RESUMO
Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.
Assuntos
Proteínas de Fusão bcr-abl/sangue , Ensaio de Proficiência Laboratorial , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ásia , Biomarcadores Tumorais/sangue , Europa (Continente) , Células HL-60/química , Humanos , Células K562/química , Laboratórios Clínicos , Modelos Lineares , América do Norte , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos TestesRESUMO
Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 µM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Indigofera/química , Antineoplásicos/isolamento & purificação , Apoptose , Sobrevivência Celular/efeitos dos fármacos , China , Cromatografia , Biologia Computacional , Humanos , Células K562/química , Células K562/efeitos dos fármacos , Estruturas Vegetais/química , Proto-Oncogene MasAssuntos
Membrana Celular/química , Núcleo Celular/química , Citoplasma/química , Janus Quinase 2/análise , Mutação de Sentido Incorreto , Mutação Puntual , Animais , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Janus Quinase 2/genética , Células K562/química , Células K562/ultraestrutura , Camundongos , Microscopia Confocal , Transtornos Mieloproliferativos/enzimologia , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , TransfecçãoRESUMO
BACKGROUND: Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. METHODS: To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. RESULTS: We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. CONCLUSIONS: This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.
Assuntos
Metilação de DNA/fisiologia , Genes do Retinoblastoma , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/fisiologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Metilação de DNA/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Decitabina , Regulação para Baixo/genética , Genes Reporter , Glioma/patologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Células K562/química , Mutação , Conformação de Ácido Nucleico , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Análise de Sequência de DNA , TransgenesRESUMO
We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.
Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteoma/química , Proteômica/métodos , Humanos , Células K562/química , Piroglutamil-Peptidase I/metabolismo , Tripsina/metabolismoRESUMO
BACKGROUND: Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells. METHODS: Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression. RESULTS: Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression. CONCLUSIONS: The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.
Assuntos
Doxorrubicina/farmacologia , Células K562/química , Proteínas de Neoplasias/análise , Proteômica , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Resistência a Múltiplos Medicamentos , Humanos , Células K562/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estatmina/análiseRESUMO
The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin alpha4beta1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins alpha4beta1, alpha4beta7 and alpha9beta1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported alpha4beta1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin alpha4beta7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with alpha4beta7. All three disintegrin domains supported alpha9beta1-dependent cell adhesion. Recognition by both alpha4beta1 and alpha4beta7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins alpha4beta1, alpha4beta7 and alpha9beta1. ADAM33 exclusively supported only alpha9beta1-dependent adhesion.
Assuntos
Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Linfócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptores de Retorno de Linfócitos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mutation or dysregulation of related homeobox genes occurs in leukemia. Using RT-PCR, we screened members of the EHG family of homeobox genes, comprising EN1 (at 2q14), GBX2 (at 2q36), and EN2, GBX1, and HLXB9 (at 7q36), for dysregulation in acute myeloid leukemia (AML) cell lines indicated by chromosomal breakpoints at these sites. Only one EHG-family gene was expressed, HLXB9, in cell line GDM-1 (AML-M4). Karyotypic analysis of GDM-1 revealed a unique t(6;7)(q23;q35), also present in the patient. Fluorescence in situ hybridization analysis showed chromosomal breakpoints close to the region upstream of HLXB9, at 7q36, a region rearranged in certain AML patients, and at 6q23 upstream of MYB, a gene activated in leukemia. Detailed expression analysis suggested ectopic activation of HLXB9 occurred via juxtaposition with regions upstream of MYB, which was highly expressed in GDM-1. Our data identified a cell line model for a novel leukemic translocation involving MYB with HLXB9, further implicating HLXB9 in leukemogenesis.
Assuntos
Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes myb/fisiologia , Proteínas de Homeodomínio/genética , Leucemia Mielomonocítica Aguda/genética , Fatores de Transcrição/genética , Translocação Genética/genética , Linhagem Celular Tumoral , Análise Citogenética/métodos , Células HL-60/química , Células HL-60/metabolismo , Células HeLa/química , Células HeLa/metabolismo , Proteínas de Homeodomínio/fisiologia , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Proteínas Proto-Oncogênicas c-myb/biossíntese , Fatores de Transcrição/fisiologia , Células U937/química , Células U937/metabolismoRESUMO
This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.
