Absence of the transcription factor CCAAT enhancer binding protein alpha results in loss of myeloid identity in bcr/abl-induced malignancy.

The lineage-determining transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is required for myeloid differentiation. Decreased function or expression of C/EBPalpha is often found in human acute myeloid leukemia. However, the precise impact of C/EBPalpha deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl-induced myeloproliferative disease. The expression of bcr/abl in C/EBPalphapos fetal liver cells led to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl-expressing C/EBPalpha-/- fetal liver cells failed to induce a myeloid disease in transplanted mice, but caused a fatal, transplantable erythroleukemia instead. Accordingly, increased expression of the transcription factors SCL and GATA-1 in hematopoietic precursor cells of C/EBPalpha-/-R01-EY-11298 ) fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl-positive cell line. Consistent with findings of the transplant model, expression of C/EBPalpha and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was identified as a critical direct target of C/EBPalpha. Down-regulation of Id1 by RNA interference impaired C/EBPalpha-induced granulocytic differentiation. Taken together, our study provides evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPalpha.

The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells.

BACKGROUND: The fusion protein RUNX1-CBFA2T1 associated with t(8;21)-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21)-positive cell lines. METHODS: The t(8;21)-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence. RESULTS: Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity. CONCLUSIONS: RUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21)-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches.

Glycosylation of FcgammaRIII in N163 as mechanism of regulating receptor affinity.

Human FcgammaRIII (CD16) is a low-affinity receptor for immunoglobulin G (IgG). There are two different isoforms of this protein: CD16a (transmembranous, expressed on natural killer cells and on macrophages) and CD16b (glycosylphosphatidylinositol-linked, expressed on neutrophilic granulocytes in two allelic forms NA1 and NA2). Both forms of the protein have a variable glycosylation pattern. The NA1 allele of CD16B has four asparagine (N)-linked glycosylation sites. One of them (N163) is localized in the ligand-binding site of domain II. This site is shared by the NA2 allele and CD16A. To examine the functional role of the glycosylation we mutated the four glycosylation sites of the NA1 allele (N39, N75, N163, N170) into glutamine (Q). HEK293 cells were stably transfected with the single mutants and wild-type CD16 as control. We determined binding of human IgG to transfected cells using immunofluorescence studies with anti-human IgG antibody. Monomeric IgG bound to N163Q transfectants with higher affinity than to other transfectants, showing that glycosylation in N163 influences the affinity of CD16 to its ligand. In addition, preincubation of WT-CD16-transfected cells with Tunicamycin (an inhibitor of N-glycosylation) resulted in an increased binding of monomeric IgG whereas N163Q-CD16-transfected cells remained unaffected. Therefore, glycosylation in N163 is a mechanism of regulating affinity of FcgammaRIII to its ligand IgG.

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome.

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.