TGF beta-induced SMAD2 phosphorylation predicts inhibition of thymidine incorporation in CD34(+) cells from healthy donors, but not from patients with AML after MDS

Cells from patients with MDS-derived AML display heterogeneous proliferativ e responses to transforming growth factor beta (TGF beta). We analyzed grow th inhibition and SMAD2 phosphorylation by TGF beta in CD34(+) cells from n ine patients, as compared to normal controls. While TGF beta consistently i nhibited thymidine incorporation of normal cells (41% of control, P < 0.05) , cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the thr ee other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 p rotein was phosphorylated in normal CD34(+) cells (n = 3), CD34(+) leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leu kemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine i ncorporation, cell proliferation and survival in all samples analyzed. In M -07e cells and CD34(+) cells from healthy donors, this inhibition was enhan ced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059) . Conversely, in CD34(+) cells from a patient with AML, both AG490 and PD09 8059 significantly enhanced TGF beta -mediated suppression of TPO-induced t hymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activat ion.