Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30
Filtrar
1.
Blood ; 123(12): 1850-9, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24470590

RESUMO

Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.


Assuntos
Linfoma Cutâneo de Células T/enzimologia , Neoplasias Cutâneas/enzimologia , Telomerase/fisiologia , Homeostase do Telômero/fisiologia , Animais , Estudos de Casos e Controles , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Xenoenxertos , Humanos , Linfoma Anaplásico de Células Grandes/enzimologia , Linfoma Anaplásico de Células Grandes/genética , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Masculino , Camundongos , Camundongos SCID , Micose Fungoide/enzimologia , Micose Fungoide/genética , Transplante de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Síndrome de Sézary/enzimologia , Síndrome de Sézary/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Telomerase/antagonistas & inibidores , Telomerase/genética , Homeostase do Telômero/genética
2.
Anal Chem ; 81(23): 9590-8, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19873978

RESUMO

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.


Assuntos
Corantes Fluorescentes/metabolismo , Espaço Intracelular/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células , Descoberta de Drogas , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Imagem Molecular , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/química , Proteína X Associada a bcl-2/metabolismo
3.
Exp Cell Res ; 314(20): 3701-11, 2008 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-18930044

RESUMO

In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation.


Assuntos
Ciclo Celular/genética , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/genética , Glioma/genética , Biossíntese de Proteínas/genética , Animais , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos SCID , Peso Molecular , Isoformas de Proteínas/genética , Ratos , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Exp Hematol ; 36(12): 1648-59, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18922616

RESUMO

OBJECTIVE: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. MATERIALS AND METHODS: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. RESULTS: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. CONCLUSION: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Citarabina/farmacologia , Diterpenos/farmacologia , Idarubicina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fenantrenos/farmacologia , Anexina A5/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Citarabina/agonistas , Citarabina/uso terapêutico , Diterpenos/agonistas , Diterpenos/uso terapêutico , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Compostos de Epóxi/agonistas , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Humanos , Proteínas I-kappa B/metabolismo , Idarubicina/agonistas , Idarubicina/uso terapêutico , Leucemia Mieloide Aguda/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Fenantrenos/agonistas , Fenantrenos/uso terapêutico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
5.
Cancer Med ; 8(11): 5173-5182, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31350815

RESUMO

PURPOSE: To assess the incidence of BCR-ABL kinase domain (KD) mutation detection and its prognostic significance in chronic phase chronic myeloid leukemia (CP-CML) patients treated with tyrosine kinase inhibitors (TKIs). PATIENTS AND METHODS: We analyzed characteristics and outcome of 253 CP-CML patients who had at least one mutation analysis performed using direct sequencing. Of them, 187 patients were early CP (ECP) and 66 were late CP late chronic phase (LCP) and 88% were treated with Imatinib as first-line TKI. RESULTS: Overall, 80 (32%) patients harbored BCR-ABL KD mutations. A BCR-ABL KD mutation was identified in 57% of patients, who progressed to accelerated or blastic phases (AP-BP), and 47%, 29%, 35%, 16% and 26% in patients in CP-CML at the time of mutation analysis who lost a complete hematologic response, failed to achieve or loss of a prior complete cytogenetic and major molecular response, respectively. Overall survival and cumulative incidence of CML-related death were significantly correlated with the disease phase whatever the absence or presence of a mutation was and for the latter the mutation subgroup (T315I vs P-loop vs non-T315I non-P-loop) (P<.001). Considering patients who were in CP at the time of mutation analysis, LCP mutated patients had a significantly worse outcome than ECP-mutated patients despite a lower incidence of T315I and P-loop mutations (P<.001). With a median follow-up from mutation analysis to last follow-up of 5 years, T315I and P-loop mutations were not associated with a worse outcome in ECP patients (P = .817). CONCLUSION: Our results suggest that early mutation detection together with accessibility to 2nd and 3rd generation TKIs have reversed the worst outcome associated with BCR-ABL KD mutations whatever the mutation subgroup in CP-CML patients.


Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Análise de Sobrevida , Adulto Jovem
6.
Hemasphere ; 2(3): e41, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31723769

RESUMO

Dasatinib is an ABL1 tyrosine kinase inhibitor (TKI) with a short in vivo plasmatic half-life but with good efficiency, which is not fully understood. We investigated the possibility that circulating erythrocytes store and then provide dasatinib to target cells. In vitro coincubation of dasatinib-treated cells with naïve leukemic cells followed by analysis of kinase inhibition, apoptosis induction, fluorescent molecule exchanges, and dasatinib dosage were performed. Cells incubated with clinically relevant concentrations of dasatinib for a short time retained, after a washout procedure, an intracellular pool of dasatinib which was transferable to naïve BCR-ABL1 expressing cells and induced their apoptosis. This was verified in total blood where the huge cellular volume of erythrocytes constituted a large reservoir of dasatinib able to induce apoptosis in naïve BCR-ABL1 cell lines and primitive chronic myeloid leukemia (CML) CD34+ cells. This dasatinib transfer necessitated a contact between donor and acceptor cells. A component exchange occurred during this contact, carrying dasatinib and other TKIs such as nilotinib or the fluorescent sunitinib. An active pool of dasatinib could be buried inside the circulating erythrocytes, out of reach of detoxifying mechanisms, but still available for target cells and thus extending the acute effect of the plasmatic pool of the drug.

7.
Cancer Biol Ther ; 6(4): 603-11, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17374988

RESUMO

Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p < 0.01). Isobologram analysis showed that both MG132 and bortezomib interacted synergistically with IDA to induce apoptosis of THP1 cells. Western blot analysis of Bax and Bim show an acumulation of these pro-apoptotic proteins in THP1 treated cells. This increase in Bim preceded the induction of apoptosis and participated in idarubicin-induced apoptosis. Proteasome inhibition also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no such effect was found on normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells or bone marrow normal myeloid cells. These data show that MG132 and bortezomib specifically sensitize leukemic cells to IDA through an increase in BIM and Bax pro-apoptotic Bcl-2 family proteins.


Assuntos
Antraciclinas/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/metabolismo , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Proteína 11 Semelhante a Bcl-2 , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Feminino , Humanos , Idarubicina/farmacologia , Leupeptinas/farmacologia , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Células Tumorais Cultivadas
8.
Leuk Res ; 61: 44-52, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28888102

RESUMO

Tyrosine kinase inhibitors (TKI) constitute the frontline treatment for chronic myeloid leukemia patients. Dasatinib, a second-generation TKI, was developed to overcome TKI resistances. However, dasatinib resistances are also described but remain less characterized. To mimic in vivo acquired dasatinib resistance, the BCR-ABL1-positive cell line K562 was transiently treated with a pharmacological concentration of dasatinib, for a short time in the presence of stem cell factor. A dasatinib resistant counterpart (K562 RES) was developed. Investigation of resistance mechanisms using kinase substrate arrays revealed that FYN was overactivated in K562 RES. The FYN inhibitor KX2-391 cooperated with dasatinib to block K562 RES proliferation. Cell tracking experiments showed that activated FYN support cell proliferation independently of BCR-ABL1 in K562 RES cells. Moreover, the MEK-ERK pathway was found hyper-phosphorylated in K562 RES cells even in the presence of dasatinib. Actually, ERK1/2 activity supported viability in K562 RES only in the absence of BCR-ABL1 activity. Finally, BCR-ABL1 and MEK inhibitor combination was sufficient to induce cell death even in non-proliferating resistant cells. Considering the conditions used to generate this dasatinib resistant cell line, such a resistance mechanism could be found in dasatinib treated patients. Consequently, it is valuable to know that inhibition of the MEK-ERK1/2 axis can overcome this resistance.


Assuntos
Antineoplásicos/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Proteínas de Fusão bcr-abl/genética , Humanos , Hibridização in Situ Fluorescente , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fyn/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/metabolismo
9.
Oncotarget ; 7(1): 845-59, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26625317

RESUMO

In spite of intensive research to improve treatment of acute myeloid leukemia (AML) more than half of all patients continue to develop a refractory disease. Therefore there is need to improve AML treatment. The overexpression of the BCL-2 family anti-apoptotic members, like BCL-2 or BCL-xL has been largely reported in lymphoid tumors but also in AML and other tumors. To counteract the anti-apoptotic effect of BCL-2, BH3 mimetics have been developed to target cancer cells. An increase in activity of ERK1/2 mitogen activated protein (MAP) kinase has also been reported in AML and might be targeted by MEK1/2 inhibitors. Hence, in the current work, we investigated whether the association of a BH3 mimetic such ABT-263 and the MEK1/2 inhibitor pimasertib (MEKI), was efficient to target AML cells. A synergistic increasing of apoptosis was observed in AML cell lines and in primary cells without affecting normal bone marrow cells. Such cooperation was confirmed on tumor growth in a mouse xenograft model of AML. In addition we demonstrated that MEKI sensitized the cells to apoptosis through its ability to promote a G1 cell cycle arrest. So, this combination of a MAP Kinase pathway inhibitor and a BH3 mimetic could be a promising strategy to improve the treatment of AML.


Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Niacinamida/análogos & derivados , Sulfonamidas/farmacologia , Doença Aguda , Compostos de Anilina/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HL-60 , Humanos , Imuno-Histoquímica , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Oncol ; 26(3): 825-34, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15703842

RESUMO

The ability to induce apoptosis in tumor cells is critical to elicit a positive response to cytotoxic chemo-therapy. In this study, we investigated the effect of the topoisomerase I inhibitors camptothecin and SN-38, known to cause an unusual form of DNA damage, on apoptotic pathways using the leukemic cell line HL-60 and its vincristine-resistant variant HL-60 VCR. Both camptothecin and SN-38 induced high levels of apoptosis in sensitive cells when compared to the multidrug-resistant ones. Interestingly, a higher BCL-2/BAX ratio was observed in HL-60 VCR at the basal state and during treatments. Moreover, these cells which did not exhibit Bcr-abl translocation or bcrp efflux pump, overexpressed topoisomerase I protein. The data provide evidence that BCL-2 protein could protect HL-60 VCR from mitochondrial membrane depolarization and block ROS production in these cells. Finally, our results suggest that dysregulation of proteins associated with DNA replication and apoptotic process could contribute to the multidrug-resistance phenotype.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Dano ao DNA , Resistência a Múltiplos Medicamentos , Replicação do DNA , Células HL-60 , Humanos , Irinotecano , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcr , Translocação Genética
12.
Exp Hematol ; 30(1): 67-73, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11823039

RESUMO

OBJECTIVE: The aim of this study was to determine whether the combination of a sizable generation of colony-forming cells (CFC) with the maintenance of their progenitors (pre-CFC) ensured by incubation in hypoxia is associated with a certain degree of cell cycling, ultimately responsible for "self-renewal" of pre-CFC. The effects of interleukin-3 (IL-3) on the cycling and CFC-generation potential of pre-CFC also was investigated. MATERIALS AND METHODS: In severely hypoxic (0.9% O(2)) murine bone marrow cell cultures containing stem cell factor, interleukin-6, and granulocyte colony-stimulating factor, pre-CFC maintenance was characterized by the culture-repopulating ability assay, an in vitro analogue of the marrow-repopulating ability assay. The proliferative history of CD34(+) cells in primary cultures was determined by PKH2 staining and related to their CFC-generation potential. In some experiments, subsets of CD34(+) cells sorted on the basis of the number of cell divisions (0, 1, >1) were independently characterized. RESULTS: In hypoxia, the numbers of CFC and CD34(+) cells were markedly reduced, whereas pre-CFC were maintained better than in air. Addition of 5-fluorouracil to hypoxic cultures for 2 days suppressed their CFC-generation potential. The CFC-generation potential of divided CD34(+) cells was maintained or increased with respect to that of undivided cells in hypoxia but not in air. IL-3 decreased CFC-generation potential at both oxygen concentrations. IL-3 also increased the number of CD34(+) cells that divided more than once in hypoxia, decreasing their CFC-generation potential. CONCLUSIONS: Maintenance of CFC-generation potential in hypoxia occurs mainly in a subset of cycling progenitors, despite their proliferation (self-renewal). IL-3 decreased CFC-generation potential by increasing the rate of pre-CFC proliferation beyond the first cycle, which probably results in their differentiation and loss of CFC-generation potential.


