ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation.

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.

Tumor invasion-inhibiting factor 2: primary structure and inhibitory effect on invasion in vitro and pulmonary metastasis of tumor cells.

The primary structure of tumor invasion-inhibiting factor 2 (IIF-2) purified from bovine liver (A. Isoai et al., Jpn. J. Cancer Res., 81:909-914, 1990) was determined. A computer homology search of the National Biomedical Research Foundation data bank revealed that IIF-2 is identical to the carboxyl-terminal region, residue number [69-89], of high mobility group 17 which is a DNA-binding non-histone protein. IIF-2 synthesized by an automated peptide synthesizer showed similar invasion-inhibitory activity as compared with the purified factor, when tested with the monolayer invasion assay system using highly invasive rat ascites tumor cells. When examined with the other in vitro assay systems using a modified Boyden chamber, the synthetic IIF-2 suppressed the chemotactic migration of highly metastatic B16 melanoma (B16FE7) cells to fibronectin or laminin and invasion through Matrigel. The IIF-2 inhibited neither the cell proliferation nor the binding of cells to fibronectin or Matrigel and also showed no significant inhibition of Mr 90,000 type IV collagenase (gelatinase) obtained from human schwannoma (YST-3) cells. The formation of lung colonies in mice given injections of B16FE7 and Lewis lung carcinoma cells was significantly reduced by the coinjection of the IIF-2. These results suggest that IIF-2 suppresses tumor invasion by impairing cell motility and inhibits the migration of metastasizing cells through extracellular matrix (extravasation steps) following their arrest in the capillary bed of the lung in vivo.

Inhibitory effects of tumor invasion-inhibiting factor 2 and its conjugate on disseminating tumor cells.

Tumor invasion-inhibiting factor 2 (IIF-2) is a polypeptide of 21 amino acids which binds to the surface of tumor cells and inhibits experimental invasion in vitro. An albumin conjugate of IIF-2 was used to examine its potential as an antimetastatic compound. The conjugate inhibits in vivo lung metastasis of various highly metastatic tumor cells, including murine melanoma, colon adenocarcinoma, squamous cell carcinoma, forestomach carcinoma, and human fibrosarcoma. In addition to the anti-lung metastasis activity of this compound, it also showed the inhibitory effects on liver and spleen metastasis of murine T-lymphoma cells. A single administration of the conjugate with melanoma cells resulted in prolonged survival times, and their lung colonization was also inhibited when the conjugate was administrated i.v. at times ranging from 6 h before to 1 h after tumor cell inoculation. Similarly, i.p. administration 1 h prior to melanoma cell injection suppressed lung colonization. Pharmacokinetic analysis revealed that the conjugate was more stable than IIF-2 peptide alone. Approximately 10% of the conjugate remained circulating 2 h postinjection and persisted 20 h without degradation, compared with rapid clearing of the unconjugated IIF-2 peptide within 5 min. Furthermore, spontaneous lung metastasis of murine melanoma and colon adenocarcinoma cell was inhibited by successive i.p. administration of the conjugate before the removal of the primary site, with no effect on primary tumor growth. The conjugate significantly reduced tumor cell arrest in the lung and both the IIF-2 peptide and its conjugate demonstrated potent inhibition of basal as well as cytokine-induced-stimulated tumor cell motility. These results suggest that one mode of IIF-2 action may be inhibition of the extravasation of metastasizing cells which have arrested in a target organ, and that the IIF-2-albumin conjugate may be a potent antimetastatic substance with utility in the prevention of artificial seeding of tumor cells during surgical removal of the primary lesions as well as inhibiting metastasis from established metastases.

Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells.

Excess endoplasmic reticulum (ER) stress induces processing of caspase-12, which is located in the ER, and cell death. However, little is known about the relationship between caspase-12 processing and cell death. We prepared antisera against putative caspase-12 cleavage sites (anti-m12D318 and anti-m12D341) and showed that overexpression of caspase-12 induced autoprocessing at D(318) but did not induce cell death. Mutation analysis confirmed that D(318) was a unique autoprocessing site. In contrast, tunicamycin, one of the ER stress stimuli, induced caspase-12 processing at the N-terminal region and the C-terminal region (both at D(318) and D(341)) and cell death. Anti-m12D318 and anti-m12D341 immunoreactivities were located in the ER of the tunicamycin-treated cells, and some immunoreactivities were located around and in the nuclei of the apoptotic cells. Thus, processing at the N-terminal region may be necessary for the translocation of processed caspase-12 into nuclei and cell death induced by ER stress. Some of the caspase-12 processed at the N-terminal and C-terminal regions may directly participate in the apoptotic events in nuclei.

