. Epigenetic modification of the binding sites for some ETS transcription factors may result in a refractory transcriptional response even in the presence of necessary trans-acting activities.
Assuntos
Metilação de DNA , Íntrons , Queratinas/genética , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Humanos , Queratinas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ribonucleases/metabolismo , TransgenesRESUMO
Cells with myogenic potential are present in many tissues, and these cells readily form skeletal muscle in culture. We here focus on menstrual blood as another cell source for regenerative medicine. Menstrual blood-derived cells have high replicative ability, similar to progenitors or stem cells, and transdifferentiate or meta-differentiate into myocytes in vitro at unexpectedly high frequencies. This unique phenotype can be explained by histological and embryological aspects of the endometrium. The remarkable myogenic capability of these cells enables us to "rescue" dystrophied myocytes of the mdx model of Duchenne muscular dystrophy through cell fusion and transdifferentiation. Endometrial cells supplied as a form of menstrual blood-tissue mixture can be used for cell-based therapy in addition to a place for embryo implantation.
Assuntos
Menstruação/sangue , Células-Tronco Mesenquimais/citologia , Distrofias Musculares/patologia , Diferenciação Celular , Transdiferenciação Celular , Feminino , Humanos , Distrofias Musculares/sangue , Mioblastos/metabolismo , Mioblastos/patologiaRESUMO
A cell line of a benign tumor, trichilemmoma, was established in vitro and has been maintained in culture for 1.5 years with more than 30 passages. Plating efficiency was less than 0.1%, and population doubling time was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) as observed in vivo. Gas chromatographic analysis revealed that extracted glycogen was composed of glucose alone. Chromosome analyses with trypsin-Giemsa banding showed an abnormal karyotype with hypodiploid modal numbers of 44 and 45. There were four marker chromosomes observed in 100% of cells in 100 metaphase cells examined. Cells did not grow on fibroblast monolayers or in soft agar in vitro but did induce tumors in athymic nude mice (12 of 15) after the s.c. injection of tissue-cultured cells (2.5 x 10(6) to 4.5 x 10(7) cells/mouse). The histological characteristics of the tumors in nude mice were similar to those of the original tumor. This is the first time, to our knowledge, that a benign human tumor cell line has been established in vitro which can induce tumors in nude mice.
Assuntos
Linhagem Celular , Neoplasias Cutâneas/patologia , Idoso , Amilases , Animais , Cromossomos , Feminino , Glicogênio/metabolismo , Humanos , Cariotipagem , Camundongos , Camundongos Nus , Microscopia Eletrônica , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/ultraestrutura , Coloração e Rotulagem , Transplante HeterólogoRESUMO
Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.
Assuntos
Apoptose , Inibinas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Ativinas , Inibidores de Caspase , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Interações Medicamentosas , Eritropoetina/farmacologia , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
We have cloned a murine homologue of the human Mcl1/EAT gene, a Bcl-2 related gene. Sequence analysis revealed that murine Mcl1/EAT (mMcl1/EAT) has three Bcl-2 homology domains, two PEST sequences, and immediate response boxes (IRB). The presence of IRB indicates that mMcl1/EAT is an immediate-early gene. mMcl1/EAT increases dramatically with exposure to retinoic acid in murine embryonal carcinoma cell lines (F9 and PCC3) as well as embryonic stem cells, both of which are models of early embryogenesis.
Assuntos
Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Regulação para Cima , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Homologia de Sequência de Aminoácidos , Células-Tronco , Células Tumorais CultivadasRESUMO
We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.
