Sequence of prolactin effects on phospholipid synthesis in Nb2 node lymphoma cells

In Nb2 node rat lymphoma cells, the effects of prolactin (PRL) on the rates of incorporation of several precursors into neutral lipids, phospholipids and proteins were determined. The onset of the PRL stimulation of radiolabeled-precursor incorporation into lipids occurred between 1 and 4 hours after PRL addition to Nb2 cells; precursors employed included [14C]-acetate, [3H]-glycerol, [32P]O4, [3H]-choline, [3H]-ethanolamine, [3H]-serine and [3H]-myoinositol. No effects were observed during the initial 60 min of culture with PRL. The effects on precursor incorporation that occur after 1 hr of PRL exposure are likely related to the stimulation of cell growth by PRL. In cells that were prelabeled with the radiolabeled precursors and subsequently incubated with PRL, PRL had no effect on the metabolism of the radiolabeled phospholipids or the accumulation of phospholipid products until several hours after hormone addition. We would conclude from these studies that the initial (60 min) effect of PRL on Nb2 node lymphoma cells does not likely use a signal transduction mechanism that involves products derived from the cellular phospholipids.     

Studies on the possible role of protein kinase C in the prolactin regulation of cell replication in Nb2 node lymphoma cells

The results of several recent studies have indicated that protein kinase C (PKC) may be involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The PKC activator 12-O-tetradeconylphorbol-13-acetate (TPA) at certain concentrations has been shown to potentiate the mitogenic effect of PRL, whereas at higher concentrations, TPA inhibits the PRL response. Several inhibitors of PKC have also been shown to impair the PRL stimulation of metabolic process in the Nb2 cells. These studies provide further evidence for the likely involvement of PKC in the PRL stimulation of mitogenesis in the Nb2 cells. A transient, time-dependent accumulation of PKC in the particulate fraction of the Nb2 cells is observed in response to PRL. TPA is also shown to elicit a similar effect, albeit at a much earlier time and with a greater magnitude. On long-term exposure (3 days), high concentrations of TPA down-regulate the PKC enzyme; this down-regulation likely accounts for the inhibitory effect of high concentrations of TPA on the PRL stimulation of cell division. In further studies, the PKC inhibitors H-7 and gossypol were shown to inhibit the PRL stimulation of cell division in a concentration-dependent fashion.     

Possible involvement of the phospholipases in the mitogenic actions of prolactin (PRL) on Nb2 node lymphoma cells

The possible role of the phospholipase enzymes in the prolactin stimulation of mitogenesis in Nb2 node lymphoma cells was investigated. Two phospholipase inhibitors including quinacrine and alpha-para-dibromoacetophenone (BPB) were employed. Quinacrine at concentrations of 1-5 microM attenuated the magnitude of the PRL stimulation of cell division; at concentrations of 10 microM and above quinacrine abolished the PRL response. BPB at concentrations of 1-10 microM also inhibited the mitogenic effect of PRL in a concentration response fashion. The polyunsaturated fatty acid arachidonic acid partially reversed the inhibitory effects of these drugs. In further studies, exogenously added phospholipase C at concentrations of 5-50 ng/ml was found to potentiate the mitogenic effect of prolactin when prolactin was employed at a concentration that evoked a half-maximal response. By itself, however, phospholipase C had no effect on the rate of cell division. Phospholipase A2 either by itself or in the presence of prolactin was without effect.     

A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.     

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

An inhibitor of ornithine decarboxylase in lactogen-deprived Nb2 node rat lymphoma cells

A previous study has shown that the activity of ornithine decarboxylase in cultured Nb2 node rat lymphoma cells falls to undetectable levels when cells become quiescent following incubation in lactogen (prolactin)-deficient medium. In the present study, it was found that addition of extracts of the lactogen-deprived, quiescent cells to extracts of log-phase cells markedly reduced the ornithine decarboxylase activity of the latter, the inhibitory activity being proportional to the amount of quiescent cell extract added. Evidence is presented that the ornithine decarboxylase-inhibitory activity in the quiescent cell extracts is due to an antizyme-like, polypeptide factor with an Mr of approx. 28,000. The activity of the inhibitor appears to be directed rather specifically against ornithine decarboxylase, since the activities of S-adenosylmethionine decarboxylase, thymidine kinase and uridine kinase were not affected. The Nb2 cell ornithine decarboxylase inhibitor may have an important role in modulating the cellular levels of ornithine decarboxylase as they change in response to the withdrawal and restoration of extracellular mitogenic lactogens.     

Growth and protein phosphorylation in the Nb2 lymphoma: effect of prolactin, cAMP, and agents that activate adenylate cyclase

The Nb2 T lymphoma is unique in that these lymphocytes proliferate in response to prolactin as well as in response to interleukin-2. In this study, we have examined the responsiveness of the adenylate cyclase system in Nb2 cells and the role of this signaling system in regulating proliferation and protein phosphorylation. An analog of cAMP inhibited prolactin-stimulated proliferation and blocked a prolactin-induced decrease in protein phosphorylation. Forskolin, a potent activator of adenylate cyclase in T lymphocytes, did not elevate cAMP levels in Nb2 cells and was not an effective inhibitor of prolactin-induced proliferation. In fact, one preparation of forskolin stimulated proliferation of quiescent Nb2 cells. Like forskolin, prostaglandin E2 did not stimulate cAMP production in Nb2 cells even though it increased cAMP in a preparation of rat peripheral blood lymphocytes. Cholera toxin appeared to ADP-ribosylate a stimulatory guanine nucleotide-binding protein in Nb2 cells, but the toxin did not increase intracellular levels of cAMP nor was it a potent anti-mitogenic agent. Pertussis toxin, an agent that can increase cAMP production through suppression of the inhibitory guanine nucleotide-binding protein, exerted only minor anti-proliferative actions on prolactin-stimulated Nb2 cells. These data suggest that cAMP inhibits Nb2 cell proliferation and prolactin-induced changes in protein phosphorylation but that the adenylate cyclase system in our clone of Nb2 cells responds poorly to agents that normally increase cAMP.     