Assuntos
Células-Tronco Hematopoéticas/citologia , Interleucina-3/farmacologia , Animais , Antígenos CD34 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Hipóxia Celular , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos CBA
13.
PLoS One ; 8(11): e78582, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223824

RESUMO

PURPOSE: BIM is essential for the response to tyrosine-kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) patients. Recently, a deletion polymorphism in intron 2 of the BIM gene was demonstrated to confer an intrinsic TKI resistance in Asian patients. The present study aimed at identifying mutations in the BIM sequence that could lead to imatinib resistance independently of BCR-ABL mutations. EXPERIMENTAL DESIGN: BIM coding sequence analysis was performed in 72 imatinib-treated CML patients from a French population of our centre and in 29 healthy controls (reference population) as a case-control study. Real-time quantitative PCR (RT qPCR) was performed to assess Bim expression in our reference population. RESULTS: No mutation with amino-acid change was found in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710). A strong statistical link was found between the presence of the T allele and the high Sokal risk group (p = 0.0065). T allele frequency was higher in non responsive patients than in the reference population (p = 0.0049). Similarly, this T allele was associated with the mutation frequency on the tyrosine kinase domain of BCR-ABL (p<0.001) and the presence of the T allele significantly lengthened the time to achieve a major molecular response (MMR). Finally, the presence of the T allele was related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy controls. CONCLUSION: These results suggest that the analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of imatinib-treated CML patients.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/genética , Benzamidas/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Membrana/genética , Piperazinas/uso terapêutico , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Pirimidinas/uso terapêutico , RNA Mensageiro/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Biomarcadores Farmacológicos/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Frequência do Gene , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Taxa de Mutação , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo
14.
Exp Hematol ; 40(5): 367-78.e2, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22240609

RESUMO

Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. We blocked the anti-apoptotic proteins of the Bcl-2 family by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions toward a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in X-linked inhibitor of apoptosis (XIAP), followed by caspase-3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines, and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34(+)CD38(-) TKI-insensitive population. Combination therapy might by an interesting strategy for the treatment of CML patients.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/efeitos dos fármacos , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Antígenos CD34/análise , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Benzamidas , Linhagem Celular Tumoral/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células-Tronco Hematopoéticas/enzimologia , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Mesilato de Imatinib , Células K562/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/enzimologia , Proteínas Proto-Oncogênicas/fisiologia , Serina Endopeptidases/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
15.
Neuro Oncol ; 14(12): 1441-51, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23104476

RESUMO

Putative cancer stem cells have been identified in glioblastoma (GBM), associated with resistance to conventional therapies. Overcoming this resistance is a major challenge to manage this deadly brain tumor. Epidermal growth factor receptor (EGFR) is commonly amplified, over-expressed, and/or mutated in GBM, making it a compelling target for therapy. This study investigates the behavior of 3 primary neurosphere (NS) cell lines and their adherent counterparts originated from human GBM resections, when treated with EGFR-tyrosine kinase inhibitor erlotinib, associated or not with cyclopamine, a hedgehog pathway inhibitor. Adherent cells cultured in the presence of serum expressed the glial fibrillary acidic protein, whereas NS-forming cells cultured in serum-free medium expressed CD133, nestin, and Oct-4, markers of neural stem and progenitor cells. For the 3 adherent cell lines, erlotinib has a moderate effect (50% inhibitory concentration [IC50], >10 µM). Conversely, erlotinib induced a strong cell growth inhibition (IC50, <1 µM) on NS-forming cells, related to the EGFR gene amplification and EGFR protein expression. A short exposure to erlotinib reduced nestin-positive cell proliferation, but NS-initiating activity and self-renewal were not altered. EGFR pathway seems essential for GBM progenitor cell proliferation but dispensable for cancer stem-like cell self-renewal. Inhibition of hedgehog pathway with cyclopamine was evaluated in association with erlotinib on NS growth. Although each drug separately had no effect on sphere initiation, their combination significantly decreased the sphere number (P < .001). Our findings show synergic efficiency for erlotinib-cyclopamine association and provide a suitable in vitro model to explore drug combinations on GBM cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/patologia , Diferenciação Celular/efeitos dos fármacos , Glioblastoma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Hibridização Genômica Comparativa , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Citometria de Fluxo , Imunofluorescência , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Neoplásicas/patologia , Quinazolinas/administração & dosagem , Alcaloides de Veratrum/administração & dosagem
16.
Cancer Biol Ther ; 11(12): 1017-27, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21508666

