RESUMO
Gene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.
Assuntos
Hematopoese/genética , Leucócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Interferência de RNA , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Feminino , Células HEK293 , Hematopoese/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/farmacologia , Antígenos de Histocompatibilidade Menor , Terapia de Alvo Molecular/métodos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.