Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.

Bioactive compounds from Psorothamnus junceus.

During a search for bioactive compounds from Psorothamnus junceus, four heterocyclic compounds, psorothamnone A (1), psorothamnone B (2), dalrubone (3), and emorydone (4) were isolated from the ethanol extract of the stem bark. Psorothamnones A (1) and B (2) demonstrated inhibitory activity against protein kinase C (PKC), a key enzyme involved in the signal transduction of cell proliferation and differentiation. Dalrubone (3) and emorydone (4) showed cytotoxicity against several human tumor cell lines.

Novel protein kinase C inhibitors: synthesis and PKC inhibition of beta-substituted polythiophene derivatives.

A series of beta-substituted polythiophene derivatives was synthesized through palladium-catalyzed coupling reaction. Their structure-protein kinase C (PKC) inhibitory activity relationship was studied. The carboxaldehyde and hydroxymethyl derivatives of alpha-terthiophene were potent PKC inhibitors (IC50 = 10(-7) M).

Novel protein kinase C inhibitors: alpha-terthiophene derivatives.

A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Inhibition of intracellular Ca2+ signalling, cytotoxicity and antitumor activity of the herbicide oryzalin and its analogues.

PURPOSE: Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca2+ ([Ca2+]i) signalling in mammalian cells. METHODS AND RESULTS: Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca2+]i signalling in Swiss 3T3 fibroblasts with IC50 values of 14, 16 and 18 microM, respectively. 45Ca2+ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC50 of 34 microM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC50 of 8 microM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC50 values in the range 3 to 22 microM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca2+]i signalling and inhibition of the growth of HT-29 human colon cancer cells (P = 0.001) and of CCRF-CEM human leukemia cells (P = 0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 microM and of its major metabolite N-depropyl oryzalin was 53 microM. CONCLUSION: It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations.

Activation of T cell Raf-1 at mitosis requires the protein-tyrosine kinase Lck.

The serine/threonine protein kinase Raf-1 is activated in response to a variety of growth factors in fibroblasts and hematopoietic cells. In T cells, Raf-1 is activated in response to stimulation through the T cell antigen receptor, the interleukin-2 receptor, and by stimulation of protein kinase C. We demonstrate here that in T cells, Raf-1 is also activated during mitosis. The mitotic activation of Raf-1 was not observed in the Lck-deficient cell line, J.CaM.1. During mitosis, Raf-1 was found to interact selectively with a mitotic form of Lck that migrated with a reduced electrophoretic mobility on SDS-polyacrylamide gels. We conclude that Raf-1 is activated during mitosis in T cells and that this mitotic activation of Raf-1 is dependent on the presence of Lck.

ras downregulation of protein kinase C mRNA in C3H 10T1/2 fibroblasts.

C3H 10T1/2 fibroblasts transformed by oncogenic ras have lower levels of protein kinase C (PKC) activity and protein. It was previously suggested that elevated levels of diacylglycerol in ras-transformed fibroblasts lead to activation-induced proteolysis of cellular PKC. We found that stable expression of T24ras in C3H 10T1/2 fibroblasts resulted in a significant decrease in levels of PKC alpha and PKC epsilon mRNA. Using C3H 10T1/2 cell lines in which the levels of activated ras can be exogenously regulated (by addition of zinc to induce the expression of a metallothionein-promoted human Ha-ras oncogene), we examined the temporal dependence of oncogenic ras expression on PKC downregulation. In these cells, downregulation of PKC protein and activity was induced but was not preceded by activation of PKC. The downregulation of PKC levels correlated with the appearance of a highly transformed morphology and was seen only at high levels of ras expression. In the inducible cells, the decrease in levels of PKC alpha mRNA had the same dependence on the levels of ras expression as did protein downregulation. These experiments provide evidence that downregulation of PKC protein levels by expression of oncogenic Ha-ras in C3H 10T1/2 fibroblasts is primarily due to altered transcriptional regulation. Because the downregulation of PKC was coupled with the onset of morphological transformation, the data suggest that this downregulation is involved in or facilitates the maintenance of a ras-transformed phenotype in C3H 10T1/2 fibroblasts.

Oncogene signal transduction inhibitors from medicinal plants.

Signal transduction is believed to be altered by cellular oncogenes or tumor suppressor genes during the transformation of normal cells into malignant cells. This proposition offers an attractive target for oncogene-based anticancer drug discovery from natural sources. Protein kinases encoded or modulated by oncogenes were used to prescreen the potential antitumor activity of medicinal plants. Protein-tyrosine kinase-directed fractionation and separation of the crude extracts of Polygonum cuspidatum and Koelreuteria henryi have led to the isolation of three different classes of protein-tyrosine kinase inhibitors, anthraquinone, stilbene and flavonoid. The anthraquinone inhibitor, emodin, displayed highly selective activities against src-Her-2/neu and ras-oncogenes.

Novel quinone antiproliferative inhibitors of phosphatidylinositol-3-kinase.

The inhibition of phosphatidylinositol-3-kinase (PtdIns-3-kinase), protein kinase C and c-Src protein tyrosine kinase by a series of halogenated naphthoquinones and quinoline quinones related to the plant-derived naphthoquinones juglone and methyljuglone, which inhibit protein kinase C, has been investigated. Some of the compounds inhibited PtdIns-3-kinase at micromolar concentrations and below. PtdIns-3-kinase inhibition was time dependent and could be prevented by endogenous thiol. The compounds were only weak inhibitors of PtdIns-4-kinase. Some of the compounds inhibited protein kinase C, but c-Src protein tyrosine kinase was only weakly inhibited. In intact cells, PtdIns-3-kinase was only partly inhibited by concentrations of the halogenated quinones that inhibited cell growth. Some halogenated quinones showed in vivo antitumor activity without accompanying toxicity, while methyljuglone was without in vivo antitumor activity. Halogenated quinones may have multiple biochemical effects in the cell that could contribute to their cytotoxic and antitumor effects. Inhibition of PtdIns-3-kinase by the halogenated quinones may provide a lead for the development of more potent and specific inhibitors.

