Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Lymphocyte activation and phospholipid pathways. 31P magnetic resonance studies

31P NMR spectra of perfused lymphocytes, embedded in alginate capsules and activated by interleukin-2, were remarkably different from those of control lymphocytes. The main differences were the appearance and gradual increase in phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occurred following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and were not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibited the effects of phytohemagglutinin, but not of interleukin-2. There were no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine and phosphatidylethanolamine is an inherent feature of the activation process. 31P NMR spectra of lymphocytes are characterized by a low signal of phosphocholine. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of cytidylyltransferase, indicated that choline kinase plays a key role in regulating phosphaditylcholine synthesis in human lymphocytes.     

Uptake of lactoferrin by mononuclear phagocytes inhibits their ability to form hydroxyl radical and protects them from membrane autoperoxidation.

Human mononuclear phagocytes do not contain the iron-binding protein lactoferrin that we have previously demonstrated inhibits the potential for human neutrophils to generate hydroxyl radical in the presence of an exogenous iron catalyst of the Haber-Weiss reaction. Previous work by other investigators has suggested that mononuclear phagocytes (monocytes and monocyte-derived macrophages (MDM] have the capacity to bind exogenous lactoferrin via lactoferrin-specific membrane surface receptors. Accordingly, we examined the possibility that uptake of iron-free (apo) lactoferrin by human mononuclear phagocytes could play a role in limiting the potential for generation of hydroxyl radical during the monocyte/MDM respiratory burst. When monocytes or MDM were incubated in the presence of apo-lactoferrin, cell-associated lactoferrin increased in proportion to the concentration of lactoferrin provided. Similar results were obtained with iron-loaded (diferric) milk lactoferrin. Consistent with the in vivo importance of these findings, we found that lactoferrin was intimately associated with human alveolar macrophages obtained by bronchoalveolar lavage. The fucose polymer fucoidan inhibited lactoferrin uptake whereas exogenous transferrin or MDM exposure to IFN-gamma was without effect. Scatchard binding analysis confirmed the presence of a lactoferrin-specific receptor with a calculated kDa of 3.56 x 10(-6) M and 3.4 x 10(7) binding sites per cell. Subcellular fractionation studies indicated that twofold more of the lactoferrin which became cell-associated over the 1-h incubation time could be found in the cytoplasmic fraction compared to the plasma membrane-containing fraction, consistent with previous evidence by others for internalization of lactoferrin by mononuclear phagocytes. When lactoferrin-loaded monocytes/MDM were incubated in lactoferrin-free media, evidence for release of lactoferrin was obtained by SDS-PAGE and immunoblot analysis, suggesting the presence of a recyclable pool of cell-associated lactoferrin. To assess the impact of lactoferrin loading on monocyte/MDM hydroxyl radical formation, lactoferrin-loaded phagocytes were stimulated with PMA in the presence of catalytic iron. Hydroxyl radical generation by lactoferrin-loaded cells was decreased to about 50% of control cells. Similarly, monocytes that had been lactoferrin-loaded demonstrated a 28% decrease in autooxidation of their membrane when stimulated in the presence of catalytic iron. These data suggest that lactoferrin binding may play an important role in maintaining optimal mononuclear phagocyte function and protecting adjacent tissue from untoward phagocyte-associated hydroxyl radical generation.     

Effect of inflammatory cytokines on the adherence of tumor cells to endothelium in a murine model

We have demonstrated that pretreatment of mouse brain microvascular endothelial cells (MBE) with tumor necrosis factor-alpha (TNF), IL-1, or LPS augmented the binding of P815 mastocytoma cells in vitro. The effect of these agents was dose and time dependent. PMA was able to mimic the influence of these factors to a limited degree. The effect of TNF on endothelium was accompanied by the appearance of changes in the expression of proteins isolated from endothelial cell membranes. The adherence of tumor cells to endothelium was not inhibited by RGD-containing peptides but could be decreased by preincubation of endothelium with high concentrations of FCS. Our data suggest that cytokines regulate the synthesis of endothelial adhesion proteins which may be involved in tumor cell adherence leading to metastasis. These results raise the possibility that cytokines may exert paradoxical effects in vivo, i.e., a cytotoxic effect that reduces tumor mass accompanied by a metastasis-enhancing effect that actually promotes dissemination of the remaining tumor cells. Definition of the molecular events involved in tumor cell-endothelial cell interactions may lead to strategies for minimizing the latter effect in therapeutic settings.     

Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities

Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder.     

Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban.

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.     

Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association