Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells.
ABSTRACT Retinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells.
Article: Inhibition of DNA methyltransferase activates tumor necrosis factor alpha-induced monocytic differentiation in acute myeloid leukemia cells.[show abstract] [hide abstract]
ABSTRACT: Transcriptional silencing via promoter methylation of genes important for cell growth and differentiation plays a key role in myeloid leukemogenesis. We find that clinically achievable levels of 5-aza-2'-deoxycytidine (5-AZA-dC), a potent inhibitor of DNA methylation, can modify chromatin and restore the ability of tumor necrosis factor alpha (TNFalpha) to induce monocytic differentiation of the acute myeloid leukemia cells NB4 and U937. Although 5-AZA-dC cannot fully induce differentiation, we show that 5-AZA-dC acts directly on TNFalpha-responsive promoters to facilitate TNFalpha-induced transcriptional pathways leading to differentiation. 5-AZA-dC regulates the expression of Dif-2, a TNFalpha target gene, by deacetylating chromatin domains in a methylation-dependent manner. Chromatin immunoprecipitation analyses of the Dif-2 promoter show histone hyperacetylation and a recruitment of the nuclear factor-kappaB transcription factor in response to 5-AZA-dC. Furthermore, 5-AZA-dC plus TNFalpha enhances the level of phosphorylated RNA Pol II at the Dif-2 promoter via synergistic recruitment of TFIIH. We conclude that nonspecific changes in chromatin can allow a specific transcriptional inducer to overcome blocks in leukemic cell differentiation. Our results support the concept of low doses of 5-AZA-dC acting in combination with other agents to target epigenetic changes that drive malignant growth in leukemic cells. [Cancer Res 2009;69(1):55-64].Cancer Research 02/2009; 69(1):55-64. · 7.86 Impact Factor
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ABSTRACT: Chemical biology, using small molecules as probes to study the cellular signaling network, has developed rapidly in recent years. The interaction between chemistry and biology not only provides new insight into the understanding of cellular activities, but also generates new lead compounds for the treatment of diseases. Transcription factors and kinases such as retinoic acid receptor-alpha (RARα), acute myeloid leukemia 1 (AML1), CAAT/enhancer-binding protein α (C/EBPα), c-myc, and c-abl play important roles in the differentiation of hematopoietic stem/progenitor cells. Abnormalities in these proteins may cause the dysregulation of hematopoiesis and even the occurrence of leukemia. Ubiquitin-mediated protein degradation represents a critical mechanism in regulating the cellular levels and functions of these proteins. Thus, targeting protein degradation has been emerging as an important strategy to conquer malignant diseases. In this review, we will summarize the recent advances in the understanding of the roles of protein degradation in leukemia, with an emphasis on the mechanisms revealed by small molecules.Frontiers of Medicine in China 12/2010; 4(4):363-70.
Article: Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells.[show abstract] [hide abstract]
ABSTRACT: Aurora kinase ensures accurate chromosome segregation during cell cycle, maintaining genetic integrity in cell division. VX-680, a small-molecule Aurora kinase inhibitor, interferes with mitotic entry and formation of bipolar spindles. Here, we evaluated VX-680 as a potential agent for treatment of all-trans retinoid acid (ATRA)-resistant acute promyelocytic leukemia (APL) in vitro. CD11b expression was utilized to assess cell differentiation by flow cytometry. Immunofluorescence staining was conducted to analyze formation of cell monopolar spindle. Cell proliferation was evaluated by MTT assay. Sub-G1 population and Annexin V/PI staining were used to measure cell apoptosis. Hoechst 33342 staining was applied for identifying morphological changes in nucleus of apoptotic cell. Aurora-A (Aur-A) activation and the signaling pathways involved in apoptosis were detected by Western blot. JC-1 probe was employed to measure mitochondrial depolarization. VX-680 inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288, accompanied by producing monopolar mitotic spindles in APL cell line NB4-R2 that was resistant to ATRA. In addition, we found that VX-680 inhibited cell proliferation as assessed by MTT assay. Flow cytometry showed that VX-680 led to apoptotic cell death in both dose- and time-dependent manners by either Sub-G1 or Annexin V/PI analysis. Hoechst 33342 staining represented typical apoptotic cells with nuclear fragmentation in VX-680 treated cells. Importantly, VX-680 inhibition of Aurora kinase suppressed Akt-1 activation and induced mitochondrial depolarization, which eventually resulted in apoptosis by activation of caspase pathway, as indicated by increasing proteolytic cleavage of procaspase-3 and poly ADP ribose polymerase (PARP) in NB4-R2 cells. Our study suggested potential clinical use of mitotic Aurora kinase inhibitor in targeting ATRA-resistant leukemic cells.Journal of Translational Medicine 01/2011; 9:74. · 3.41 Impact Factor