L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphoma cells

Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL-dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitric oxide (NO) was recently identified as a downstream regulator of PRL action, and as an inhibitor of apoptosis in immune cells. In the present study, the role of NO in PRL-regulated Nb2 cell function was investigated. Nb2 cells expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The eNOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells but decreased by at least 3-fold at 3-24 h following PRL treatment. Downregulation of eNOS was not accompanied by a corresponding decrease in the eNOS protein, the level of which remained constant for at least 24 h after PRL treatment. PRL had no effect on the phosphorylation state or subcellular redistribution of the eNOS enzyme, or on production of NO by Nb2 cells. However, increasing concentrations of L-arginine (NOS substrate) alone increased NO production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanced Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.125 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survival and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone significantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Expression of the anti-apoptotic gene bcl-2, which was stimulated within 1 h by PRL, was upregulated by L-arginine or DEA/NO alone at 2 h and 8 h, respectively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prolactin-independent, Bcl-2-mediated pathway.     

Studies on DNA content, RNA synthesis, and DNA template activity in aging cells of Paramecium aurelia.

Investigations by Feulgen microspectrophotometry in Paramecium aurelia indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [³H]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of [³H]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Effects of mezerein and diglycerides on the PRL stimulation of cell replication in Nb2 node lymphoma cells

These studies provide further support for the thesis that the activation of protein kinase C is likely involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The diterpene mezerein is shown to potentiate the mitogenic effect of PRL at a hormone concentration which elicits a less than maximum response. A similar response was observed with two diglycerides, diolein and dicaprin. Neither mezerein nor the diglycerides affected the magnitude of response to a maximum stimulatory concentration of PRL.     

Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.     

Effect of 2 MHz ultrasound on DNA, RNA and protein synthesis in Pisum sativum root meristem cells.

The amounts of DNA, RNA and protein synthesis were determined in Pisum sativum root meristem cells at various times after a 1 min exposure to 1 MHz ultrasound at a power density of 30 W/cm2. Immediate depressions in all three macromolecular syntheses occurred after sonication, followed by an apparent recovery several hours later. These events appear to correlate in time with the subsequent reduction and recovery in mitotic index in Pisum sativum root meristem cells exposed to 2 MHz ultrasound.     

Studies on the mechanism by which glutamine and heat shock increase lactate synthesis by L929 cells in the presence of insulin

Both glutamine and heat shock increase lactate production in the L929 cell system. Glutamine is now shown to increase hexose uptake in the presence of insulin, to inhibit pyruvate oxidation, and to provide reducing equivalents to the cytosolic compartment. The relative contribution of these processes to lactate production depends on the availability of pyruvate. When ample pyruvate is available from the culture medium, stimulation of lactate synthesis by glutamine and heat shock is transaminase dependent, suggesting that shuttling of reducing equivalents from mitochondria to cytoplasm is involved. In the absence of medium pyruvate, stimulation of glycolysis by both glutamine and heat shock is largely responsible for increased lactate synthesis. None of the observed effects of glutamine appears to be sufficient to explain the observed stimulation of glycolysis.     

DNA, RNA, and protein synthesis by porcine oocyte-cumulus complexes during expansion

The experiments described in this report were designed to determine three biosynthetic functions of oocyte-cumulus complexes during expansion. The events investigated were DNA, RNA, and protein synthesis during a 24-h in vitro culture; these were determined by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation into oocyte-cumulus complexes, respectively. The quality of proteins produced was also determined by slab-gel electrophoresis. Results indicated that, during follicle-stimulating hormone-induced cumulus expansion, total DNA synthesis was significantly (P less than 0.05) reduced whereas RNA synthesis remained unchanged. Overall protein synthesis was markedly increased (P less than 0.05), with one major band (Mr = 22,000) and two minor bands (Mr = 19,500 and 78,000) being produced during expansion.     

Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.     

Protective effect of prolactin and placental lactogen on NO-induced Nb2 lymphoma cell apoptosis

Nitric oxide (NO) is an important modulator involved in immune regulation. Here, we describe conditions under which NO-donors induce apoptosis on Nb2 lymphoma cells, as evidenced by decreased cell viability and increased hypodiploid DNA content determined by flow cytometry. In addition, DNA fragmentation typical of apoptosis was shown by agarose gel electrophoresis. This apoptosis was accompanied by a significant increase of caspase-3-like enzymatic activity. Both ovine prolactin (oPRL) and ovine placental lactogen (oPL) exerted a protective effect on the NO-donor-induced apoptosis. Furthermore, dexamethasone (Dex)-induced cell death was also associated with caspase-3-like activity and oPL had the same potency as oPRL in its protective effect on Dex-induced apoptosis of Nb2 cells.