RESUMEN
Dysfunction of genes that control mitosis and are responsible for the correct segregation of sister chromatids in anaphase is often accompanied by aneuploidy, which is frequently detected in leukemia. One of the components of the kinetochore complex, namely, the AF15q14/KNL1/CASC5 protein, is an important factor ensuring the correct binding of the pericentromeric region of chromosomes with the spindle microtubules. As shown recently, in some leukemias, the gene of this protein can be involved in the generation of the chromosomal translocation t(11;15)(q23;q14) or a variant of the chimeric MLL-AF15Q14 oncogene, which serves as a biomarker of poor prognosis. Despite the implication of mRNA of the CASC5 gene in oncogenesis of solid tumors, expression of this gene in hematopoietic neoplasms has not been studied. We analyzed expression levels of the CASC5 gene and the nearest regulatory genes, including WT1, APOBEC3A (A3A), and N-MYC. A pronounced decrease in CASC5 expression in bone marrow cells of primary leukemia patients compared with healthy donors was found. It was also shown that reduced expression of the CASC5 gene correlates with the detection of targeted mutations in patients composed two prognostic subgroups (favorable, unfavorable) with a significance level (p <0.05). It was noted that the change in the expression level of the CASC5 gene in acute myeloid leukemia is associated with overexpression of the genes WT1, A3A, and in some cases N-MYC and SPT16, which is consistent with the resistance to chemotherapy and leukemia progression. However, the question of which regulatory gene initiates leukemogenesis remains open.
Asunto(s)
Leucemia Mieloide Aguda , Leucemia , Proteínas de Ciclo Celular/genética , Citidina Desaminasa , Expresión Génica , Humanos , Leucemia/genética , Proteínas Asociadas a Microtúbulos/genética , Proteína de la Leucemia Mieloide-Linfoide , Proteínas , Translocación GenéticaRESUMEN
The aim of this study was to study interactions between stromal bone marrow microenvironment and leukemic cells. We tested the hypothesis that stromal cells prevent apoptosis of AML cells by up-regulating anti-apoptotic proteins in leukemic blasts. In HL-60 and NB-4 cells, serum deprivation- and ara-C-induced apoptosis was diminished when cells were cocultured with murine MS-5 stromal cells (P < 0.02). This effect was reproduced with conditioned medium from MS-5 cells. Cocultivation with stromal cells induced Bcl-2 expression levels, both by PCR analysis and flow cytometry. In primary AML (n = 14), ara-C-induced apoptosis was significantly lower in cells cocultured with MS-5 cells than in controls (P < 0.001). This effect was partially preserved when leukemic cells were separated from stromal cells by a microporous insert (in 5/9 samples, P = 0.04). In addition, Bcl-2 levels were significantly higher in stroma-supported than in control CD34(+) AML cells (P < 0.01). Bcl-X(L) levels were higher in 5/7 samples grown on stromal layers. Of note, in AML patients resistant to induction chemotherapy (n = 6), Bcl-2 increased significantly after cultivation with stromal cells, but no such increase was noted in cells from chemotherapy-sensitive patients. In conclusion, MS-5 stromal cells prevented apoptosis in HL-60 cells and in primary AML blasts via modulation of Bcl-2 family proteins. The observed association of high Bcl-2 expression in stroma-supported AML blasts in vitro with resistance to chemotherapy in vivo suggests that the same mechanisms may be operational in vivo.
Asunto(s)
Apoptosis/efectos de los fármacos , Células de la Médula Ósea/patología , Leucemia Mieloide/metabolismo , Compuestos Orgánicos , Células del Estroma/fisiología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antimetabolitos Antineoplásicos/farmacología , Células de la Médula Ósea/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Citarabina/farmacología , Cartilla de ADN/química , Femenino , Colorantes Fluorescentes , Células HL-60/efectos de los fármacos , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Masculino , Ratones , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Major leucocyte subpopulations were isolated from peripheral blood of healthy donors, and patients with chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). In vitro UV irradiation was performed at the wavelength of 257nm (UVC band). DNA double-stranded breaks (DNAdsbs) were detected immediately after UV-irradiation, by means of agarose gel electrophoresis. Cell viability was estimated after 18h in culture, as relative numbers of residual non-apoptotic cells. Evaluation of the dose-response curves revealed that normal CLL lymphoid cells showed only moderate damage after UV-irradiation, as assessed by DNAdsbs and cell viability criteria. However, normal granulocytes and myeloid blasts from CML patients expressed a sharp increase in DNAdsbs, even at lower doses of UV-radiation. UV-induced amplification of endogenous oxidative systems (e.g. NADPH-dependent oxidase) is suggested as a probable reason for enhanced DNA breakage and apoptosis in cells of the granulocytic lineage.