RESUMO

Gliomas are the most common malignant primary brain tumors in adults. The median survival never exceeds 12 months, owing to inherent resistance to both radio and chemotherapies. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in glioblastomas (GBM), making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. Inhibition of cell proliferation and induction of apoptosis by erlotinib were investigated in U87-MG and DBTRG-05MG, two human glioblastoma cell lines. The expression of several apoptosis-related proteins was investigated in these cell lines and in tumoral tissue from glioblastomas. Both cell lines expressed wild-type EGFR but were deficient for PTEN. Erlotinib induced a marked accumulation of the BIM protein, but the activation of caspase-3 machinery was missing, regardless of the decrease in XIAP. Moreover, in U87-MG, erlotinib promoted accumulation of αB-crystallin a small heat shock protein capable to impair caspase activation. DBTRG-05MG was found deficient for procaspase 3 and constitutively overexpressed αB-crystallin. Similarly, deficiencies in PTEN and procaspase 3 were constantly found in samples from glioblastoma samples, while αB-crystallin expression was inconsistent. In cell lines, high concentrations of erlotinib induced cell death through a caspase independent process and an autophagic process was evidenced in U87-MG. Inhibition of autophagy induced a marked increase in the death-inducing activity of erlotinib. These results confirm that glioblastoma cell lines exhibit several anti-apoptotic mechanisms, and underline that EGFR targeted therapy must be associated to other inhibitors to achieve an antitumoral effect.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Glioblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Glioblastoma/ultraestrutura , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Cancer Res ; 68(23): 9809-16, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19047160

RESUMO

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Proteínas de Fusão bcr-abl/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Pirimidinas/farmacologia , Quinases da Família src/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Tiazóis/farmacologia , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
18.
Cancer Biol Ther ; 6(6): 912-9, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17538248

RESUMO

It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48 h. Bim accumulation preceded apoptosis induction which was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL dephosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Citocinas/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Membrana/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/farmacologia , Animais , Antígenos CD34/biossíntese , Proteína 11 Semelhante a Bcl-2 , Benzamidas , Linhagem Celular Tumoral , Humanos , Mesilato de Imatinib , Células K562 , Camundongos , Modelos Biológicos
19.
J Biol Chem ; 282(22): 16413-22, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17400550

RESUMO

Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor sensitive to oxygen levels, which responds to stress factors under both hypoxic and nonhypoxic conditions. UV irradiation being a common stressor of skin, we looked at the effect of UVB on HIF-1alpha expression in keratinocytes. We found that UVB induces a biphasic HIF-1alpha variation through reactive oxygen species (ROS) generation. Whereas rapid production of cytoplasmic ROS down-regulates HIF-1alpha expression, delayed mitochondrial ROS generation results in its up-regulation. Indeed, activation of p38 MAPK and JNK1 mediated by mitochondrial ROS were required for HIF-1alpha phosphorylation and accumulation after UVB irradiation. Our experiments also revealed a key role of HIF-1alpha in mediating UVB-induced apoptosis. We conclude that the broad impact of the HIF-1 transcription factor on gene expression could make it a key regulator of UV-responsive genes and photocarcinogenesis.


Assuntos
Apoptose/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Citoplasma/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Queratinócitos/metabolismo , Raios Ultravioleta , Regulação para Cima/efeitos da radiação , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Citoplasma/patologia , Humanos , Queratinócitos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Stem Cells ; 24(1): 65-73, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16123391

RESUMO

Physiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cell survival, cycle, and functionality in serum-free cultures for 72 hours with or without interleukin-3 (IL-3). As from 24 hours, IL-3 improved cell survival and proliferation in all conditions. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2, respectively, than at 20%; transforming growth factor-beta signaling seemed not to be involved. To explore cell cycle further, fresh CD34+ cells were stained with PKH26 and cultured for 72 hours, and then undivided and divided cells were sorted. At 0.1% O2, 46.5%+/-19.1% of divided cells returned to G0 compared with 7.9%+/-0.3% at 20%. Colony formation and nonobese diabetic/severe combined immunodeficient mice engraftment efficiency were similar after 3 days at 20% and 0.1% O2 concentrations but lower than at T0. In conclusion, a low O2 concentration, close to those found in bone marrow stem cell niches, induces the G0 return of CD34+ cells without impairing their functional capacity.


Assuntos
Antígenos CD34/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Interleucina-3/farmacologia , Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular , Animais , Ciclo Celular , Sobrevivência Celular , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxigênio/química , Fator de Células-Tronco/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA