Abstract
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.
Highlights
Addition of nanomolar concentration of the phorbol to either mature granulocytes or macrophages by treatment ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) to with various chemical compounds (2-7)
Because the growth rate of mammalian cells is extracts of the seed of Croton tigliumand have been identified tightly coupled to the functionosf the protein synthetic machinery, we have examined whether TPA induaces change in HL-60 translational functions
Because the growth rate andDNA replication in mammalian cells is closely associated with changes in protein synthetic machinery (40-45), we investigated whether TPA can affect the translational functionosf HL-60 cells
Summary
HL-60 cells were generously supplied by Dr Judith Christman (Departments of Biochemistry and Pediatrics, Mount Sinai School of Medicine). ~ - [ ~ ~ S ] Methion(1i n10e0Ci/mmol) was from Amersham Corp. [U-'4C]Leucine (310 Ci/mol) and Enhance were obtained from New England Nuclear. [y3'P]ATP (4000-5000 Ci/mmol) was from ICN Radiochemicals. Threepl aliquots of the NEM-treated (0 or 15 min) fractions were tested for their protein synthesis inhibitory activity by using rabbit reticulocyte lysates. 2.5-111 aliquots of DEAE-100 (0.35 mg/ml) and DEAE-350 (0.57 mg/ml) fractions (an equal amount of proteins from control or TPA-treated cell extracts as used), 2.5 p1 of eIF-2 (2 mg/ml protein) or eIF-2B (1 mg/ml protein), or 2.5 pl of HCI (0.6 mg/ml protein) were added to 20plof master mix which contained 5 pl of buffer A (675mM KOAc, 10mM Mg(OAc)*, 100mM Hepes-KOH, 7.5 mM DTT, pH 7.4), 1 p1 of buffer B (1 mM ATP/ Mg(OAc)z, pH 7.4), 0.1pl of [y3'P]ATP (1.2 pCi,4286 Ci/mmol, ICN Radiochemicals),and 13.9 plof HzO.Each reaction mixture was raised to the final volume of p1 by the addition of H20 (if necessary). The reaction was stopped by the addition of25plof SDSsample buffer, and each sample was boiled for 15 min and subjected to 10% SDS-polyacrylamidegel electrophoresis
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