Assuntos
Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Medicina de Precisão , OncologiaRESUMO
Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.
Assuntos
Apoptose , Grupo dos Citocromos c/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Indução de Apoptose , Caspases/metabolismo , Citosol/metabolismo , Citosol/fisiologia , Ativação Enzimática , Flavoproteínas/metabolismo , Proteínas de Choque Térmico HSP27 , Humanos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Células U937RESUMO
OBJECTIVES: Although high-throughput sequencing is revolutionising medicine, data on the actual cost of whole exome sequencing (WES) applications are needed. We aimed at assessing the cost of WES at a French cancer institute in 2015 and 2018. METHODS: Actual costs of WES application in oncology research were determined using both micro-costing and gross-costing for the years 2015 and 2018, before and after the acquisition of a new sequencer. The entire workflow process of a WES test was tracked, and the number and unit price of each resource were identified at the most detailed level, from library preparation to bioinformatics analyses. In addition, we conducted an ad hoc analysis of the bioinformatics storage costs of data issued from WES analyses. RESULTS: The cost of WES has decreased substantially, from 1921 per sample (i.e. cost of 3842 per patient) in 2015 to 804 per sample (i.e. cost of 1,608 per patient) in 2018, representing a decrease of 58%. In the meantime, the cost of bioinformatics storage has increased from 19,836 to 200,711. CONCLUSION: This study suggests that WES cost has decreased significantly in recent years. WES has become affordable, even though clinical utility and efficiency still need to be confirmed.
Assuntos
Neoplasias , Patologia Molecular , Custos e Análise de Custo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Sequenciamento do ExomaRESUMO
The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.
Assuntos
Caspases/metabolismo , Eritrócitos/enzimologia , Eritropoese/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Diferenciação Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/enzimologia , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacosRESUMO
Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Macrófagos/citologia , Macrófagos/fisiologia , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Caspases play important roles in apoptotic cell death and in some other functions, such as cytokine maturation, inflammation, or differentiation. We show here that the 5'-flanking region of the human CASP-2 gene contains three functional response elements for sterol regulatory element binding proteins (SREBPs), proteins that mediate the transcriptional activation of genes involved in cholesterol, triacylglycerol, and fatty acid synthesis. Exposure of several human cell lines to statins, lipid-lowering drugs that drive SREBP proteolytic activation, induced the CASP-2 gene to an extent similar to that for known targets of SREBP proteins. Adenoviral vector-mediated transfer of active SREBP-2 also induced expression of the CASP-2 gene and the caspase-2 protein and increased the cholesterol and triacylglycerol cellular content. These rises in lipids were strongly impaired following small interfering RNA-mediated silencing of the CASP-2 gene. Taken together, our results identify the human CASP-2 gene as a member of the SREBP-responsive gene battery that senses lipid levels in cells and raise the possibility that caspase-2 participates in the control of cholesterol and triacylglycerol levels.
Assuntos
Cisteína Endopeptidases/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologia , Região 5'-Flanqueadora , Sítios de Ligação , Caspase 2 , Linhagem Celular Tumoral , Colesterol/biossíntese , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , RNA Interferente Pequeno/genética , Elementos de Resposta , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Triglicerídeos/biossínteseRESUMO
Mitochondria are involved in hematopoietic cell homeostasis through multiple ways such as oxidative phosphorylation, various metabolic processes and the release of cytochrome c in the cytosol to trigger caspase activation and cell death. In erythroid cells, the mitochondrial steps in heme synthesis, iron (Fe) metabolism and Fe-sulfur (Fe-S) cluster biogenesis are of particular importance. Mutations in the specific delta-aminolevulinic acid synthase (ALAS) 2 isoform that catalyses the first and rate-limiting step in heme synthesis pathway in the mitochondrial matrix, lead to ineffective erythropoiesis that characterizes X-linked sideroblastic anemia (XLSA), the most common inherited sideroblastic anemia. Mutations in the adenosine triphosphate-binding cassette protein ABCB7, identified in XLSA with ataxia (XLSA-A), disrupt the maturation of cytosolic (Fe-S) clusters, leading to mitochondrial Fe accumulation. In addition, large deletions in mitochondrial DNA, whose integrity depends on a specific DNA polymerase, are the hallmark of Pearson's syndrome, a rare congenital disorder with sideroblastic anemia. In acquired myelodysplastic syndromes at early stage, exacerbation of physiological pathways involving caspases and the mitochondria in erythroid differentiation leads to abnormal activation of a mitochondria-mediated apoptotic cell death pathway. In contrast, oncogenesis-associated changes at the mitochondrial level can alter the apoptotic response of transformed hematopoietic cells to chemotherapeutic agents. Recent findings in mitochondria metabolism and functions open new perspectives in treating hematopoietic cell diseases, for example various compounds currently developed to trigger tumor cell death by directly targeting the mitochondria could prove efficient as either cytotoxic drugs or chemosensitizing agents in treating hematological malignancies.
