P120 GAP REQUIREMENT IN NORMAL AND MALIGNANT HUMAN HEMATOPOIESIS

There is evidence to suggest that the p120 GAP (GAP), originally descr ibed as an inhibitor of p21ras, may also serve as a downstream effecto r of ras-regulated signal transduction. To determine whether GAP expre ssion is required for the growth of human normal and leukemic hematopo ietic cells, we used GAP antisense oligodeoxynucleotides to inhibit it and analyzed the effects of this inhibition on the colony-forming abi lity of nonadherent, T lymphocyte-depleted mononuclear cells and of hi ghly purified progenitors (CD34+ MNC) obtained from the bone marrow an d peripheral blood of healthy volunteers or chronic myeloid leukemia ( CML, bcr-abl-positive) patients. The acute myelogenous leukemia cell l ine MO7, the Philadelphia1 BV173 cell line, and the acute promyelocyti c leukemia NB4 and HL-60 cell lines were similarly examined. GAP antis ense treatment inhibited colony formation from normal myelo-, erythro- , and megakaryopoietic progenitor cells as well as from CML progenitor cells. Proliferation of MO7 (growth factor-dependent) and BV173 (bcr- abl-dependent) cells, but not that of NB4 and HL-60 (growth factor-ind ependent) cells, was also inhibited, even though a specific downregula tion of GAP was observed in each cell line, as analyzed by either or b oth mRNA and protein expression. Stimulation of MO7 cells with hematop oietic growth factors increased the expression of GAP as well as the l evels of active GTP-bound p21ras. Stimulation of GAP expression was in hibited upon GAP antisense treatment. These data indicate that p120 GA P is involved in human normal and leukemic hemopoiesis and strongly su ggest that GAP is not only a p21ras inhibitor (signal terminator), but also a positive signal transducer.