Assuntos
Metilação de DNA , Genes , Impressão Genômica , Proteínas/genética , Espermatozoides/metabolismo , Adulto , Animais , Sequência de Bases , Ilhas de CpG , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RNA Longo não Codificante , RNA não Traduzido , RatosRESUMO
SOX is a family of genes related to the testis-determining gene, SRY. We have isolated and sequenced an hSOX20 cDNA from a cell line of human embryonic carcinoma. This cDNA contains an open reading frame (ORF) encoding 233 amino acids. The protein encompasses an SRY-type HMG box exhibiting strong homologies to those of mouse Sox15 and Sox16. Various adult and fetal tissues were tested for hSOX20 mRNA by Northern analysis. Its expression is restricted to the fetal testis and the size of the transcript is 1.5 knt. Electrophoretic mobility shift assay indicated that recombinant hSOX20 polypeptide is capable of binding to AACAAT sequence.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas Nucleares , Fatores de Transcrição , Adulto , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência Molecular , Fatores de Transcrição SOX , Processos de Determinação Sexual , Proteína da Região Y Determinante do SexoRESUMO
EAT/mcl-1 (EAT), a bcl-2-related immediate early gene, is up-regulated at an early stage of differentiation of human embryonal carcinoma cells. Recent studies have revealed that EAT inhibits apoptosis both in vitro and in vivo. In the present study, we demonstrated that the EAT gene was up-regulated in the early stage of rat myocardial infarction. This pattern of up-regulation was apparently different from that of another immediate early gene, c-fos. EAT, an anti-apoptotic molecule, was strongly up-regulated in the non-ischemic region. In contrast, the expression of c-fos was induced in both ischemic and non-ischemic regions, and was higher in the ischemic region. Apoptosis of cardiomyocytes is currently thought to significantly contribute to acute myocardial infarction. We detected cardiomyocyte apoptosis by gel electrophoresis of genomic DNA and in situ nick end labeling in the ischemic region, but not in the non-ischemic region. As an inhibitor of apoptosis, EAT may play a role in the protection of cardiomyocytes in the early stage of acute myocardial infarction.
Assuntos
Apoptose/genética , Infarto do Miocárdio/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Masculino , Proteína de Sequência 1 de Leucemia de Células Mieloides , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Regulação para CimaRESUMO
Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Óxidos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Óxidos/uso terapêutico , Células Tumorais CultivadasRESUMO
Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3 on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1x10(7)) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Leucemia Promielocítica Aguda/patologia , Óxidos/farmacologia , Tretinoína/farmacologia , Animais , Antineoplásicos/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Óxidos/uso terapêutico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Transplante Heterólogo , Tretinoína/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
Bone marrow is one of the most radiosensitive organs. Irradiation causes a marked decrease in the total number of hematopoietic cells in the bone marrow. The reticular meshwork structure of marrow stromal cells, however, is relatively resistant to irradiation. Unimpaired stromal cell structure has been thought to be a prerequisite for the repopulation of hematopoietic cells during recovery from the effects of irradiation. The reticular framework is maintained by cell adhesion apparatuses such as gap junctions. The in vitro radiobiologic survival values of a cloned stromal cell line, H-1/A, were studied (ñ = 1.8, D0 = 138 cGy). Radiation doses of up to 4000 cGy had no detectable effects on the production of colony-stimulating factor 1. H-1/A cells communicate with each other via gap junctions as determined by the sensitive dye-transfer method. Gap-junctional communication between H-1/A cells was resistant to different levels of irradiation (500 to 10,000 cGy), but it was lost during adipocyte differentiation of H-1/A cells. Marrow stromal cells, which are important in the recovery of hematopoiesis, seemed capable of coordination with each other through gap junctions even when exposed to radiation.
Assuntos
Medula Óssea/efeitos da radiação , Comunicação Celular/efeitos da radiação , Tolerância a Radiação , Medula Óssea/ultraestrutura , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Fatores Estimuladores de Colônias/biossíntese , Fator Estimulador de Colônias de MacrófagosRESUMO
The genetic structure and regulation of the c-myc gene was comprehensively studied for the first time in Burkitt's lymphoma with t(2;8) translocation. In a Burkitt's lymphoma cell line, KOBK101, the immunoglobulin kappa-encoding gene on chromosome 2, accompanied by its enhancer, was translocated to the pvt-1 locus located about 250 kb downstream from c-myc on chromosome 8. Only the c-myc allele on the translocated chromosome carried aberrant SalI and KpnI sites in the first intron, so the two c-myc alleles and their transcripts were analyzed separately. The c-myc allele on the untranslocated chromosome conserved the normal c-myc sequence and was transcriptionally silent. In contrast, the c-myc allele on the translocated chromosome was actively transcribed at three- to fivefold higher levels, as compared with non-malignant B-cell lines. Additionally, it carried predominant multiple mutations consisting of 64 nucleotide substitutions, three short deletions, and a one-base insertion, most of which clustered in the first exon and intron. The 24-base deletion in the first intron completely overlapped the binding site of a putative negative transcriptional factor of the 138-kDa phosphoprotein, MIF. Thus, the multiple mutations and the deregulated, allele-specific expression of c-myc were associated with the chromosomal translocation in cis. Together activation by the long-distance immunoglobulin kappa enhancer, and the alleviation of negative regulation by the mutations, seemed to cause the allele-specific activation of c-myc.