Diet, signal transduction and carcinogenesis.

The defects in the regulation of cell growth and differentiation that manifest themselves as cancer result from multiple defective genes and their products, which are involved in the processes of cellular signaling, regulation of gene expression and control of the cell through its replication cycle. Each of these molecular defects represents a new target for development of novel therapeutic agents and prophylactic interventions. Evidence suggests that such therapeutic agents will show great efficacy for cells made cancerous by the single targeted defect. However, poor anticancer efficacy for clinically presenting cancer may occur as a result of the multiple molecular lesions. A combined-agent approach seems likely to be more successful, but this will require diagnosis of each tumor in substantially greater detail, down to the molecular level. When such molecular diagnosis becomes generally feasible, it should be possible to use combinations of highly specific agents at very low doses for therapy and ultimately for prevention of tumor metastasis. Chemoprevention in general may be achieved more easily than therapy with mechanism-based interventions, as certain individual lesions, in theory, may be rate limiting for carcinogenesis but may not be a significant contributor to the neoplastic phenotype by the time the tumor presents in the clinic.

Advances with phospholipid signalling as a target for anticancer drug development.

The phosphatidylinositol-3-kinases (PtdIns-3-kinase) are a family of enzymes involved in the control of cell replication. One member of the family, the mammalian p110/p85 PtdIns-3-kinase, is a potential target for anticancer drug development because of its role as a component of growth factor and oncogene activated signalling pathways. There are a number of inhibitors of this PtdIns-3-kinase, the most potent being wortmannin (IC50 4 nM). Wortmannin inhibits cancer cell growth and has shown activity against mouse and human tumor xenografts in mice. Other inhibitors of the PtdIns-3-kinase are halogenated quinones which also inhibit cancer cell growth and have some in vivo antitumor activity. Some D-3-deoxy-3-substituted myo-inositol analogues and their corresponding PtdIns analogues have been synthesized. They may act as myo-inositol antimetabolites in the PtdIns-3-kinase pathway and they can inhibit cancer cell growth.

Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver.

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.

A multiwell assay for inhibitors of phosphatidylinositol-3-kinase and the identification of natural product inhibitors.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol-3-kinase (PtdIns-3-K) has been developed. Four natural product inhibitors of Ptdlns-3-K have been identified with IC50 values for hypericin 0.18 microM, emodin 3.3 microM, asperuloside 2.0 microM and uttronin A 1.1 microM.

A multisample assay for inhibitors of phosphatidylinositol phospholipase C: identification of naturally occurring peptide inhibitors with antiproliferative activity.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol specific phospholipase C (PtdInsPLC) has been developed. Three naturally occurring peptide inhibitors of PtdInsPLC have been identified, myroridin K, streptothricin B and edeine, with IC50 values of 8.3, 6.7 and 16.1 microM, respectively. All three peptides inhibited colony formation of HT-29 human colon adenocarcinoma cells, with IC50 values of 7.2, 3.9 and 13.0 microM, respectively. The compounds also inhibited the growth of other human cancer cells in culture. One of the peptides, myroridin K, has previously been reported to have in vivo antitumour activity. It is possible that inhibition of PtdInsPLC is responsible for the cell growth inhibition and antitumour properties of the peptide compounds.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.

Phosphatidylinositol-3-kinase is an important enzyme for intracellular signaling. The microbial product wortmannin and some of its analogues have been shown to be potent inhibitors of phosphatidylinositol-3-kinase. The 50% inhibitory concentration for inhibition by wortmannin is 2 to 4 nM. Kinetic analysis demonstrates that wortmannin is a noncompetitive, irreversible inhibitor of phosphatidylinositol-3-kinase, with inactivation being both time- and concentration-dependent. Wortmannin has previously been reported to be an inhibitor of myosin light chain kinase but with an inhibitory concentration of 0.2 microM. Wortmannin was found not to be an inhibitor of phosphatidylinositol-4-kinase, protein kinase C, or protein tyrosine kinase. Wortmannin inhibited the formation of phosphatidylinositol-3-phosphates in intact cells. The results of the study suggest that wortmannin and its analogues may have utility as pharmacological probes for studying the actions of phosphatidylinositol-3-kinase.

Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues.

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genus Helenium, caused a marked potentiation of the increases in intracellular free Ca2+ concentration ([Ca2+]i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca2+ partly attenuated the increased [Ca2+]i responses caused by helenalin. The increased [Ca2+]i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca2+]i responses. No change in resting [Ca2+]i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca2+]i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca2+]i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca2+ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca2+]i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca2+ from these stores.

Kinase inhibitors from Polygonum cuspidatum.

Bioassay-directed fractionation of a medicinal plant, Polygonum cuspidatum (Polygonaceae), has led to the discovery of a hydroxystilbene, resveratrol [1], as an inhibitor of a protein-tyrosine kinase (p56lck) partially purified from bovine thymus. Both trans and cis isomers of resveratrol possess comparable protein-tyrosine kinase inhibitory activity. Comparison of the IC50 values of resveratrol for protein-tyrosine kinase inhibitory activity with those of piceid (resveratrol-O3-beta-glucoside) [2] and resveratrol-O4'-beta-glucoside [3] shows the requirement of free hydroxyl groups on both phenyl rings for the protein-tyrosine kinase inhibition. Protein kinase C inhibitory analysis suggests the requirements of two free hydroxyl groups on one phenyl ring only.