p21Waf1 inhibits granulocytic differentiation of 32Dcl3 cells

Defining the molecular mechanisms that prevent myeloid progenitor cells from maturing is important because defects in maturation contribute to the development of myeloproliferative and myelodysplastic diseases. IL-3 is an important developmental factor for myeloid progenitor cells in vivo and is required to maintain the undifferentiated state in the 32Dcl3 cell line. The mechanisms employed by IL-3 to block differentiation, however, are not well understood. 32Dcl3 cells are myeloid progenitor cells of murine origin with high basal levels of p21waf1/cip1 (p21) expression. Our laboratory has previously reported that p21 levels decreased as CD34+-derived myeloid progenitor cells underwent terminal granulopoiesis in vitro. The effect of p21 upon the expression of genes associated with granulocytic differentiation has been unexplored, however. Since IL-3 maintains high levels of p21 in 32Dcl3 cells, we tested the hypothesis that p21 is an inhibitor of myeloid differentiation. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3, proteins not abundant in cells maintained as myeloblasts by IL-3. Rescue with human p21 in these cells suppressed premature granule protein expression. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF. Since high expression levels of p21 and overexpression of the IL-3 receptor have been correlated with poor outcomes in acute myeloid leukemias (AML), differentiation blockade by p21 may be one mechanism that contributes to AML pathogenesis.