The immunomodulatory role of angiocidin, a novel angiogenesis inhibitor.

The observation that many tumors exist in a microenvironment comprised of immune cells has led to the hypothesis that the immune system may play a significant role in the suppression of tumor growth. It is now clear that immune effector cells are capable of recognizing and destroying some cancer cells. However, tumors have developed numerous mechanisms by which they avoid immune recognition and death. Cancer immunotherapy attempts to harness the power of the immune system and direct it against tumor growth, while circumventing the immune-evasion strategies utilized by tumors. Many approaches are currently being investigated, including the re-infusion of autologous immune effector cells (i.e. cytotoxic T lymphocytes and macrophages) back into hosts after ex vivo expansion and activation. The therapeutic effects of specific cytokines are also being evaluated for their impact on tumor growth. Our lab has discovered a novel thrombospondin-1 (TSP-1) binding protein, termed "angiocidin", with potent anti-tumor and anti-angiogenic capabilities. To further investigate the anti-tumor activity of angiocidin, we examined whether angiocidin could play a role in immune system modulation. We have found that the monocytic leukemia cell line THP-1, as well as freshly isolated human peripheral blood monocytes, differentiate into macrophage-like cells when treated with angiocidin. These cells underwent dramatic morphological changes and became more phagocytic. Angiocidin-treated monocytes also activated T lymphocytes in co-culture conditions. Angiocidin-treated THP-1 cells upregulated cytokine mRNA expression and secretion via NF-kappaB, MAPK, and PI3-K. Based on these data, we hypothesize that angiocidin's ability to elicit tumor cell death may be mediated in part by it's pro-inflammatory effects on immune cells in the tumor microenvironment.

Tumor necrosis factor (TNF) and phorbol ester induce TNF-related apoptosis-inducing ligand (TRAIL) under critical involvement of NF-kappa B essential modulator (NEMO)/IKKgamma.

We show that tumor necrosis factor (TNF) and phorbol 12-myristate 13-acetate (PMA) induce TNF-related apoptosis-inducing ligand (TRAIL) in T cells. In cells deficient for NF-kappaB essential modulator (NEMO)/IKKgamma, an essential component of the NF-kappaB-inducing I-kappaB kinase (IKK) complex, induction of TRAIL expression was completely abrogated but was recovered in cells restored for IKKgamma expression. In cells deficient for receptor-interacting protein expression TNF, but not PMA-induced TRAIL expression was blocked. Inhibition of protein synthesis with cycloheximide blocked PMA, but not TNF-induced up-regulation of TRAIL. As both TNF and PMA rapidly induce NF-kappaB activation this suggests that NEMO/IKKgamma-dependent activation of the NF-kappaB pathway is necessary but not sufficient for up-regulation of TRAIL in T cells. The capability of the NF-kappaB pathway to induce the potent death ligand TRAIL may explain the reported proapoptotic features of this typically antiapoptotic pathway.

Protein kinase C mu selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway.

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.

Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1.

Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32.

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu.

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.

Protein kinase C mu is negatively regulated by 14-3-3 signal transduction proteins.

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3tau isoform has been shown to associate with protein kinase C theta and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau interacts with protein kinase C mu (PKCmu), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCmu and 14-3-3tau can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCmu-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCmu deletion mutants, the 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau to PKCmu is significantly enhanced upon phorbol ester stimulation of PKCmu kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3tau is not a substrate of PKCmu. In contrast 14-3-3tau strongly down-regulates PKCmu kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCmu kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCmu in T cells.

Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells.

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

An immobilized two-enzyme system (fungal alpha-amylase/glucoamylase) and its use in the continuous production of high conversion maltose-containing corn syrups.

Fungal alpha-amylase (E.C. 3.2.1.1) and glucoamylase (E.C. 3.2.1.3) were chemically attached to separate reactor modules made from Microporous Plastic Sheet (MPS). Immobilization of enzymes and subsequent chemical reactions were accomplished by pumping reactants through the sheet, i.e., perpendicular to the surface. The expressed activity of the reactor modules was ca. 800 U/g for both fungal alpha-amylase and glucoamylase. The kinetics and short-term effects of pH and temperature on the expressed activity of the immobilized enzymes were investigated. Using commercially available DE-42 corn syrup at 50% dissolved solids, half-lives of 2000 and 5000 h were achieved for glucoamylase and fungal alpha-amylase, respectively. The reactors were operated at 50 degrees C and at pH 4.3 for glucoamylase and 5.5 for fungal alpha-amylase. A typical DE-62 corn syrup product was continuously produced in a two-stage reactor system by pumping the feedstock through the glycoamylase reactor and then through the fungal alpha-amylase reactor. Saccharide distributions at each stage were controllable to +/-0.2%.