Assuntos
Linfoma de Burkitt/genética , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 8 , Regulação Neoplásica da Expressão Gênica , Genes myc , Translocação Genética , Alelos , Sequência de Bases , Northern Blotting , DNA de Neoplasias , Humanos , Dados de Sequência Molecular , Mutação , Mapeamento por Restrição , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The EAT/mcl-1 gene was isolated during the early differentiation of a retinoic acid-induced human embryonal carcinoma cell line to the trophectoderm lineage. EAT/mcl-1, a bcl-2 related gene, is involved in the genetic pathway of apoptosis; this suggests a role for apoptosis and the involvement of this gene in early placental development. In the current investigation, we analysed expression of EAT/mcl-1 at the mRNA level by Northern blot analysis and in situ hybridization, as well as at the protein level, by immunoblot analysis and immunohistochemistry. Our results identified constant expression of this gene in the placenta throughout pregnancy as well as a shift in its localization from the cytotrophoblast in the first trimester to the syncytiotrophoblast in the third trimester. In addition, there was an inverse correlation between EAT/mcl-1 expression and TaT-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) reactivity in trophoblasts in the first trimester. These results suggest a role for EAT/mcl-1 in both early placental development in regulating trophoblast differentiation as well as a role for this gene in placental maintenance in regulating the process of trophoblast turnover.
Assuntos
Apoptose/genética , Genes bcl-2 , Proteínas de Neoplasias/genética , Placenta/citologia , Placentação , Proteínas Proto-Oncogênicas c-bcl-2 , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Placenta/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
Jumping translocations (JTs) are unbalanced chromosomal translocations in which an identical chromosomal region is translocated to the telomeric region of different chromosomes. JTs are rare in hematological malignancies where they are second translocations and may be an indicator of poor prognosis. We report a case of acute myeloid leukemia with t(16;21) and a JT in which the long arm of chromosome 1 distal to q21 is translocated to the terminal region of chromosome 10. The leukemic cells exhibit high expression of EAT/mcl1, an anti-apoptotic Bcl-2 related gene. Since EAT/mcl1 is mapped to 1q21 near the breakpoint in the JTs, high level expression of EAT/mcl1 may be associated with the poor prognosis of leukemia with JTs.
Assuntos
Apoptose/genética , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 21/genética , Cromossomos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Translocação Genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Cromossomos/ultraestrutura , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 16/ultraestrutura , Cromossomos Humanos Par 21/ultraestrutura , DNA de Neoplasias/genética , Humanos , Cariotipagem , Leucemia Mieloide Aguda/patologia , Masculino , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/biossíntese , Prognóstico , Telômero/genética , Telômero/ultraestruturaRESUMO
This CPC concerns a 47-year-old male patient with acquired immunodeficiency syndrome (AIDS). The patient became symptomatic when he developed Pneumocystis carinii pneumonia, but recovered sufficiently to be treated as an outpatient. Two years after falling ill, he developed septic shock and died within a short time. During this period, he failed to respond to HIV drugs, and there was no improvement in his immunodeficient status. The HIV retrieved from the patient's organs at autopsy was found to be type E and to have acquired resistance to Zidovudine. It was also possible to determine the route of infection. HIV treatment guidelines are continuously being revised on the basis of HIV research and the development of new treatment plans, and at the present time, when no definitive method of treatment has yet been established, it is essential for the clinician to keep abreast of the latest information. Since HIV patients are compromised hosts, it is important to diagnose and treat other infectious complications, not only complications unique to AIDS, and we have briefly described the latest HIV therapy.