ABSTRACT
The transcription factor Wilms' tumor gene 1 (WT1) is highly expressed in the majority of leukemias, suggesting a role in leukemogenesis. Acquired WT1 mutations are reported as an independent predictor of poor clinical outcome, and mutations resulting in deletion of the entire DNA-binding zinc-finger domain (WT1delZ), is the most common type. The aim of this study was to study cellular effects of WT1(delZ) that may contribute to an oncogenic phenotype. We found that expression of WT1(delZ) supported proliferation of human hematopoietic CD34(+) progenitor cells. Moreover, WT1(delZ) transduced cells expressed erythroid markers, including raised levels of STAT5, independently of addition of erythropoietin. At the global gene expression level, WT1(delZ) caused upregulation of genes related to cell division and genes associated with erythroid maturation, in the absence of added erythropoietin. Our results indicate that WT1(delZ) promotes cell proliferation and expansion of progenitor cells, consistent with a possible role in leukemogenesis.
Subject(s)
Hematopoietic Stem Cells/metabolism , Leukemia/genetics , Leukemia/metabolism , Mutant Proteins , WT1 Proteins/genetics , WT1 Proteins/metabolism , Antigens, CD34/metabolism , Cell Culture Techniques , Cell Proliferation , Colony-Forming Units Assay , Erythropoietin/metabolism , Gene Expression , Gene Expression Profiling , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Hemoglobin A/metabolism , Humans , STAT5 Transcription Factor/metabolism , Transduction, GeneticABSTRACT
OBJECTIVE: Secreted enzymatically inactive proforms of hematopoietic serine proteases proteinase 3 (PR3), azurocidin, and granzymes A, B, H, K, and M are able to reduce the fraction of granulopoietic progenitors (CFU-GM) in S-phase, whereas human leukocyte elastase (HLE) and cathepsin G lack this ability. The objective of the present study was to map the specific sequence(s) of PR3 and other hematopoietic serine proteases responsible for the downmodulation of S-phase. METHODS: Synthetic peptides corresponding to N-terminal sequences of PR3, purified recombinant PR3, and HLE, as well as hybrid proteins constructed by interchanging the N-terminal regions of PR3 and HLE, thus creating PR3/HLE and HLE/PR3, respectively, were tested for their ability to reduce the fraction of human marrow CFU-GM killed by cytosine arabinoside. In addition, we measured the effect of synthetic peptides on bromodeoxyuridine (BrdU) incorporation in common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP) isolated by cell sorting. RESULTS: The common N-terminal motif of PR3 and other serine proteases (i.e., IVGG or IIGG) downmodulate the S-phase of CFU-GM at 40 to 80 nM concentration. Tetrapeptide IVGG, but not IVGR, significantly reduces BrdU incorporation in GMP within the CD34+ population. When the N-terminal of HLE is presented by the HLE/PR3 hybrid protein it is fully active. CONCLUSION: These findings demonstrate that the downmodulatory effect on CFU-GM in S-phase is an S-phase arrest mediated by the first four N-terminal amino acids of PR3, and also suggest that this activity is dependent on the configuration of the proform providing the correct presentation of this N-terminal motif.