Assuntos
Antígenos de Neoplasias/farmacologia , Células Dendríticas/efeitos dos fármacos , Células K562/química , Antígenos de Neoplasias/isolamento & purificação , Vacinas Anticâncer/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , ImunofenotipagemRESUMO
DNAzymes are nucleic acid enzymes that can recognise specific RNA substrate via Watson-Crick base pairing and cleave it with multiple turnovers. We have designed and examined the effects of DNAzymes targeting the PML/RARalpha fusion gene in acute promyelocytic leukaemia (APL). The DNAzymes (DZ1 and DZ3) were designed to cleave the PML/RARalpha transcript at the GC nucleotides at the fusion point and three nucleotides upstream of that respectively. Disabled DNAzymes were synthesised and used as controls. Cell-free cleavage reactions were performed on total RNA from NB4 cell line and PML/RARalpha and RARalpha-amplified RNA fragments (aRNA). Postcleavage examination showed that DZ1 and DZ3 cleave PML/RARalpha efficiently and specifically. NB4 APL cells transfected with DZ1 or DZ3 showed a significant suppression of PML/RARalpha protein expression. These DNAzymes also inhibited the proliferation of NB4 cells, reduced the viability rate, and induced apoptosis in these cells. The disabled DNAzymes showed no effect on NB4 cells. The two DNAzymes did not produce any significant effect on K562 cells, which were used as control cells. DNAzymes are more resistant to serum than ribozymes. These data show that targeting the PML/RARalpha fusion gene with DNAzymes can induce apoptosis in APL cells and may have a role in the treatment of APL. They also show DNAzymes are promising tools for targeting specific genes in leukaemia.
Assuntos
DNA Catalítico/farmacologia , Leucemia Promielocítica Aguda/patologia , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/antagonistas & inibidores , RNA Neoplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pareamento de Bases , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/efeitos dos fármacos , Sistema Livre de Células , DNA Catalítico/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Marcação de Genes , Humanos , Hidrólise , Células K562/química , Células K562/efeitos dos fármacos , Lipossomos/administração & dosagem , Proteínas de Neoplasias/antagonistas & inibidores , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/antagonistas & inibidores , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Transfecção , Tretinoína/farmacologiaRESUMO
The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.
Assuntos
Genoma , Nanotecnologia/métodos , Proteínas de Bactérias/genética , Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Cromossomos Humanos/química , Cromossomos Humanos/genética , Clostridium perfringens/genética , Biologia Computacional/métodos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Genoma Bacteriano , Genoma Humano , Humanos , Fatores Hospedeiros de Integração/deficiência , Fatores Hospedeiros de Integração/genética , Células K562/química , Células K562/metabolismo , Microscopia de Força Atômica/métodos , Mitose/genética , Especificidade da Espécie , Staphylococcus aureus/genéticaRESUMO
A large amount of metaphase chromosomes were isolated from synchronized human cell lines by a polyamine procedure. All the chromosomal proteins extracted by an acetic acid extraction method were fully dissolved into the sample solutions for isoelectric focusing (IEF) or radical free and highly reduced (RFHR) two-dimensional electrophoreses (2-DEs). As a result, well-separated and highly reproducible 2-DE patterns were obtained. This could not be attained by an ordinary acetone precipitation method. The 2-DE patterns visualized using Coomassie Brilliant Blue (CBB) staining indicated that more than one hundred proteins were involved in the isolated metaphase chromosomes, although the most abundant proteins, histones, occupied a greater part of the chromosomal proteins. It was also shown that colcemid treatment for cell cycle synchronization had little effect on the 2-DE pattern compared to that obtained without the treatment. Furthermore, no significant differences were observed in the 2-DE patterns among the chromosomal proteins prepared from two different human cell lines, BALL-1 and K562. However, 2-DE analysis of isolated metaphase chromosomes from HeLa cells apparently showed a smaller number of proteins than the BALL-1 and K562 cell lines at a neutral pI range. The present study paves the way for elucidating protein composition of human metaphase chromosomes.
