RESUMO
Background: The incidence of noncommunicable diseases (NCDs) has been rapidly ramped up worldwide. Hence, there is an urgent need to non-invasively detect NCDs possibly by exploiting saliva as a 'liquid biopsy' to identify biomarkers of the health status. Since, the absence of standardized procedures of collection/analysis and the lack of normal ranges makes the use of saliva still tricky, our purpose was to outline a salivary proteomic profile which features healthy individuals. Methods: We collected saliva samples from 19 young blood donors as reference population and the proteomic profile was investigated through mass-spectrometry. Results: We identified 1,004 proteins of whose 243 proteins were shared by all subjects. By applying a data clustering approach, we found a set of six most representative proteins across all subjects including Coronin-1A, F-actin-capping protein subunit alpha, Immunoglobulin J chain, Prosaposin, 78 kDa glucose-regulated protein and Heat shock 70 kDa protein 1A and 1B. Conclusion: All of these proteins are involved in immune system activation, cellular stress responses, proliferation, and invasion thus suggesting their use as biomarkers in patients with NCDs.
RESUMO
Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Glicoproteínas/metabolismo , Humanos , Camundongos , Células-Tronco Neoplásicas/citologia , Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transplante de Células-TroncoRESUMO
Neural stem cells appear to be best suited for regenerative therapy in neurological diseases. However, the effects of high levels of potentially toxic substances such as sulfatides--which accumulate in metachromatic leukodystrophy (MLD)--on this regenerative ability are still largely unclear. To start addressing this question, in vitro and in vivo experiments were used to examine the behavior of multipotential neural precursors exposed to abnormally high levels of sulfatides. Following transplantation of dissociated neurospheres into the brain of presymptomatic MLD pups, the majority of donor-derived cells were distributed in a caudal to rostral direction, with higher numbers in the cortex. Most if not all of the donor cells acquired an astroglial phenotype. We found no evidence of oligodendrocyte or neuronal commitment of transplanted cells in long-term-treated MLD mice (e.g. up to 1.5 years of age). This was in line with our in vitro findings of sulfatides blocking oligodendrocyte formation after induction of differentiation in sulfatide-treated epidermal growth factor/fibroblast growth factor responsive neurospheres. Transplanted MLD mice showed an improved arylsulfatase A (ARSA) activity and a significant amelioration of sulfatide metabolism, neurodegeneration and motor-learning/memory deficits. Furthermore, transplanted cells were shown to act as a source of ARSA enzyme that accumulated in endogenous brain cells, indicating the occurrence of enzyme cross-correction between transplanted and host cells. These results provide a first insight into the effect of sulfatides on the stemness properties of neural stem cells and on the effects of the MLD environment on the in vivo expectations of using neural stem cells in cell therapy.
Assuntos
Encéfalo , Leucodistrofia Metacromática , Neurônios/fisiologia , Oligodendroglia/fisiologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/fisiopatologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leucodistrofia Metacromática/fisiopatologia , Leucodistrofia Metacromática/terapia , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Neurônios/citologia , Oligodendroglia/citologia , Células-Tronco/citologia , Sulfoglicoesfingolipídeos/metabolismoAssuntos
Antineoplásicos Fitogênicos/farmacologia , Paclitaxel/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Transformada , Códon , Resistencia a Medicamentos Antineoplásicos/genética , Heterozigoto , Humanos , Camundongos , Mutação , Tolerância a Radiação/genética , Células Tumorais CultivadasRESUMO
The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.
Assuntos
Apoptose/genética , Ciclo Celular/genética , Síndrome de Li-Fraumeni/genética , Mutação , Proteína Supressora de Tumor p53/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/efeitos da radiação , Ciclo Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Ciclinas/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Fase G1/genética , Fase G2/genética , Fase G2/efeitos da radiação , Raios gama , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Síndrome de Li-Fraumeni/metabolismo , Linfócitos/patologia , Linfócitos/efeitos da radiação , Mitose/genética , Mitose/efeitos da radiação , Fosforilação , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Chemokines are a bipartite family of chemotactic proteins that bear the structural hallmark of four cysteine residues, the first two of which are in tandem. The spectrum of action of C-C chemokines, monocyte chemotactic protein-1 (MCP-1), MCP-2, and MCP-3, in particular, encompasses, in addition to monocytes, other leukocyte populations. Evidence is presented that MCP-1, MCP-2, and MCP-3 are active on natural killer cells. Available information on receptor usage by MCP-1 and related chemokines and signal transduction pathways is reviewed. A better understanding of signaling mechanisms will provide a new basis for therapeutic strategies.