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.

Molecular cloning of a putative serotonin receptor gene from barnacle, Balanus amphitrite.

We isolated a putative serotonin receptor gene from a genomic library of the barnacle, balanus amphitrite Darwin, using an Ncol fragment of the barnacle G protein-coupled receptor gene that is homologous to the alpha 2-adrenoceptor. The cloned genomic DNA had no intron and specified an open reading frame of 1137 base pairs encoding 379 amino acids (aa). The predicted aa sequence has a typical seven hydrophobic transmembrane spanning region and a consensus G protein-binding motif. This receptor was most homologous to the human 5HT1A receptor and closely related to other 5HT1 receptor subtypes.

Molecular cloning of a new member of the putative G protein-coupled receptor gene from barnacle Balanus amphitrite.

An intronless gene encoding a putative G protein-coupled receptor was isolated from the genomic library of barnacle Balanus amphitrite Darwin, with probes obtained from degenerate polymerase chain reaction (PCR) primers used to amplify putative transmembrane regions. The cloned genome DNA specifies an open reading frame of 1431 bp encoding 476 amino acids with seven hydrophobic transmembrane (TM)-spanning regions. The predicted protein contains potential asparagine-linked glycosylation and serine/threonine phosphorylation sites in the N-terminal and intracellular loops, respectively. Moreover, the protein has a consensus G protein-binding motif (Ala-Ile-Ser-Leu-Asp-Arg-Tyr-Leu-Ala) in TM domain III. This receptor is most closely related to human alpha 2-adrenergic receptor with 36.9% identity in 409 amino acids overlap. It is also homologous to human serotonin1A (5HT), snail pond 5HT and mouse D2-dopamine receptors with 33-36% identities. Within TM regions among these biogenic amine receptors, the cloned receptor shows considerable amino acid homology with more than 40% overall identities.

How platelet aggregation affects B16BL6 melanoma cell trafficking.

In blood-borne metastasis, intravasated metastatic tumor cells are thought to localize at the target site via a series of processes involving platelet aggregation, adhesion to endothelium, and invasion through the basal membrane. In the present study, we examined how platelet aggregation contributes to the trafficking of metastatic tumor cells in vivo by use of an inhibitor of platelet aggregation. Highly invasive B16BL6 melanoma cells were labeled with [2-18F]2-fluoro-2-deoxy-D-glucose and injected into mice to determine cell trafficking non-invasively by positron emission tomography. Both platelet aggregation inhibitor cyclo(RSarDPhg), which could not inhibit metastasis, and metastatic inhibitor cyclo(GRGDSPA) suppressed the accumulation of B16BL6 cells in the lung by about 12%, suggesting that platelet aggregation partly affects cell trafficking but not to a great extent, and that platelet aggregation is not the essential step for B16BL6 cell arrest in targets.

Invasion-inhibiting factor 2-albumin conjugate inhibits invasion and spontaneous metastasis of MKL-4 human breast cancer cells transplanted into female nude mice.

Invasion-inhibiting factor 2 (IIF-2) and its albumin conjugate have been reported to inhibit spontaneous metastasis of highly metastatic cancer cells with no effect on primary tumor growth. To confirm the inhibitory effects of the IIF-2 conjugate on tumor invasion and spontaneous metastasis, we administered the conjugate intra-peritoneally (i.p.) to female nude mice bearing transplanted tumors with MKL-4 cells, which are MCF-7 human breast cancer cells cotransfected with fibroblast growth factor 4 and lacZ. Neither 10 nor 20 mg/kg doses of the conjugate caused any inhibition of primary tumor growth, but 20 mg/kg significantly inhibited tumor invasion and spontaneous metastasis. Tumor invasion was measured by a novel computer-assisted image analysis. Spontaneous microscopic metastases into lymph nodes and distant organs were measured by whole organ staining for beta-galactosidase activity and observed with a dissecting microscope. The dose of 10 mg/kg significantly inhibited tumor invasion but not metastasis. Interestingly, the number of factor VIII-positive microvessels in the tumors was not reduced by treatment at either dose level. These findings suggest that the anti-invasive effect of the IIF-2 conjugate may reduce both lymphatic and hematogenous metastases in this MKL-4 metastasis model without affecting angiogenesis.

