Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium.
Incorporation of [3H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulate significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 5 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures.
Sandru
G
G
Department of Radio-Oncology, University of Bern, Inselspital, Switzerland.
Greiner
R
R
D000328:Adult; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology; D007376:Interleukin-2 / Q000502:physiology; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000502:physiology; D008934:Mitogens / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism*; D014158:Transcription, Genetic / Q000528:radiation effects
D017077:Culture Media, Conditioned; D007375:Interleukin-1; D007376:Interleukin-2; D008934:Mitogens; D011691:Puromycin; D013936:Thymidine