Growth Regulation in Hematopoietic Cells Is Partially Mediated by Double Stranded RNA-Dependent Protein Kinase

Abstract 862 Poster Session II, Sunday, 5/2 (poster 141)

An understanding of the mechanisms which control quiescence of early hematopoietic stem cells remains an elusive but essential element in the development of strategies to expand hematopoietic stem cells for transplantation. Double-stranded RNA-dependent protein kinase (PKR) downregulates protein synthesis and inhibits cell proliferation in virally infected cells. It has been postulated that PKR is also involved in the regulation of proliferation of normal cells. To test the possible role of PKR in the regulation of hematopoietic cell proliferation, we characterized interferon-induced expression and activity of PKR in two IL-3 dependent cell lines of hematopoietic origin, TF-1, and FDC-P1, both of which are sensitive to the growth inhibitory effects of interferon. First, constitutive expression of PKR was detected in both cell lines by fluorescence-activated cell sorting and western blot, and was increased 5-10 fold by exposure to interferon (500 IU/ml). Secondly, PKR activity, detected by measurement of intracellular elF-2α (PKR substrate) phosphorylation, was increased in FDC-P1 and TF-1 cell lines by 40% and 100% respectively upon exposure to interferon. Third, PKR activity (as measured both by substrate phosphorylation and by PKR autophosphorylation) was similarly increased in both cell lines when cells were deprived of IL-3. Finally and most significantly, the inhibitory effects of interferon (30-50% inhibition by 500 IU/ml interferon) on both FDC-P1 and TF-1 cell lines could be reversed by exposure of cells to antisense oligonucleotides (ODNs) directed at the PKR mRNA. Specifically, treatment of TF-1 or FDC-P1 cells with 10 µM species-specific ODNs resulted in abrogation of the growth inhibitory effects of interferon. Taken together with published data linking PKR to growth control in other normal cells, these findings suggest a regulatory role for PKR in normal hematopoiesis, and identify PKR as a potential therapeutic target for strategies to expand hematopoietic stem cells.