A novel Arg-Gly-Asp containing peptide specific for platelet aggregation and its effect on tumor metastasis: a possible mechanism of RGD peptide-mediated inhibition of tumor metastasis.

A novel cyclic tetrapeptide containing L-arginine-glycine-L-aspartic acid-L-phenylglycine (cyclo-RGDPhg) was synthesized and found to be a potent inhibitor of platelet aggregation induced by highly metastatic murine squamous cell carcinoma (SCCVII) cells (IC50 = 3.3 microM) as well as ADP (1.5 microM). This cyclic peptide, however, showed similar or less inhibitory activities on adhesion of SCCVII cells to fibronectin, vitronectin and type IV collagen as compared with those of parent linear tetrapeptide, RGDS. These results show that cyclo-RGDPhg peptide is a highly specific antagonist for gpIIb/IIIa on platelets. Moreover, this peptide failed to suppress pulmonary metastasis of SCCVII cells in an experimental metastasis model. These results indicate that RGD peptide-mediated inhibition of tumor metastasis is attributed to the suppression of cell adhesion but not platelet aggregation. These also suggest that platelet aggregation is not an essential step during blood circulation of tumor cells for the completion of metastasis.

Production and characterization of monoclonal antibodies to rat liver arginase.

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.

Detection of caspase-9 activation in the cell death of the Bcl-x-deficient mouse embryo nervous system by cleavage sites-directed antisera.

Caspases, which play crucial roles during apoptosis, are activated from their inactive proforms in a sequential cascade of cleavage by other members of the caspase family. Caspase-9 is autoprocessed by the Apaf-1/cytochrome c pathway and acts at an early point in this cascade, whereas Bcl-xL, an antiapoptotic member of the Bcl-2 family, prevents activation of caspases in vitro. Little is known, however, about the relation between caspase-9 and Bcl-xL during development of the mammalian nervous system. We used antisera against two cleavage sites in mouse caspase-9 that recognize only the activated form of mouse caspase-9, and we examined immunohistochemically the activation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse embryos. Mouse caspase-9 is processed at both D(353) and D(368), but it is processed preferentially at D(368) during apoptosis of cultured cells induced by various stimuli and in the nervous system of Bcl-x-deficient mouse embryos. We show that Bcl-xL protects against caspase-9- and/or caspase-3-dependent apoptosis in the caudal portion of the ventral hindbrain, anterior horn cells, and dorsal root ganglia neurons of the normal mouse embryos and against caspase-9/caspase-3-independent apoptosis in the dorsal region of the nervous system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation of caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5.

Inhibition of motility and invasion of human lung cancer cells by invasion inhibiting factor 2.

The motility and invasion of cancer cells are basic requirements for the establishment of distant metastases. In this study, we examined the effect of invasion inhibiting factor 2 (IIF2), a motility/invasion regulatory agent, on the motility, invasion, growth and basement membrane attachment of human lung cancer cells. IIF2 significantly reduced cell dissociation, colony scattering and invasion induced by the motogenic factor, HGF/SF. Western and Northern analyses showed these cells to be positive for the HGF/SF receptor c-met. These effects were blocked by an anti-IIF2 antibody. IIF2 did not affect the growth and attachment of lung cancer cells to the basement membrane. It is concluded therefore that invasion inhibiting factor 2 is an inhibitor of human lung cancer cell motility and invasion in vitro and this may bear some importance in the construction of anti-metastatic therapies.

ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos.

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5'-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.

Purification and characterization of tumor invasion-inhibiting factors.

