RESUMO
Iron is required for the differentiation of HL-60 cells along the monocyte lineage in vitro, reflecting a requirement for iron in the transcriptional induction of the p21(WAF1/CIP1) gene. To determine if the same requirement holds true for differentiation in other cell lineages and for primary human CD34(+) bone marrow precursor cells, we induced granulocyte differentiation by treating HL-60 cells with dimethyl sulphoxide, and erythroid or megakaryocytic differentiation by treating K562 cells with butyrate or phorbol myristate acetate, respectively. Nitro blue tetrazolium reduction, expression of haem, or expression of CD41 was used to assess granulocytic, erythroid, or megakaryocytic differentiation respectively. Purified CD34(+) cells were cultured with granulocyte/macrophage-colony stimulating factor and stem cell factor to induce myelomonocytic differentiation. Iron deprivation was induced by desferrioxamine. p21(WAF1/CIP1) antisense oligonucleotides were used to inhibit p21 expression. Iron deprivation blocked p21 induction as judged by real-time polymerase chain reaction assays. In addition, both iron deprivation and p21 antisense blocked CD34(+) cell differentiation. These observations were not explained by induction of widespread apoptosis under conditions of iron deprivation. We concluded that both iron and functional p21(WAF1/CIP1) are required for in vitro differentiation of human haematopoietic precursors along multiple cell lineages.
Assuntos
Linhagem Celular Tumoral/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Hematopoese/genética , Ferro/metabolismo , Antígenos CD34/genética , Elementos Antissenso (Genética)/genética , Apoptose/fisiologia , Butiratos/farmacologia , Carcinógenos/farmacologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Desferroxamina/farmacologia , Dimetil Sulfóxido/farmacologia , Regulação Leucêmica da Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HL-60/efeitos dos fármacos , Células HL-60/fisiologia , Hematopoese/efeitos dos fármacos , Humanos , Células K562/efeitos dos fármacos , Células K562/fisiologia , Sideróforos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/genéticaRESUMO
We compared the gene expression profile of adult acute lymphoblastic leukemia (ALL) to normal hematopoietic and non-ALL samples using oligonucleotide arrays. Connective tissue growth factor (CTGF) was the highest overexpressed gene in B-cell ALL compared with the other groups, and displayed heterogeneous expression, suggesting it might have prognostic relevance. CTGF expression was examined by quantitative reverse transcriptase-polymerase chain reaction (ORT-PCR) on 79 adult ALL specimens. CTGF expression levels were significantly increased in ALL cases with B-lineage (P < .001), unfavorable cytogenetics (P < .001), and blasts expressing CD34 (P < .001). In a multivariate proportional hazards model, higher CTGF expression levels corresponded to worsening of overall survival (OS; hazard ratio 1.36, for each 10-fold increase in expression; P = .019). Further studies are ongoing to confirm the prognostic value of CTGF expression in ALL and to investigate its role in normal and abnormal lymphocyte biology.
Assuntos
Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Sequência de Bases , Linfoma de Burkitt/genética , Linfoma de Burkitt/mortalidade , Estudos de Casos e Controles , Fator de Crescimento do Tecido Conjuntivo , Primers do DNA/genética , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
Iron is required for monocyte/macrophage differentiation of HL-60 leukaemia cells. Differentiation requires induction of the cyclin-dependent kinase inhibitor p21 (WAF1/CIP1), and cell cycle arrest at the G1/S checkpoint. With iron depletion, p21 induction and differentiation are blocked. To establish the roles of iron and p21 in normal monocyte/macrophage differentiation, we examined generation of dendritic cells (DCs) and macrophages from peripheral monocytes. Monocytes were cultured with interleukin 4 and granulocyte-macrophage colony-stimulating factor (GM-CSF), then treated with lipopolysaccharide to produce DCs or with M-CSF to produce macrophages. Iron deprivation was induced by desferrioxamine (DF). Monocyte-derived DCs had characteristic phenotype and morphology, and stimulated proliferation of naïve allogeneic T lymphocytes. In contrast, DCs generated under iron deprivation were phenotypically undifferentiated and did not stimulate T cells. Similarly, macrophages expressed a characteristic phenotype and morphology, and phagocytosed latex beads, but macrophages generated under iron deprivation failed to develop a mature phenotype and had impaired phagocytosis. Iron deprivation blocked induction of p21 (WAF1/CIP1) expression in both DC and macrophage cultures. Furthermore, p21 antisense oligonucleotides, but not sense oligonucleotides, inhibited both DC and macrophage differentiation. These data indicate that a key role of iron in haematopoiesis is to support induction of p21 which, in turn, is required for DC and macrophage differentiation.