RESUMO
Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Fusão bcr-abl/genética , Fatores de Transcrição/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Divisão Celular , Ensaio de Unidades Formadoras de Colônias , Primers do DNA , Regulação para Baixo , Amplificação de Genes , Regulação da Expressão Gênica , Genes Reporter , Humanos , Sequências Repetidas Invertidas/genética , Células K562 , Luciferases/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.
Assuntos
Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/fisiologia , Benzamidas , Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Dasatinibe , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos C3H , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Interferência de RNA , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Fator de Transcrição CHOP/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/farmacologia , Proteínas de Fusão bcr-abl/biossíntese , Fator de Transcrição GATA2/genética , Genes myb/efeitos dos fármacos , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Primers do DNA/genética , Proteínas de Fusão bcr-abl/genética , Genes abl , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , RNA Interferente Pequeno/genética , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPalpha, which were more potent than those of K298E C/EBPalpha, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation.
