ISOLATION AND CHARACTERIZATION OF HL-60 CELLS RESISTANT TO NITROPRUSSIDE-INDUCED DIFFERENTIATION

Sodium nitroprusside and sodium nitrite, which generate nitric oxide a nd increase the intracellular cGMP concentration, and 8-bromo-cGMP, a membrane-permeable cGMP analog, induce myelomonocytic differentiation of HL-60 cells (Boss, G. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7174-7178). We have selected HL-60 cells resistant to nitroprusside-i nduced differentiation as assessed by acquisition of the OKM-1 antigen , reduction of nitro blue tetrazolium, and morphologic maturation. The variant cells were also resistant to differentiation induced by sodiu m nitrite and two cGMP analogs but still differentiated in response to other inducing agents such as dimethyl sulfoxide and cAMP analogs and showed the same changes in c-myc and c-fos expression in response to the latter drugs as occurred in parental cells. We studied the early s teps of the NO/cGMP signal transduction pathway in the variant cells a nd found that basal and nitroprusside-stimulated guanylate cyclase act ivity was similar in parental and variant cell extracts and that nitro prusside increased the intracellular cGMP concentration to the same ex tent in parental and variant cells. As part of these studies we found that HL-60 cells expressed only alpha(2) and beta(2) guanylate cyclase mRNA; the abundance of these two mRNA species was similar in parental and variant cells. Neither nitroprusside nor 8-bromo-cGMP changed the intracellular calcium concentration in parental or variant cells. The data suggest that the defect in the variant cells is after guanylate cyclase activation in the NO/cGMP transduction pathway and that the cG MP and cAMP transduction pathways operate independently in inducing di fferentiation of HL-60 cells.