Assuntos
Doenças Hematológicas/fisiopatologia , Hematopoese/fisiologia , Mitocôndrias/fisiologia , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Senescência Celular/genética , DNA Mitocondrial/genética , Doenças Hematológicas/patologia , Humanos , Ferro/metabolismo , Mitocôndrias/metabolismo , MutaçãoRESUMO
Anticancer drugs can induce tumor cell death by caspase-dependent apoptosis. The observation that procaspase-10 expression decreased in leukemic cells from acute myeloblastic leukemia patients at first relapse led us to explore the role of caspase-10 in cytotoxic drug-induced apoptosis. We show that caspase-10 is activated in etoposide-treated cells in a dose- and time-dependent manner. A caspase-10 peptide inhibitor, a caspase-10 dominant-negative mutant or a small interfering RNA (siRNA)-mediated downregulation of the enzyme negatively interfere with drug-induced cell death and caspase-2, -3, -8 and -9 activation. The extrinsic pathway to apoptosis is not involved in drug-induced caspase-10 activation that occurs downstream of Bax redistribution to mitochondria and cytochrome c release from this organelle. siRNA-mediated downregulation of Apaf-1 prevents etoposide-mediated activation of caspase-10. In a cell-free assay, cytochrome c and dATP treatment of cell extracts after immunodepletion of either caspase-3 or caspase-9 indicates that caspase-10 is activated downstream of caspase-9. Then, caspase-10 is involved in a feedback amplification loop that amplifies caspase-9 and -3 activities. Altogether, these data indicate an active role for caspase-10 in cytotoxic drug-induced tumor cell death, downstream of the mitochondria.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Caspase 10/fisiologia , Etoposídeo/farmacologia , Transdução de Sinais , Apoptossomas/efeitos dos fármacos , Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Leucemia Mieloide Aguda/diagnóstico , Mitocôndrias/metabolismo , RecidivaRESUMO
We determined the number and functional status of CD4+ CD25(high) regulatory T cells (Treg) in blood samples from patients with metastatic carcinoma, and evaluated their sensitivity to a single intravenous infusion of cyclophosphamide. Treg numbers were significantly higher in 49 patients with metastatic cancer (9.2% of CD4+ T cells) compared to 24 healthy donors (7.1%). These cells expressed the transcription factor forkhead box P3 (FoxP3), glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) and intracellular CD152, and demonstrated a suppressive activity in vitro against CD4+ CD25- autologous proliferation. At a single intravenous infusion, cyclophosphamide failed, in association with a non-specific immunotherapy by intratumoral bacille Calmette-Guérin (BCG), to modulate significantly Treg numbers or function. Metastatic cancer is associated with an expansion of peripheral blood CD4+ CD25(high) FoxP3+ GITR+ CD152+ Treg cells whose immunosuppressive properties do not differ from those of healthy subjects. Moreover, cyclophosphamide administration may not represent an optimal therapy to eliminate Treg, which further underlines the need to identify specific agents that would selectively deplete these cells.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Vacina BCG/uso terapêutico , Ciclofosfamida/uso terapêutico , Metástase Neoplásica/terapia , Linfócitos T Reguladores/imunologia , Idoso , Terapia Combinada , Feminino , Fatores de Transcrição Forkhead/sangue , Humanos , Tolerância Imunológica , Imunofenotipagem , Antígenos Comuns de Leucócito/sangue , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