Assuntos
Fatores Quimiotáticos/fisiologia , Citocinas/fisiologia , Receptores de Citocinas/fisiologia , Transdução de Sinais , Cálcio/fisiologia , Quimiocina CCL2 , Quimiotaxia , Humanos , Monócitos/fisiologia , Proteínas RecombinantesRESUMO
The possible role of protein kinase C in cholesterol-induced resistance to ether lipids was investigated. The enrichment of HL60 cells in cholesterol (CHOL) (HL60-CHOL) resulted in a significant increase in the ID50 values for 1-octadecyl-2-methyl-rac-glycero- 3-phosphocholine (ET-18-OMe) (3.75 +/- 0.7 microM and 6.69 +/- 0.5 microM for HL60 and HL60-CHOL, respectively). In the same conditions, HL60 and HL60-CHOL cells showed comparable levels of both cytosolic and membrane-associated protein kinase C activity. Phorbol ester (PMA) stimulation induced protein kinase C to translocate from the cytosol to the plasma membrane in both cell types and with similar kinetics (272 +/- 32% and 299 +/- 41% increase in HL60 and HL60-CHOL, respectively after 100 ng/ml PMA for 10 min). Pretreatment of the two cell types with 50 microM ET-18-OMe resulted in comparable levels of PKC inhibition after phorbol ester stimulation. These results suggested that alterations in plasma membrane lipid composition induced by CHOL do not result in major changes in protein kinase C activity. Thus, protein kinase C does not appear to be involved in cholesterol-induced resistant phenotype in HL60 cells.
Assuntos
Colesterol/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Lipídeos de Membrana/metabolismo , Éteres Fosfolipídicos/farmacologia , Proteína Quinase C/metabolismo , Resistência a Medicamentos , Humanos , Ésteres de Forbol/farmacologia , Células Tumorais CultivadasRESUMO
Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1-Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer.
Assuntos
Apoptose/fisiologia , Mitocôndrias/fisiologia , Proteínas Quinases/fisiologia , Proteína bcl-X/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Autofagia/fisiologia , Proteína Beclina-1 , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células HEK293 , Humanos , Proteínas de Membrana/fisiologia , Fosforilação/fisiologia , Transdução de Sinais/fisiologiaAssuntos
Patógenos Transmitidos pelo Sangue , Vacinas contra Hepatite B/imunologia , Controle de Infecções Dentárias/métodos , United States Occupational Safety and Health Administration , Recursos Humanos em Odontologia/educação , Resíduos Odontológicos , Humanos , Eliminação de Resíduos de Serviços de Saúde , New Jersey , Exposição Ocupacional/prevenção & controle , Roupa de Proteção , Estados UnidosRESUMO
In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Gangliosídeo G(M3)/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Megacariócitos/enzimologia , Proteínas de Neoplasias/biossíntese , Neuraminidase/biossíntese , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Gangliosídeo G(M3)/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Neoplasias/genética , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
BACKGROUND AND OBJECTIVE: Clozapine is a diabenzodiazepine derivative characterized by a high therapeutic index in schizophrenic patients resistant to traditional neuroleptic drugs, because of the rarity of any extrapyramidal side effects, and its particular hematologic toxicity. According to the international literature, clozapine-induced neutropenia occurs mainly during the first 4-6 months of treatment, and its incidence decreases considerably over time. This neutropenic effect is not dose-dependent and normally clears up after drug discontinuation, although it may evolve into agranulocytosis. The aim of this study is to evaluate the in vitro toxic effect of clozapine and N-desmethylclozapine on both committed and immature human hematopoietic progenitor cells. DESIGN AND METHODS: Cytotoxic assays were performed in vitro on normal human bone marrow samples treated with clozapine or with its metabolite N-desmethylclozapine. The clonogenic potential after treatment with both compounds was assessed on low density mononuclear cells (LD-MNC), purified CD34+ cells, cytokine driven liquid cultures and long term culture initiating cell (LTC-IC). RESULTS: Clozapine and N-desmethylclozapine had a dose-dependent inhibitory effect on in vitro growth of CFU-GM and BFU-E from normal bone marrow. The two drugs had toxic effects on purified CD34+ progenitor cells but no significant effect on LTI-IC. INTERPRETATIONS AND CONCLUSIONS: Our data indicate a cytotoxic effect, which is more pronounced with N-desmethylclozapine and at high doses, on the committed progenitor cell compartment but not on primitive hematopoietic cells. Furthermore, our data show that clozapine and N-desmethylclozapine have a direct effect on treated cells and do not induce apoptotic death.
