Craig V. Byus

Type I and type II cyclic AMP-dependent protein kinase as opposite effectors of lymphocyte mitogenesis

THE role of cyclic AMP in the regulation of cell growth and differentiation has been extensively investigated in many cell types following various growth stimuli1,2. In recent years, peripheral blood lymphocytes treated with antigens3, plant lectins4, lipopolysaccharides5, or periodate oxidation6 have served as a model system for assessing whether cyclic AMP is involved in the regulation of the mitogenic response promoted in these cells by the above classes of agents7,8. There are two conflicting hypotheses, as outlined by Friedman1, concerning the role of cyclic AMP in lymphocyte proliferation. The first hypothesis suggests that the increase in cyclic AMP levels following mitogen stimulation8 and the subsequent increase in 32P incorporated into F1 histones9 and certain cystosol proteins10 implicates cyclic AMP as a positive mediator of mitogenesis in lymphocytes. The other hypothesis reasons that cyclic AMP inhibits proliferation, and this hypothesis is supported primarily by observations from several laboratories that raising the intracellular cyclic AMP level through the use of phosphodiesterase inhibitors11, prostaglandins12, or cyclic AMP analogues11,13–15 effectively inhibits mitogen-stimulated RNA and DNA synthesis. We present here evidence that may help resolve this apparent paradox. Incubation of Ficoll–Hypaque purified human peripheral blood lymphocytes with concanavalin A (conA) leads to the activation of only type I cyclic AMP-dependent protein kinase in these cells even though there are significant amounts of both type I and type II kinase present. But, the addition of dibutyryl cyclic AMP (DBcAMP) to the conA-stimulated lymphocytes at a concentration sufficient to block the synthesis of RNA and DNA results in the activation of both type I and type II cyclic AMP-dependent protein kinase. It therefore seems likely that while the activation of type I protein kinase represents a positive component in the progression of events promoted in a lymphocyte by a mitogenic signal (as it does in other cell types in which a trophic response is evoked), the activation of type II protein kinase (or perhaps types I and II in concert) represents a mechanism by which a negative influence can be imposed on the proliferative process by the generation of abnormally high levels of intracellular cyclic AMP.

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

Identification and characterization of a diamine exporter in colon epithelial cells.

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

Effect of immobilization and concurrent exposure to a pulse-modulated microwave field on core body temperature, plasma ACTH and corticosteroid, and brain ornithine decarboxylase, Fos and Jun mRNA.

Abstract Stagg, R. B., Hawel, L. H., III, Pastorian, K., Cain, C., Adey, W. R. and Byus, C. V. Effect of Immobilization and Concurrent Exposure to a Pulse-Modulated Microwave Field on Core Body Temperature, Plasma ACTH and Corticosteroid, and Brain Ornithine Decarboxylase, Fos and Jun mRNA. Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (±0.3°C) during the initial 30 min of loose- tube restraint in conditioned animals. When conditioned/tube- trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2–3 min, ACTH increased to 83.2 ± 31.0 pg/dl, compared to 28.1 ± 7.7 pg/dl for cage controls, reaching a maximum at 15–30 min (254.6 ± 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 ± 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3–10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 ± 54.8 compared to 31.2 ± 10.2 pg/dl; corticosterone, 12.2 ± 6.2 μg/dl compared to 37.1 ± 6.4 μg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10–20 fold higher (541.8 compared to 27.2–59.1 pg/dl for ACTH) and 2–5 fold higher (45.7 compared to 8.4– 20.0 μg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Selective putrescine export is regulated by insulin and ornithine in Reuber H35 hepatoma cells

Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10(-7) M insulin yielded intracellular putrescine levels that remained elevated for 36 along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0-12 h) and 1.0 (12-36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC50 approx. 1 microM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.

Induction of ornithine decarboxylase by 12-0-tetradecanoylphorbol-13 acetate in rat tissues

Summary Injection of 100μ g of 12-0-tetradecanoylphorbol-13-acetate (TPA) intraperitoneally led to a marked increase in ornithine decarboxylase (ODC) activity in the brain, liver, and lung of male rats. The enzyme activity was increased 45-fold, 9-fold, and 8-fold above controls in the liver, brain, and lung respectively. Maximal induction of the enzyme occurred at 5 hr and returned to control levels by 12 hr. Tumor promotor-induced ornithine decarboxylase activity was also observed in the liver and lung of hypophysectomised animals but not in the brain. The implications of these data on future studies involving the promotion of TPA of tumors initiated in tissues other than the epidermis are discussed.

A kinetic characterization of putrescine and spermidine uptake and export in human erythrocytes.

Using human erythrocytes as a model system for the study of mammalian polyamine transport, detailed kinetic parameters regarding the uptake and export of putrescine and spermidine were determined. The putrescine uptake data indicated a multi-component uptake system comprised of a low-capacity saturable component and a non-saturable component. The saturable putrescine uptake component demonstrated a calculated Km of 21.0 microM and a V(max) of only 6.52 x 10(-13) M/s. The non-saturable linear putrescine uptake rate was defined by a significant pH dependence, a lack of uptake inhibition by related polyamines, and a permeability pi of 3.19 x 10(-8) s-1. These findings suggested that non-saturable putrescine uptake involved a process of simple diffusion. Spermidine uptake exhibited Michaelis-Menten kinetics with a Km and Vmax of 12.5 microM and 1.36 x 10(-12) M/s, respectively. Spermidine uptake did not demonstrate pH dependence and was not significantly inhibited by any of the tested polyamines. The Arrhenius plot of spermidine uptake was determined to be biphasic with calculated activation energies of spermidine uptake of 135.2 kJ/mol for 19-21 degrees C and 59.3 kJ/mol for 21-35 degrees C. These data suggest the possibility of multiple spermidine uptake processes which are not mediated by simple diffusion across the cell membrane. The putrescine export process demonstrated both saturable and non-saturable components. The calculated Km, V(max) and pi for putrescine export were 33.8 microM, 1.19 x 10(-11) M/s and 2.81 x 10(-7) s-1, respectively. The spermidine export process was non-saturable up to intracellular spermidine concentrations of 4 microM. At similar intracellular and extracellular concentrations of putrescine and spermidine, however, export processes displayed rates which were an order of magnitude greater than their respective uptake rates. This finding supports the possible presence of mediated putrescine and spermidine export processes different than simple diffusion.

Characterization of putrescine and cadaverine export in mammalian cells

We characterized the mechanism(s) involved in the efflux of putrescine/cadaverine from cultured mammalian cells using various pharmacological agents. Verapamil and quinine inhibited putrescine and cadaverine export in monocytic-leukemic RAW 264 and H35 hepatoma cells in a concentration-dependent manner with an IC50 in the micromolar range. Verapamil, which inhibits L-type calcium channels, inhibited putrescine export, regardless of whether calcium was present in the extracellular medium or not. Furthermore, the export of putrescine in the absence of verapamil did not appear to depend upon extracellular calcium. Neither intracellular calcium, external sodium, changes in intracellular pH nor phosphorylation affected the levels of putrescine export independently from changes in intracellular putrescine levels. The data suggest that verapamil and quinine inhibit putrescine/cadaverine efflux from the cell by binding directly to an integral membrane protein.

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]