THE WILMS-TUMOR GENE IS EXPRESSED IN A SUBSET OF CD34(-REGULATED EARLY IN THE COURSE OF DIFFERENTIATION IN-VITRO() PROGENITORS AND DOWN)

The Wilms' tumor gene (wt1) is strongly expressed in malignant blasts of acute myeloid leukemia (AML) in approximately 80% of all cases. How ever, the role of wt1 expression in nonmalignant hematopoietic cells r emains unclear. To characterize the expression of wt1 in differentiati ng hematopoietic progenitors, we isolated and cultured CD34(+) progeni tor cells from four healthy bone marrow donors with stern cell factor (SCF) and granulocyte colony stimulating-factor (G-CSF) to induce diff erentiation into granulocytes. Four different cultures were carried ou t for 12 days, During culture: wt1 mRNA expression was analyzed by def ining its ratio relative to beta-actin using reverse transcriptase pol ymerase chain reaction (RT-PCR). To monitor the stage of differentiati on, expression of cell surface markers and peroxidase was analyzed dai ly. The initial purity of CD34(+) cells ranged between 80% and 90%; af ter 12 days, the frequency of neutrophil bands and segment ed neutroph ils was approximately 60%. Using RT-PCR to determine the ratio of wt1 to beta-actin expression, we reproducibly detected maximum expression of wt1 mRNA at day 0 in two cultures and at day 1 in two other CD34(+) cell cultures; at both these time points nearly all cells fulfilled t he morphological and immunephenotypical criteria of early hematopoieti c blast cells. Wt1 expression dropped rapidly at day 1. and 2, respect ively, in these two pairs of cultures, and was accompanied by an incre ase of cells expressing CD33 surface antigen. Our data suggest that wt 1 expression is restricted to a subset of CD34(+) progenitors and down regulated in later stages of differentiation in vitro.