Tumor invasion-inhibiting factors were purified from bovine liver using an in vitro system for estimating the tumor invasion ability. The acid-ethanol extract of liver was subjected to ultrafiltration (Amicon PM10 membrane), and the fraction corresponding to the molecular weight range below 10,000 was further fractionated by ion-exchange (DEAE-Toyopearl), gel filtration (Bio-Gel P6), and reverse-phase (C18) chromatographies. Two types of active polypeptides with molecular weights of about 5,000 and 2,000 were purified and named IIF-1 and IIF-2, respectively. Both peptides inhibited tumor invasion with half-maximum concentrations of 2-6 ng/ml in vitro. The amino acid compositions of both peptides were determined.

Effects of methylthiodeoxyadenosine and its analogs on in vitro invasion of rat ascites hepatoma cells and methylation of their phospholipids.

The relationship between tumor invasiveness in vitro and methylation of plasma membrane phospholipids was investigated. For this purpose, two hepatoma cell lines, C1-30 and LC-AH, were used which show specific penetration to below cultured monolayers of mesothelial cells from rat mesentery and endothelial cells from calf pulmonary artery, respectively. Methylthiodeoxyadenosine (MTA) and five of its analogs, difluoro-MTA, deoxyadenosine, sinefungin, phenylthiodeoxyadenosine and fluorophenylthiodeoxyadenosine, inhibited the invasion of the tumor cells without affecting their proliferation. This inhibition was associated with reduction in the incorporation of radioactivity of [methyl-3H]methionine into cellular phosphatidylethanolamine derivatives without changes in the labelings of RNA and DNA and carboxylmethylation of protein. These compounds also decreased the membrane fluidity of the tumor cells, measured by a steady-state fluorescence polarization method. Three other MTA analogs (fluorodideoxyadenosine, fluoroazidodideoxyadenosine and fluoroaminodideoxyuridine) did not affect the invasiveness of the tumor cells or alter their phospholipid methylation or membrane fluidity at concentrations that did not inhibit proliferation. These results suggest that the decrease in invasiveness of tumor cells by MTA and its analogs is due to alterations in the phospholipid composition and fluidity of the tumor cell membranes.

Inhibition of in vitro tumor cell invasion by ginsenoside Rg3.

The effect of plant glycosides on tumor cell invasion was examined. Among the glycosides tested, ginsenoside Rg3 was found to be a potent inhibitor of invasion by rat ascites hepatoma cells (MM1), B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN-1) cells, when examined in a cell monolayer invasion model. Structurally analogous ginsenosides, Rb2, 20(R)-ginsenoside Rg2 and 20(S)-ginsenoside Rg3 (a stereoisomer of Rg3), showed little inhibitory activity. Neither Rh1, Rh2, 20(R)-ginsenosides Rh1, Rb1, Rc nor Re had any effect. The effective ginsenoside, Rg3, tended to inhibit experimental pulmonary metastasis by highly metastatic mouse melanoma B16FE7 cells as well. Taking account of our previous finding that 1-oleoyl-lysophosphatidic add (LPA) induced invasion by MM1 cells in the monolayer invasion model, the effect of Rg3 on molecular events associated with the invasion induced by LPA was analyzed in order to understand the mechanism of the inhibition. Rg3, which suppressed the invasion induced by LPA, dose-dependently inhibited the LPA-triggered rise of intracellular Ca2+. Protein tyrosine phosphorylation triggered by LPA was not inhibited by Rg3.

A potent anti-metastatic activity of tumor invasion-inhibiting factor-2 and albumin conjugate.

Tumor invasion-inhibiting factor-2 (IIF-2) peptide was chemically conjugated to albumin with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride. The molar ratio of the covalently linked IIF-2 peptide and albumin in the purified conjugate was 1.4 +/- 0.4, as determined with a radioactive peptide. The conjugate inhibited the invasion of HT1080 and B16FE7 cells in vitro at 40- to 60-fold lower concentrations than IIF-2 peptide. Scatchard analysis of binding data demonstrated that the IIF-2-albumin conjugate bound to HT1080 and B16FE7 cells with Kd values of 240 and 340 nM, respectively. The conjugate suppressed the lung colonization of B16FE7 cells more effectively than the IIF-2 peptide in an experimental metastasis. These results indicate that the covalent linkage of the IIF-2 peptide to a carrier macromolecule provides a structure that enables this peptide to exhibit increased inhibitory action in cancer cell invasion and metastasis, and that IIF-2 exerts its inhibitory action on invasion by binding to a specific binding site on the tumor cell surface.