BACKGROUND: Patients diagnosed with therapy-related myeloid neoplasms (TRMN) with concomitant active neoplastic disorder (CAND) are usually proposed for best supportive care (BSC). We evaluated the feasibility of using 5-azacytidine (AZA) in this setting. METHODS: All patients referred to Gustave Roussy between 2010 and 2015 for TRMN diagnosis (less than 30% blast) and eligible for AZA treatment were included. Patients with CAND proposed for BSC were also described. Patient's outcomes were analyzed based on the presence or not of a CAND. RESULTS: Fifty-two patients with TRMN were analyzed, including 19 patients with CAND (14 eligible for AZA) and 33 without CAND eligible for AZA. The 5 patients with CAND ineligible for AZA had a worst performance status (p=0.016) at diagnosis and a shorter overall survival (OS) (0.62 months). Baseline characteristics of patients eligible for AZA were similar in the 2 groups except a trend for best performance status in patients with CAND (p=0.06). Overall response rate (71.4% vs 60.3%), transfusion independence (50.0% vs 45.5%) and OS (12.7 months vs 10.8 months) were similar between patients with and without CAND respectively (p=ns). CONCLUSION: Here we report the feasibility and efficacy of AZA for selected patients with TRMN and a CAND.
Assuntos
Azacitidina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Segunda Neoplasia Primária/tratamento farmacológico , Neoplasias/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/administração & dosagem , Feminino , Humanos , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/mortalidade , Neoplasias/patologia , Segunda Neoplasia Primária/complicações , Segunda Neoplasia Primária/mortalidade , Indução de Remissão , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Sterol Regulatory Element Binding Proteins (SREBP) are transcription factors that regulate lipid synthesis. We have shown recently that the human CASP-2 gene, encoding procaspase-2, was under the positive control of SREBP-2 that transactivates several responsive elements in the promoter region. We describe here the function of an additional SREBP-responsive element located in the first intron. Remarkably, this site is uniquely responsive to SREBP-1c that is mainly involved in fatty acids synthesis. This observation, together with our recent findings, strengthens the notion that the CASP-2 gene belongs to the SREBP-responsive gene battery in human cells.
Assuntos
Caspases/genética , Íntrons , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Sítios de Ligação , Caspase 2 , Caspases/metabolismo , Células Cultivadas , DNA/metabolismo , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Ativação TranscricionalRESUMO
BACKGROUND: The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. PURPOSE: This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. METHODS: Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. RESULTS: Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. CONCLUSION AND IMPLICATIONS: In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Glicoproteínas de Membrana/fisiologia , Receptor fas/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Proteína Ligante Fas , Citometria de Fluxo , Células HT29/efeitos dos fármacos , Células HT29/metabolismo , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Receptor fas/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.