Assuntos
Cromossomos Humanos/química , Cromossomos Humanos/genética , Eletroforese em Gel Bidimensional/métodos , Metáfase/genética , Nucleoproteínas/metabolismo , Linhagem Celular Tumoral , Células HeLa/química , Células HeLa/metabolismo , Histonas/metabolismo , Humanos , Focalização Isoelétrica/métodos , Células K562/química , Células K562/metabolismo , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
NALP1 (also called DEFCAP, NAC, CARD7) has been shown to play a central role in the activation of inflammatory caspases and processing of pro-IL1b (pro-interleukin-1b). Previous studies showed that NALP1 is highly expressed in peripheral blood mononuclear cells. In the present study, we report that expression of NALP1 is absent from CD34+ haematopoietic blast cells, and its levels are upregulated upon differentiation of CD34+ cells into granulocytes and to a lesser extent into monocytes. In peripheral blood cells, the highest levels of NALP1 were observed in CD3+ (T-lymphocytes), CD15+ (granulocytes) and CD14+ (monocytes) cell populations. Notably, the expression of NALP1 was significantly increased in the bone marrow blast cell population of some patients with acute leukaemia, but not among tissue samples from thyroid and renal cancer. A search for consensus sites within the NALP1 promoter revealed a sequence for CREB (cAMP-response-element-binding protein) that was required for transcriptional activity. Moreover, treatment of TF1 myeloid leukaemia cells with protein kinase C and protein kinase A activators induced CREB phosphorylation and upregulated the mRNA and protein levels of NALP1. Conversely, ectopic expression of a dominant negative form of CREB in TF1 cells blocked the transcriptional activity of the NALP1 promoter and significantly reduced the expression of NALP1. Thus NALP1 is transcriptionally regulated by CREB in myeloid cells, a mechanism that may contribute to modulate the response of these cells to pro-inflammatory stimuli.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Leucemia Mieloide/genética , Transcrição Gênica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Reguladoras de Apoptose , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562/química , Células K562/metabolismo , Células K562/patologia , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Dados de Sequência Molecular , Proteínas NLR , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: Some abnormalities in porphyrin metabolism have been described in erythrocytes from patients with end-stage renal failure. A peptidic fraction of 56.2 kD isolated from plasma of these patients was previously identified as an aminolevulinate dehydratase inhibitor. The aim of this study was to examine the in vitro effect of this peptide on heme synthesis in the erythroleukemia K562 cells. METHODS: The 56.2-kD fraction was purified from uremic plasma by protein electroelution and, its action on the mitochondrial rate-limiting steps of heme synthesis, as well as the hemoglobin content during erythroid differentiation induced by sodium butyrate, was investigated in K562 cells. RESULTS: Two hours after addition of the 56.2-kD peptide, the activities of aminolevulinate acid synthase and aminolevulinate dehydratase were reduced while the activity of the ferrochelatase was enhanced, indicating that this peptide easily across the membranes. A 3-day incubation with this peptide enhanced approximately twofold the hemoglobin and porphyrin levels during erythroid differentiation of K562 cells without variation of cell growth. CONCLUSION: This study shows that the addition of the 56.2-kD uremic factor to K562 cells was clearly implicated in heme disturbances existing in chronic renal failure but it did not play a negative role in the pathogenesis of the uremic anemia.
Assuntos
Hemoglobinas/análise , Falência Renal Crônica/sangue , Peptídeos/sangue , Uremia/sangue , Células Cultivadas , Eritrócitos/enzimologia , Humanos , Células K562/química , Sintase do Porfobilinogênio/antagonistas & inibidores , Sintase do Porfobilinogênio/fisiologiaRESUMO
Leukemias are considered malignant clonal disorders arising from the accumulation of mutations in hematopoietic cells; the majority of these mutations are thought to be acquired somatically. Measurement of mutation frequency (Mf) at the hypoxanthine phosphoribosyltransferase (HPRT) locus has been developed as a method for estimating genomic instability. We investigated the Mf in 16 leukemic cell lines to determine whether these cell lines showed evidence of genomic instability. Although some leukemic cell lines had markedly elevated Mfs, the Mfs at the HPRT locus in leukemic cell lines were not always higher than those of B-lymphoblastoid cell lines and T lymphocytes from normal individuals. We were able to identify the HPRT mutation for 159 of 160 individual HPRT mutants. The HPRT mutations were characterized at a molecular level and classified as either gross chromosomal rearrangements (GCRs) or point mutations, such as single-nucleotide substitutions, insertions, or deletions. With rare exceptions, individual leukemic cell lines showed either point mutations or GCR, but not both. Of note, all the cell lines that primarily showed point mutations are known to be defective in mismatch repair machinery.
