No 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 ...

In view of the frequent contamination of tissue cultures with organisms of the pleuropneumonia group (PPLO), we are reporting the effectiveness of tylosin tartrate in elminating such a contaminant from our culture.During the attempt to plate in agar one of our lines of murine leukemia cells(l), we detected a filterable microorganism which may be related to Mycoplasma granu1arum.f Several chemotherapeutic and antibiotic agents, namely tetracyclin, kanamycin, erythromycin, chloromyce tin, polymixin, s tovarsol and iodine had been previously tested with uniformly negative results. Although the cultures frequently seemed ('cured" when examined immediately after treatment, PPLO reappeared in the subsequent passages.Materials and methods. Purina tylan soluble (tylosin tartrate), a product of Eli Lilly and Co., is distributed by Ralston Purina Co. Prior to the use of tylan in tissue cultures, it was tested with bacteriologic media for activity against M . pulmonis of murine infectious catarrh and M . granularurn, recovered from tissue cultures. These two species are readily cultivable in 20% horse serum-nutrient bouillon and 48 hours-old cultures were employed. Transfers (.OS ml) were made to serum-bouillon and serum-agar containing remectively 100, 50. and 2 5 pg/ml of tylan. The two higher concentrations completely suppressed growth of both mycoplasmas in both media, as did also the lowest one with M . pulmonis. Growth of M . granularum was not completely inhibited, however, in the presence of 25 pg/ml and a few atypical colonies aDpeared on serum-agar. A concentration of 50 pg/ml was used, therefore, as a triaI *