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A protein with a molecular mass of 35-37 kDa has been isolated and partially purified from the postribosomal supernatant of wheat germ by ammonium sulfate precipitation (60-90%), Sephadex G-75, and DEAE-cellulose chromatography. It inhibited endogenous protein synthesis in rabbit reticulocyte lysates but had no effect on translation in wheat germ extracts. At low concentrations (0.34-1.36 ng/15 microliter assay), inhibition was limited to initiation of peptide synthesis. At higher concentrations (13.6 ng/15 microliter assay), elongation was also suppressed.

Evidence is presented of ET-1 release by cultured human fibroblasts. Such a conclusion is supported by the parallelism in displacement curves obtained using dilutions of extracts prepared from fibroblast conditioned media and the synthetic ET-1 standards, and the significant time dependent increase in ET-1 content in media from IFN-beta treated human fibroblasts. An increase in cell density significantly elevated the total amount of ET-1 in the conditioned media, although a linear relationship between these two variables was not observed.

Exposure of human leukemic HL-60 cells to monocyte-inducing compounds is accompanied by numerous newly synthesized protein changes in subfractions of the cell. Two proteins with molecular weights of 42,000 and 38,000 (p42, p38), present in the ribosomal salt wash (RSW) of proliferating cells, were markedly elevated and suppressed, in PMA-induced differentiated cells. Actin was identified as a component of p42. P38 cross-reacted with an actin-specific monoclonal antibody in Western blot analysis. Sequencing of gel-purified p38 showed that it was not N-terminally blocked and represented a truncated actin cleaved between val 43 and met 44. The reduction of p38 accompanying proliferating to differentiated cell transition may be indicative of proteolytic differences between the two cellular phenotypes.

The alkaloid nicotine induces a dose-dependent inhibition of cell proliferation and morphological change when added to BALB/C 3T3 cells. Significant differences were observed between control and nicotine-treated cells with respect to the newly synthesized secreted proteins by using heparin-agarose column chromatography. Both the anticellular and protein synthesis modulating activities of nicotine were affected by the degree of confluence of cells, suggesting a complex mode of action of nicotine in mammalian cells.

PMA treatment of human leukemic cells resulted in a significant increase in the phosphorylation of a 72-kDa protein, which was abrogated by treating the nuclear extracts with DNase I, but additionally stimulated by adding DNA. To be active, DNA must be double-stranded with an average size of 300 base pairs, but shows no apparent species- or sequence-specificity. NP-72 isolated from control or PMA-treated nuclei with 1 mM ATP lacked phosphorylating activity, suggesting it to be a substrate for a dsDNA-stimulated protein kinase(s). Simultaneous exposure of HL-60 cells to PMA and the protein kinase C inhibitor staurosporine diminished the phosphorylation of NP-72. These data suggest that leukemia cell differentiation is accompanied by the induction and/or activation of a dsDNA-stimulated protein kinase whose protein substrates include NP-72 and whose activity is directly or indirectly influenced by protein kinase C.

Human promyelocytic leukemia cells (HL-60) acquire monocytic biochemical characteristics when exposed to the alkaloid nicotine. We report herein that the nicotine-induced phenotype change is accompanied by the transient reduced expression of mRNA for the protooncogene c-myc. The suppressive effect of nicotine on c-myc mRNA levels is not reversed by the addition of ADP-ribosyl transferase inhibitor, 3-aminobenzamide.

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.

Staurosporine, an inhibitor of protein kinase C (PKC), has been shown to inhibit the induction of two cytosolic proteins, p42 and p91, during the differentiation of HL-60 cells initiated by PMA treatment. Differentiation of these cells into macrophages was also blocked. p42 has been identified as actin by Western Blot analysis and previous studies have demonstrated p91 to be the Ca+(+)-dependent actin-binding protein gelsolin. Increased levels of these proteins may be important for the attainment of macrophage functions, e.g. phagocytosis and secretion. These studies may therefore establish a link between PKC activation and the induction of specific cellular proteins, such as actin and gelsolin, as important monocyte maturation products without which the cells will be restricted from acquiring certain macrophage activities.

