Isolation and characterization of HL-60 cells resistant to nitroprusside-induced differentiation.

Sodium nitroprusside and sodium nitrite, which generate nitric oxide and 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-induced 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 sodium 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 steps of the NO/cGMP signal transduction pathway in the variant cells and found that basal and nitroprusside-stimulated guanylate cyclase activity was similar in parental and variant cell extracts and that nitroprusside increased the intracellular cGMP concentration to the same extent 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 cGMP and cAMP transduction pathways operate independently in inducing differentiation of HL-60 cells.

Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.

The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).

Double mixing stopped-flow method for the study of equilibria and kinetics of dimer-tetramer association of hemoglobins: studies on Hb Carp, Hb A, and Hb Rothschild.

A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp beta 37 (C3)----Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 M urea solutions (0.1 M Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers (K(Hb4L4 in equilibrium with 2Hb2) = 37.3 x 10(-6) M) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A.

Double-mixing kinetic studies of the reactions of methyl isocyanide and CO with diliganded intermediates of hemoglobin: alpha 2CO beta 2 and alpha 2 beta 2CO.

Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.

Selective cytotoxicity of L-canavanine in tumorigenic Madin-Darby canine kidney T1 cells.

L-canavanine, an analog of L-arginine, was examined for toxicity in a normal canine kidney epithelial cell line, Madin-Darby canine kidney (MDCK), and in its chemically transformed derivative MDCK-T1. Under conditions where the analog reversibly arrested growth of MDCK cells, more than 90% of the tumorigenic cells were killed. This selective cytotoxicity was not due to any difference in growth rate (both lines doubled every 24 h). Nor was it caused by the inhibition of protein synthesis or DNA replication, since amino acid deprivation and drugs which inhibited replication directly and indirectly did not kill the transformed cells preferentially. To the contrary, cycloheximide killed MDCK cells preferentially. Although the mechanism for the selective cytotoxicity of canavanine in the tumorigenic cells remains obscure, one clue was afforded by the observation that the analog caused a loss in plating efficiency of the T1 cells prior to their detachment from plates. Selective sensitivity to canavanine may therefore reside in cell surface proteins, which are known to differ both qualitatively and quantitatively between normal and transformed cells. The present results support our previous findings suggesting the possible value of using canavanine as an agent for cancer chemotherapy.