Assuntos
Antipsicóticos/toxicidade , Clozapina/análogos & derivados , Clozapina/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Antipsicóticos/efeitos adversos , Células Cultivadas , Clozapina/efeitos adversos , Ensaio de Unidades Formadoras de Colônias , Humanos , Esquizofrenia/tratamento farmacológicoRESUMO
The effect of the increasing degree of human gingival inflammation on adenylate cyclase (basal, fluoride stimulated) and low Km and high Km cAMP phosphodiesterase activities were evaluated in separate studies. Human gingival biopsies were classified by the Löe Bleeding Index as mildly, moderately, and markedly inflamed. Basal and F- stimulated adenylate cyclase (cAMP synthesis) activities were found to be unaltered by the increasing degree of inflammation when the data were expressed on either a mg wet wt, or mg protein basis. A significant loss of F- stimulated adenylate cyclase (mg protein) activity was observed in the moderately inflamed group when the data were compared with either the mildly or markedly inflamed groups of tissue. The low Km, and high Km cAMP phosphodiesterase activities (cAMP degradation) were found to be unaffected by gingival inflammation. This suggests that neither cAMP synthesis, nor degradation are stimulated in human gingiva by inflammation.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Gengivite/enzimologia , Humanos , Masculino , Índice PeriodontalRESUMO
Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis.
Assuntos
Quimiocina CCL2/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Fosfolipases A/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Sequência de Bases , Complemento C5a/farmacologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ácido Oleico , Ácidos Oleicos/metabolismo , Oligonucleotídeos Antissenso/genética , Fosfolipases A/genética , Fosfolipases A/fisiologia , Fosfolipases A2 , FosforilaçãoRESUMO
We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34+ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-alpha, TGF-beta and Flt-3 ligand (fold increase of CD1a+ cells = 102 +/- 32 after 2 weeks of culture). CD34+ cells were also grown under continuous flow conditions in an artificial capillary system: after 14d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 +/- 2 v 18.9 +/- 4) but the percentage of CD1a+/CD83+/ CD80+ cells was considerably higher, whereas the CD14+ cells were significantly reduced (8.9 +/- 2 v 26 +/- 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a+/S100+/ CD8 3+/CD80+/CD14- 'large cells' with great internal complexity (mature DCs); the second including 'small cells' either CD33+/CD14+, CD33+/CD15+ or CD33+/CD13-/CD14. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.
Assuntos
Antígenos CD34 , Técnicas de Cultura de Células/métodos , Citocinas/farmacologia , Células Dendríticas/citologia , Divisão Celular/fisiologia , Combinação de Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Imuno-Histoquímica , Imunofenotipagem , Proteínas de Membrana/farmacologia , Microscopia Eletrônica , Perfusão , Fator de Células-Tronco/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin-dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre-treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34(+) bone marrow progenitors because the 50% growth inhibition of colony-forming unit granulocyte macrophage (CFU-GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and of beta-catenin, and was antagonized by ectopic expression of Bcl-2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl-2, a decrease of Bax but no changes in Bcl-X(L) protein expression at any time point. In Ph(+) cell lines BCR-ABL protein was only down-regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti-tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.
Assuntos
Apoptose/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Transativadores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Linhagem Celular Transformada , Cisplatino/administração & dosagem , Proteínas do Citoesqueleto/análise , Relação Dose-Resposta a Droga , Proteínas de Fusão bcr-abl , Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Genes p53 , Células HL-60/efeitos dos fármacos , Células HL-60/ultraestrutura , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Leucemia Mieloide/patologia , Glicoproteínas de Membrana/análise , Microscopia Eletrônica , Paclitaxel/administração & dosagem , Fatores de Tempo , beta CateninaRESUMO
The A-Myb transcription factor is structurally related to the c-myb proto-oncogene and is involved in the control of proliferation and/or differentiation of mature B lymphocytes. We have shown previously by PCR analysis that A-myb is preferentially expressed in CD38+ CD39- sIgM- mature B cells. We demonstrate here, using in situ hybridization, that A-myb expression is restricted to the dark zone of human tonsils and lymph nodes. Furthermore, we show that A-Myb expression is cell cycle regulated both in tonsillar B cells and in Burkitt's lymphoma cell lines, being detectable only in the S and G2/M phases of the cell cycle and not in G0/G1 phase. Strong proliferation of resting human B cells induced in vitro by a variety of physiologic signals, including anti-mu, CD40 ligand, IL-2, IL-4, IL-6, IL-13, IFN-gamma, TNF-alpha, anti-CD19, and anti-CD20, failed to induce A-myb expression, suggesting that proliferation alone is not sufficient for A-myb expression in the absence of induction of a true centroblast phenotype. Finally, we show that differentiation of germinal center B cells in vitro toward either memory or plasma cells is accompanied by rapid down-regulation of A-myb expression. We conclude that A-myb is a marker of centroblasts generated in vivo.