Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60 leukemic cells.
Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.
Konno
S
S
Borelli
T J
TJ
Wu
J M
JM
D000818:Animals; D002457:Cell Extracts / Q000302:isolation & purification / Q000494:pharmacology; D002460:Cell Line; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2; D005636:Fructosephosphates / Q000494:pharmacology; D005914:Globins / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism / Q000494:pharmacology; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002457:Cell Extracts; D015852:Eukaryotic Initiation Factor-2; D005636:Fructosephosphates; D010448:Peptide Initiation Factors; D010704:Phorbols; D011506:Proteins; D012333:RNA, Messenger; C027618:fructose-6-phosphate; D005914:Globins; D013755:Tetradecanoylphorbol Acetate