Induction of differentiation of murine erythroleukemia cells by aminonucleoside of puromycin and inhibition of this induction by purines and purine derivatives.

Abstract

The effect of the aminonucleoside of puromycin (AMS) on Friend erythroleukemia cells in culture was investigated, because purines and purine analogues are known to act as inducers of differentiation. After treatment with 20-30 psM AMS for 4 days, the cultures contained between 80 and 90% benzidine-positive cells. Stimulation of hemoglobin synthesis was dose and time dependent. Inosine had no stimulatory activity; however, when it was added to the medium together with AMS, erythroid differentiation was almost completely inhibited. The inhibitory effect of inosine on this potent inducer was also dose and time dependent. No cytotoxicity was observed with either compound, alone or in combination. Inhibition of AMS stimulation of erythroid differentiation was also observed in the presence of inosine monophosphate and poly(inosinic acid). Hypoxanthine had a dual effect. At high concentrations (500 jsg/ml) it acted as an inducer, but when added at low concentrations (20 ;&g/ml) together with AMS it inhibited differentiation. These findings suggest there is a link between purine biosynthesis and the event(s) required to trigger differentiation. Agonist-antagonist activity of closely related biological compounds has thus been revealed in the erythroleukemia cells. Various compounds have the ability to induce differentiation of Friend erythroleukemia (FL) cells, but the mechanism(s) of their action remains unclear (1). Because purine and purine analogues are efficient inducers (2), we were led to investigate the effect of the aminonucleoside of puromycin (AMS) on FL cells. This semisynthetic antibiotic [6-dimethylamino-9-3'-amino-3'-deoxyribosyl)purine] is an inhibitor of cell proliferation and RNA synthesis, particularly in normal diploid cultured mammalian cells, and its inhibitory effect can be prevented by inosine (3). The results of the present study demonstrate that these two closely related biological compounds also have an agonist-antagonist relationship on the differentiation of FL cells. AMS was found to be a potent inducer of erythrodifferentiation, and its effect was inhibited by inosine. Other compounds such as adenosine, hypoxanthine, inosine monophosphate (IMP), and poly(inosinic acid) [poly(I)] were also inhibitory, but to a lesser extent. MATERIALS AND METHODSCultures of 3-day-old FL cells of clone 745 were seeded at a concentration of 1 X 105 cells per ml in BHK21 medium (Eurobio, Paris, France) supplemented with fetal calf serum and maintained at 370C in a humidified CO2 incubator. To determine the number of cells synthesizing hemoglobin in each culture, the cells were stained with benzidine as described (4) and the number of benzidine-positive cells was scored.AMS, IMP, and hypoxanthine (obtained from Sigma), inosine (from P-L Biochemicals), and poly(I) (from Boehringer) were added to the cultures at the concentrations and the times indicated in the text. RESULTSIn order to determine the effect of AMS on FL cells, various concentrations of the compound were added to the cultures at the time of ...

Characterization of the metabolic forms of 6‐thioguanine responsible for cytotoxicity and induction of differentiation of HL‐60 acute promyelocytic leukemia cells

“…This possibility is supported by studies with kinase-deficient mutants that provide evidence for a link between the formation of cytotoxic analog nucleotides and the induction process. Thus, the differentiation inducing ability of xylosyladenine in the presence of an inhibitor of adenosine deaminase is abolished in an adenosine kinase-deficient clone of the Friend erythroleukemia (Lacour et al, 1980). Similarly, bromodeoxyuridine, a moderately effective initiator of the maturation of HL-60 cells, is unable to induce differentiation in a thymidine kinase deficient clone of this tumor (Koeffler et al, 1983).…”

“…A variety of purine and pyrimidine analogs have been shown to be inducers of differentiation in both the Friend (Ebert et al, 1976) and HL-60 (Bodner et al, 1981) leukemias. Some of the active inducers include the aminonucleoside of puromycin (Lacour et al, 1980), 5-bromo-2'-deoxyuridine (Koeffler et al, 19831, 3-deazauridine, pyrazofurin, virazole (Bodner et al, 1981) and xylosyladenine (Garrett and Kredich, 1981). The concentration of these antimetabolites required to initiate maturation is exceedingly close to the level required to produce cytotoxicity, implying that the metabolic actions involved in the production of cytotoxicity and differentiation may be the same.…”

Modulation of cyclic AMP levels and differentiation by adenosine analogs in mouse erythroleukemia cells

“…Similar studies were performed to determine whether PIA also inhibited induction of MEL differentiation by agents such as HMBA (Reuben et al, 1976), butyric acid , diazepam (Clarke and Ryan, 1980;Wang et al, 19841, hypoxanthine (Gusella and Housman, 1976), and the aminonucleoside analog of puromycin (AMS) (Lacour et al, 1980). The effects of PIA were dependent upon the inducer (Table 3).…”

Activation of lymphocytes by brominated nucleoside and cyclic nucleotide analogues: implications for the "second messenger" function of cyclic GMP.

“…Purines have been shown to induce and inhibit induction of differentiation in Friend erythroleukemia cells (25). Conjugation of nucleosides to IgG showed that guanosine was dominant, determining either tolerization or immunization, depending on the experimental protocol (26).…”