Assuntos
Hipoxantina Fosforribosiltransferase/genética , Leucemia/genética , Mutação/genética , Adolescente , Adulto , Idoso , Linfócitos B/química , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Marcadores Genéticos/genética , Instabilidade Genômica/genética , Células HL-60/química , Células HL-60/metabolismo , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Leucemia/patologia , Masculino , Mutagênese/genética , Projetos Piloto , Splicing de RNA/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Recombinação Genética/genética , Células U937/química , Células U937/metabolismoRESUMO
The t(9;11) has been described in patients with acute myeloid leukemia (AML), and two genes [AF9 (at 9p21) and FBP17 (at 9q34)] have been cloned as fusion partners of the MLL gene. From an AML-M5 with a t(9;11)(q34;q23), we identified a novel MLL fusion partner, AF9Q34. The AF9Q34 protein shows high homology with nGAP, a RAS GTPase-activating protein (RASGAP), and contains the highly conserved GRD and FLR motifs characteristic of RASGAPs. Recently, the rat homologue (DAB2IP) also was identified and reported to act as a RASGAP both in vivo and in vitro. RASGAPs negatively regulate the activity of RAS proteins that modulate diverse cellular processes by cycling between an inactive GDP-bound and an active GTP-bound state. In addition, the NH(2) terminus harbors an amino acid stretch with homology to the pleckstrin homology (PH) domain implicated in regulating the interaction between RAS and the catalytic domain of RASGAP. As a result of the breakpoint in the AF9Q34-MLL fusion protein, this PH domain is disrupted. This suggests that because of the translocation, the normal function of the AF9Q34 gene is aborted. Thus, AF9Q34 encodes a novel RASGAP gene that appears to be deregulated as a result of the translocation. The identification of this RASGAP protein in a novel MLL fusion implies that an indirect RAS-deregulating mechanism could be involved in leukemic transformation.
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Monocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proto-Oncogenes , Fatores de Transcrição , Proteínas Ativadoras de ras GTPase/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Quebra Cromossômica/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 9/genética , Proteínas de Ligação a Ácido Graxo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HL-60/química , Células HL-60/metabolismo , Células HeLa/química , Células HeLa/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Especificidade de Órgãos/genética , Translocação Genética/genética , Células U937/química , Células U937/metabolismoRESUMO
In this study, the human multienzyme aminoacyl-tRNA synthetase "core" complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three-dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl-tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three-dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.
Assuntos
Aminoacil-tRNA Sintetases/química , Núcleo Celular/química , Citocinas/química , Citoplasma/química , Proteínas de Neoplasias/química , Proteínas de Ligação a RNA/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/ultraestrutura , Animais , Fracionamento Celular , Cromatografia Líquida de Alta Pressão , Citocinas/isolamento & purificação , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Células K562/química , Células K562/enzimologia , Cinética , Microscopia Eletrônica , Modelos Moleculares , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Coelhos , Homologia Estrutural de ProteínaRESUMO
DNA microarrays produced by deposition (or 'spotting')of a single long oligonucleotide probe for each gene may be an attractive alternative to other types of arrays. We produced spotted oligonucleotide arrays using two large collections of approximately 70-mer probes, and used these arrays to analyze gene expression in two dissimilar human RNA samples. These samples were also analyzed using arrays produced by in situ synthesis of sets of multiple short (25-mer) oligonucleotides for each gene (Affymetrix GeneChips). We compared expression measurements for 7344 genes that were represented in both long oligonucleotide probe collections and the in situ-synthesized 25-mer arrays. We found strong correlations (r = 0.8-0.9) between relative gene expression measurements made with spotted long oligonucleotide probes and in situ-synthesized 25-mer probe sets. Spotted long oligonucleotide arrays were suitable for use with both unamplified cDNA and amplified RNA targets, and are a cost-effective alternative for many functional genomics applications. Most previously reported evaluations of microarray technologies have focused on expression measurements made on a relatively small number of genes. The approach described here involves far more gene expression measurements and provides a useful method for comparing existing and emerging techniques for genome-scale expression analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular , Biologia Computacional/métodos , Biologia Computacional/estatística & dados numéricos , Perfilação da Expressão Gênica/estatística & dados numéricos , Genes/genética , Humanos , Células K562/química , Células K562/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Sondas de Oligonucleotídeos/genética , Sensibilidade e EspecificidadeRESUMO
The interaction of the alpha(5)beta(1) integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the alpha(5)beta(1)/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between alpha(5)beta(1) and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an alpha(5)beta(1) expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the alpha(5)beta(1)/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual alpha(5)beta(1)/FN7-10 interactions. The dynamic rupture force of the alpha(5)beta(1)/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the alpha(5)beta(1)/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.
Assuntos
Fibronectinas/química , Integrina alfa4beta1/química , Microscopia de Força Atômica/métodos , Elasticidade , Fibronectinas/metabolismo , Humanos , Integrina alfa4beta1/metabolismo , Células K562/química , Células K562/metabolismo , Substâncias Macromoleculares , Micromanipulação/métodos , Modelos Químicos , Movimento (Física) , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estresse Mecânico , Resistência à Tração , Suporte de CargaRESUMO
OBJECTIVE: We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity. MATERIALS AND METHODS: Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose. RESULTS: The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism. CONCLUSION: The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.