Cell growth and response to interferon (IFN) have been used to assess alteration in properties of cultured skin fibroblasts from patients with familial Alzheimer's Disease (AD). A comparison of the proliferative characteristics between "low" (7-11) and "high" (18-21) passage diploid fibroblasts from control and AD individuals shows that AD cells become severely restricted in their proliferative potential earlier than matched controls as a result of "in vitro aging". In addition, while control cells grow adequately in a serum-free, chemically-defined media (Nutridoma) (90-95% compared to FBS-supplemented media), AD cells grow poorly in Nutridoma (45-53% compared to FBS-supplemented media). An interferon (IFN)-inducible enzyme, 2'5'-oligoadenylate (2-5A) synthetase, is significantly reduced in IFN-treated AD cells compared to age-matched control cells in two cell passage-dependent manner. These data suggest that an intrinsic property of AD cells is the progressive reduced ability to interact with normal biologic signals provided by proteins such as interferons which in turn may contribute to the pathogenesis of AD.

The effects of highly purified human immune interferon (IFN-gamma) on the differentiation of human promyelocytic HL-60 leukemic cells have been studied. The addition of 100 units/ml interferon to HL-60 cells for 5 days results in morphological changes characteristic of macrophages. At the biochemical level, there is a 3-fold increase in the specific activity of the enzyme NADase. Kinetic analysis shows that IFN-gamma causes an increase in the Vmax of NADase without affecting the apparent Km. Pulse labeling experiments with [35S] methionine show a marked change in the de novo synthesis of several proteins in the course of interferon treatment. Chromatography on DNA-agarose show that after treatment with interferon for 24 or 48 h, there is a 60-70% decrease in newly synthesized proteins which bind DNA-agarose and can be subsequently displaced from the column with 2% SDS containing buffer (from 7.7-8.7% bound in control cell extracts to 2.6-3.1% bound in interferon treated cell extract).

Heparin, but not a number of other acid mucopolysaccharides, inhibits (2'-5')An synthetase partially purified from rabbit reticulocytes. The effect of heparin was apparent at 10 micrograms/ml (21%). Maximum inhibition was observed at 200 micrograms/ml (80%). At the latter concentration, the dsRNA-dependent (2'-5')An synthetase activity was inhibited 80% whereas the dsRNA-independent enzyme activity was inhibited only 30%. The formation of the dimer, trimer, tetramer and high oligomers of (2'-5')An was inhibited to the same extent with the addition of heparin. Heparin covalently bound to agarose beads was shown to be a useful affinity resin for the purification of (2'-5')An synthetase from rabbit reticulocyte lysates.

The binding of p3A4,3'-32P [pCp] to rabbit reticulocyte RNase L can be displaced by the trimer and tetramer triphosphates of 2',5'-oligoadenylates (2-5A). Using assay conditions of protein synthesis, 2-5A trimer or tetramer triphosphates are shown to be equally effective when the displacement is done at 4 degrees C (on ice). In contrast, at 30 degrees C, the tetramer triphosphates still displace whereas the trimer triphosphates become ineffective. When lysates are preincubated at temperature ranging from 4 degrees-37 degrees C, the same results are obtained even when the subsequent displacement is done on ice. Incubation temperature also significantly affects the ability of metabolically stable dyes cibacron blue and aurintricarboxylic acid to inhibit RNase L binding activity. Taken together, these results suggest that rabbit reticulocyte RNase L may assume multiple conformations which are differentially affected by various forms of 2-5A or other compounds.

By a radiobinding assay, an affinity labeling technique, and a column procedure which quantitates mRNA intactness, evidence is presented on the induction of the (2'-5')An-dependent endoribonuclease by interferon in cultured guinea pig macrophages.

Polyadenylated RNA has been isolated from control and interferon-treated HL-60 cells by centrifugation through cesium chloride and oligo(dT)-cellulose column chromatography. The affinity column-purified RNA is poorly translated in the mRNA-dependent rabbit reticulocyte lysates but is an excellent template for in vitro protein synthesis using the wheat germ cell extracts. The discrepancy in the efficiency of HL-60 mRNA utilization in the two commonly used cell-free protein synthesizing systems is attributable to an inhibitory component present in the polyadenylated RNA. This contaminant is most likely double-stranded RNA based on (i) the ability of 2-aminopurine (3-5 mM) or high concentrations of penicillium chrysogenum double-stranded RNA (10-15 micrograms/ml) to overcome the inhibition exerted by the component, and (ii) the ability of the component to promote the enzymatic conversion of ATP into 2-5A by the highly purified rabbit reticulocyte 2-5A synthetase.