C/EBPalpha S248A mutation reduces granulocytic differentiation in human leukemic K562 cells.

Phosphorylation of C/EBPalpha can either lead to granulocytic differentiation or a block in granulopoiesis. This dichotomy in effect is dependent on the upstream signaling pathway and the phosphorylation site in C/EBPalpha. Ras signaling induced phosphorylation of S248 residue of C/EBPalpha is known to enhance granulocytic differentiation. In this study, using beta-estradiol inducible stable cell lines we show that the point mutation of phosphorylation site S248 in C/EBP disrupts the CD11b and GCSFr expression and subsequently reduces the differentiation of leukemic K562 cells. Based on our observations in the present study, we conclude that S248A mutation of C/EBPalpha leads to a reduction of granulocytic differentiation markers and a block in differentiation at the morphological level.

Downregulation of c-Jun expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation.

In the present study, we investigated the mechanism of CD44 ligation with the anti-CD44 monoclonal antibody A3D8 to inhibit the proliferation of human acute myeloid leukemia (AML) cells. The effects of A3D8 on myeloid cells were associated with specific disruption of cell cycle events and induction of G0/G1 arrest. Induction of G0/G1 arrest was accompanied by an increase in the expression of p21, attenuation of pRb phosphorylation and associated with decreased Cdk2 and Cdk4 kinase activities. Since c-Jun is an important regulator of proliferation and cell cycle progression, we analysed its role in A3D8-mediated growth arrest. We observed that A3D8 treatment of AML patient blasts and HL60/U937 cells led to the downregulation of c-Jun expression at mRNA and protein level. Transient transfection studies showed the inhibition of c-jun promoter activity by A3D8, involving both AP-1 sites. Furthermore, A3D8 treatment caused a decrease in JNK protein expression and a decrease in the level of phosphorylated c-Jun. Ectopic overexpression of c-Jun in HL60 cells was able to induce proliferation and prevent the antiproliferative effects of A3D8. In summary, these data identify an important functional role of c-Jun in the induction of cell cycle arrest and proliferation arrest of myeloid leukemia cells because of the ligation of the cell surface adhesion receptor CD44 by anti-CD44 antibody. Moreover, targeting of G1 regulatory proteins and the resulting induction of G1 arrest by A3D8 may provide new insights into antiproliferative and differentiation therapy of AML.

Elevated c-Jun expression in acute myeloid leukemias inhibits C/EBPalpha DNA binding via leucine zipper domain interaction.

Transcription factor C/EBPalpha induces normal myeloid differentiation, inactivation of C/EBPalpha leads to a differentiation block in acute myeloid leukemias (AML), and overexpression of C/EBPalpha results in AML growth arrest and differentiation. Recent reports suggest that C/EBPalpha is activated or inactivated via protein-protein interactions. We previously reported that C/EBPalpha needs to inactivate the proto-oncogene c-Jun via leucine zipper domain interaction in order to induce granulocytic differentiation. We, therefore, hypothesized that c-Jun expression might be elevated in AML and subsequently inactivate C/EBPalpha. In fact, compared to normal bone marrow mononuclear cells, c-Jun expression is increased in AML patient samples (Affymetrix expression microarray analysis, n=166). c-Jun binds to C/EBPalpha via the leucine zipper domains and prevents C/EBPalpha from DNA binding. Inactivation of C/EBPalpha by c-Jun is necessary for c-Jun to induce proliferation because c-Jun-induced proliferation can be prevented by ectopic overexpression of C/EBPalpha. The dominant-negative 30-kDa C/EBPalpha protein, found in AML, fails to downregulate c-Jun mRNA expression in AML patient samples. Thus, our data suggest a model for AML in which c-Jun promotes proliferation and prevents differentiation by inhibiting C/EBPalpha DNA binding via leucine zipper domain interaction. It might depend on the expression levels of C/EBPalpha and c-Jun, if inhibition of C/EBPalpha by c-Jun or if inhibition of c-Jun by C/EBPalpha is more predominant: proliferation versus differentiation; AML versus normal myeloid development.

Proteomic analysis of transcription factor interactions in myeloid stem cell development and leukaemia.

Recent results indicate that interactions of transcription factors with other nuclear proteins play an important role in stem cell development, lineage commitment and differentiation in the haematopoietic system, and the pathogenesis of myeloid leukaemias. High-throughput proteomics by mass spectrometric analysis of gel-separated proteins can identify multi-protein complexes and changes in the expression of multiple proteins simultaneously. This review describes an application of proteomic methods (2D gel electrophoresis (GE) and mass spectrometry (MS)), which can be used to identify regulated protein targets of transcription factors important in myeloid differentiation and leukaemia. This global high-throughput functional proteomics approach could lead to new insights into the network of protein-protein interactions and target proteins involved in myeloid stem cell development and leukaemia as well as provide new targets for rational pathogenesis-based therapies of leukaemia and cancer.

The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia.

The transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1(-/-) mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion gene AML1-ETO. Here we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1. AML1-ETO physically binds to PU.1 in t(8;21)(+) Kasumi-1 cells. AML1-ETO binds to the beta(3)beta(4) region in the DNA-binding domain of PU.1 and displaces the coactivator c-Jun from PU.1, thus down-regulating the transcriptional activity of PU.1. This physical interaction of AML1-ETO and PU.1 did not abolish the DNA-binding capacity of PU.1. AML1-ETO down-regulates the transactivation capacity of PU.1 in myeloid U937 cells, and the expression levels of PU.1 target genes in AML French-American-British (FAB) subtype M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits antiproliferative function of PU.1. Overexpression of PU.1, however, differentiates AML1-ETO-expressing Kasumi-1 cells to the monocytic lineage. Thus, the function of PU.1 is down-regulated by AML1-ETO in t(8;21) myeloid leukemia, whereas overexpression of PU.1 restores normal differentiation.

Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.

The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.