Assuntos
Inibidores de Caspase , Caspases/biossíntese , Caspases/química , Isoformas de Proteínas , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Sequência de Bases , Southern Blotting , Western Blotting , Caspase 2 , Caspases/genética , Morte Celular , Linhagem Celular , Sistema Livre de Células , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Relação Dose-Resposta a Droga , Ativação Enzimática , Etoposídeo/farmacologia , Humanos , Cinética , Linfoma/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death. The relationships between cell cycle regulation and apoptosis triggering remain poorly understood. The gadd153 gene encodes the nuclear protein CHOP 10 that acts as a negative modulator of CCAAT/enhancer binding protein transcriptional factors and inhibits cell cycle progression. We have investigated the relationships between gadd153 gene expression and apoptosis induction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibitor. The gadd153 gene was constitutively expressed in the four studied cell lines. In U937 and HL-60 cells that were very sensitive to apoptosis induction by the drug, VP-16 induced a time- and dose-dependent increase of gadd153 gene mRNA expression. Using agarose gel electrophoresis and a quantitative filter elution assay, apoptotic DNA fragmentation was observed to begin when gadd153 gene expression increased. Equitoxic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis induction by the drug. Nuclear run-on and mRNA stability experiments demonstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells. Cycloheximide did not prevent gadd153 expression increase. Both gadd153 mRNA level increase and internucleosomal DNA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethylketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2. Z-VAD and Z-DEVD peptides that inhibit interleukin 1beta-converting enzyme-like proteases suppressed DNA fragmentation without preventing gadd153 mRNA increase in VP-16-treated U937 cells. These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-tosyl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1beta-converting enzyme-related proteases activation in leukemic cells in which treatment with VP-16 induces rapid apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Leucemia/tratamento farmacológico , Leucemia/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Camptotecina/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Daunorrubicina/farmacologia , Células HL-60/efeitos dos fármacos , Humanos , Leucemia/metabolismo , Proteínas Nucleares/genética , Inibidores de Serina Proteinase/farmacologia , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias do Colo/patologia , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspase 8 , Caspase 9 , Membrana Celular/metabolismo , Cisplatino/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/biossíntese , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais CultivadasRESUMO
The REG and PRO cell clones were obtained from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. When injected s.c. into syngeneic hosts, REG cells induce tumors that regress in less than 3 weeks, whereas PRO cells, like parental cells, induce progressive tumors. Here, we show that compared to PRO cells, REG cells are more sensitive to cell death induced by anticancer drugs. The small heat shock protein (HSP) 27 is not expressed or inducible in REG clones, whereas it is abundantly expressed and inducible by heat shock in PRO clones. The expression of HSP27 in REG cells increases their resistance to apoptosis in vitro and dramatically enhances their tumorigenicity when injected s.c. into syngeneic rats. HSP27 expression in REG cells both increases tumor size and delays tumor regression. This increased tumorigenicity is associated with a substantial decrease of in vivo tumor cell apoptosis. We conclude that HSP27 expression in malignant cells increases their tumorigenicity in syngeneic animals. In combination with the role of HSP27 in tumor cell resistance to cytotoxic agents, its contribution to tumorigenicity makes this protein a potential target for antitumoral therapy.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas de Choque Térmico/biossíntese , Adenocarcinoma/imunologia , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Células Clonais , Neoplasias do Colo/imunologia , Proteínas de Choque Térmico/fisiologia , Camundongos , Camundongos Nus , Ratos , Ratos Endogâmicos , Células Tumorais CultivadasRESUMO
Graft versus host disease (GvHD), which is the primary complication of allogeneic bone marrow transplantation, can alter the intestinal barrier targeted by activated donor T-cells. Chemical inhibition of the stress protein HSP90 was demonstrated in vitro to inhibit T-cell activation and to modulate endoplasmic reticulum (ER) stress to which intestinal cells are highly susceptible. Since the HSP90 inhibitor 17-allylamino-demethoxygeldanamycin (17AAG) is developed in clinics, we explored here its ability to control intestinal acute GvHD in vivo in two mouse GvHD models (C57BL/6î²BALB/c and FVB/Nî²Lgr5-eGFP), ex vivo in intestine organoids and in vitro in intestinal epithelial cultures. We show that 17AAG decreases GvHD-associated mortality without impairing graft versus leukemia effect. While 17AAG effect in T-cell activation is just moderate at the dose used in vivo, we observe a striking intestinal integrity protection. At the intestine level, the drug promotes the splicing of the transcription factor X-box binding protein 1 (XBP1), which is a key component of the ER stress. This effect is associated with a decrease in intestinal damage and an increase in Lgr5(+) stem cells, Paneth cells and defensins production. The importance of XBP1 splicing control is further confirmed in cultured cells and organoids of primary intestinal epithelium where XBP1 is either shRNA depleted or inhibited with toyocamycin. In conclusion, 17AAG has a protective effect on the epithelial intestinal barrier in mouse models of acute GvHD. This compound deserves to be tested in the therapeutic control of acute GvHD.
Assuntos
Benzoquinonas/farmacologia , Citoproteção/efeitos dos fármacos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Intestinos/patologia , Lactamas Macrocíclicas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Animais , Benzoquinonas/uso terapêutico , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestinos/efeitos dos fármacos , Lactamas Macrocíclicas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Splicing de RNA/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Cromatina/ultraestrutura , Precursores Enzimáticos/metabolismo , Fosfatidilserinas/metabolismo , Motivos de Aminoácidos , Apoptose/efeitos dos fármacos , Sequência de Bases , Caspase 2 , Caspases/genética , Cisteína , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/genética , Etoposídeo/farmacologia , Humanos , Isoenzimas , Leucemia , Dados de Sequência Molecular , Mutação , Inibidores da Topoisomerase II , Transfecção , Células Tumorais CultivadasRESUMO
The present study explored the role of the cell surface receptor Fas (CD95/APO-1) in apoptosis induced by camptothecin (CPT) in the HT29 colon carcinoma cell line. CPT-induced apoptosis was associated with high molecular weight DNA fragmentation as measured by filter elution. This fragmentation was inhibited by the caspase inhibitor, z-VAD-fmk and by cycloheximide, which also prevented proteolytic activation of caspase-3 and poly(ADP-ribose)polymerase cleavage. Under such conditions, Fas, Fas ligand, Bax, and p21 expression were increased and Fas recruited the FADD adaptor. Fas expression increase was blocked by cycloheximide but not by z-VAD-fmk, consistent with caspase activation downstream from Fas. Treatment of HT29 cells with FasL or with the CH-11 agonistic anti-Fas antibody potentiated the apoptotic response of cells treated with CPT. The anti-Fas blocking antibody ZB4 and the Fas-ligand inhibitor failed to protect HT29 cells from CPT-induced apoptosis. Such a protection was obtained by transient expression of constructs encoding a dominant-negative mutant of FADD, FADD in an antisense orientation and E8 or MC159 viral proteins that inhibit Fas-induced apoptosis at the level of FADD and procaspase-8, respectively. Together, these data show that topoisomerase I-mediated DNA damage-induced apoptosis involves activation of the Fas pathway without detectable Fas-ligand requirement in CPT-treated cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Neoplasias do Colo/patologia , Glicoproteínas de Membrana/fisiologia , Receptor fas/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Proteínas de Transporte/fisiologia , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Proteína Ligante Fas , Proteína de Domínio de Morte Associada a Fas , Genes p53 , Células HT29 , Humanos , MutaçãoRESUMO
HSP27 form oligomeric structures up to 800 Kda. In cultured cells, the equilibrium between small and large oligomers shifted towards smaller oligomers when phosphorylated on serine residues. To further explore HSP27 structural organization and its repercussion in HSP27 antiapoptotic and tumorigenic properties, we transfected colon cancer REG cells with wild type HSP27 and two mutants in which the phosphorylatable serine residues have been replaced by alanine (to mimic the non phosphorylated protein) or aspartate (to mimic the phosphorylated protein). In growing cells, wild type and alanine mutant formed small and large oligomers and demonstrated antiapoptotic activity while aspartate mutant only formed small multimers and had no antiapoptotic activity. In a cell-free system, only large oligomeric structures interfered with cytochrome c-induced caspase activation, thereby inhibiting apoptosis. The inability of the aspartate mutant to form large oligomers and to protect tumor cells from apoptosis was overcome by growing the cells in vivo, either in syngeneic animals or nude mice. These observations were reproduced by culturing the cells at confluence in vitro. In conclusion (1) large oligomers are the structural organization of HSP27 required for its antiapoptotic activity and (2) cell-cell contacts induce the formation of large oligomers, whatever the status of phosphorylatable serines, thereby increasing cell tumorigenicity.