Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial.

In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A. nancymai system. The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.

Differential signaling by alternative HGF isoforms through c-Met: activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis.

HGF/NK2, a naturally occurring truncated HGF isoform, antagonizes the mitogenic and morphogenic activities of full length HGF, but stimulates cell scatter, or the motogenic response to HGF. We studied postreceptor signaling by these HGF isoforms in the human breast epithelial cell line 184B5, and in murine myeloid progenitor 32D cells transfected with c-Met, the human HGF receptor (32D/c-Met). HGF stimulated DNA synthesis in 184B5 and 32D/c-Met cells, while HGF/NK2 was mitogenically inactive, despite the ability of HGF/NK2 to stimulate c-Met autophosphorylation, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) in both cell systems. In 184B5 cells, HGF stimulated sustained MAPK activation, while activation by HGF/NK2 declined rapidly. In contrast, both isoforms activated MAPK with rapidly attenuated kinetics in 32D/c-Met cells. In both cell systems the increased motility observed in response to either HGF or HGF/NK2 treatment was more potently blocked by the PI3 kinase inhibitor wortmannin, than by PD98059, an inhibitor of MAPK kinase (MEK1). These data suggest that (1) alternative HGF isoforms signaling through c-Met generate both common and distinct biological responses, (2) the extent and duration of ligand-stimulated c-Met and MAPK activities are dependent on the cellular context and are not predictive of mitogenic signaling, and (3) in at least some HGF target cells, the activation of both MAPK and PI3K signaling pathways is insufficient for mitogenesis elicited through c-Met.

Functional and biophysical characterization of recombinant human hepatocyte growth factor isoforms produced in Escherichia coli.

Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2). We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF. All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps. Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations. Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays. These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.

Hepatocyte growth factor (HGF)/NK1 is a naturally occurring HGF/scatter factor variant with partial agonist/antagonist activity.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF is structurally related to plasminogen, including an amino-terminal hairpin loop, four kringle domains, and a serine protease-like region. A truncated HGF/SF isoform, designated HGF/NK2, which extends through the second kringle domain and behaves as a competitive HGF/SF antagonist, was previously shown to be encoded by an alternative HGF/SF transcript. In this study, we describe a second naturally occurring HGF/SF variant, HGF/NK1, consisting of the HGF/SF amino-terminal sequence and first kringle domain. This product is encoded by a 2-kilobase alternative transcript containing intronic sequence that was contiguous with exon K1b. Analysis of baculovirus-expressed HGF/NK1 revealed that this isoform possesses the heparin binding properties of HGF/SF and modest mitogenic and scattering activity relative to HGF/SF. However, at a 40-fold molar excess, HGF/NK1 inhibited HGF/SF-dependent DNA synthesis. HGF/NK1 stimulated tyrosine phosphorylation of Met, and covalent affinity cross-linking demonstrated a direct HGF/NK1-receptor interaction. These findings establish that the HGF/SF gene encodes multiple alternative products, which include not only a mitogenic agonist (HGF/SF) and a pure antagonist (HGF/NK2) but also a molecule with partial agonist/antagonist properties.

Interaction between the 67 kilodalton metastasis-associated laminin receptor and laminin.

Normal and neoplastic cells interact with laminin via a variety of cell surface proteins. The specific binding sites on laminin for each particular cell surface laminin-binding protein have not yet been identified. In this study, the interaction between laminin and the high affinity metastasis-associated 67 kD laminin receptor (67 LR) was investigated by electron microscopy using the rotary shadowing technique. Laminin receptor that was purified from human colon carcinoma metastases appeared as a globular structure with a diameter of 5.2 +/- 0.8 nm. The 67 LR specifically bound to laminin on its long arm close to the intersection of the long and the short arms. There was no specific interaction of bovine serum albumin with laminin. Biochemical confirmation of the rotary shadowing experiments included slot blot solid phase assays in which [I125]-labeled 67 LR bound in a dose dependent manner to laminin as well as to the chymotrypsin resistant (C1) fragment of laminin that contains a short piece of the long arm. [I125]-labeled 67 LR did not bind to the pepsin resistant (P1) fragment of laminin that did not contain that segment on the long arm. This study therefore identifies the binding site on laminin for the 67 kD metastasis-associated laminin receptor as a region on the long arm of laminin close to the intersection of the four arms.

Inverse modulation of steady-state messenger RNA levels of two non-integrin laminin-binding proteins in human colon carcinoma.

BACKGROUND: Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin-binding proteins, including the 67-kd laminin receptor (67 LR) and a 31-kd human laminin-binding protein (HLBP31) homologous to the 31-kd human IgE-binding protein/galactoside-binding lectin. PURPOSE: To investigate whether various laminin-binding proteins are differentially expressed in human colon carcinoma, we studied messenger RNA (mRNA) levels of the 67 LR and HLBP31 in matched tumor and adjacent normal mucosa samples from a series of 21 patients. METHODS: Total cellular RNA from tumor and normal mucosa was isolated and analyzed by Northern and slot blot hybridization. In addition, HLBP31 protein levels were assessed by the immunoblot technique. Quantitative laminin affinity chromatography was also used to measure the synthesis of HLBP31 protein in five human cancer cell lines. RESULTS: The steady-state mRNA level of HLBP31 was downregulated (i.e., decreased) in 18 of 21 human colon carcinomas compared with the level in their corresponding normal colonic mucosa. On average, the level of HLBP31 mRNA was decreased 50% +/- 30% (+/- SD) in the colon cancers. The mean ratio of colon cancer HLBP31 mRNA to adjacent normal mucosa HLBP31 mRNA was twofold lower in primary tumors of patients with metastases (0.3 +/- 0.2 SD) than in primary tumors of patients free of metastatic lesions (0.6 +/- 0.2 SD). The differences between the two groups of patients were statistically significant (P less than .05, Wilcoxon-Mann-Whitney test). We have previously shown that the ratio of colon cancer 67 LR mRNA to corresponding normal mucosa 67 LR mRNA was increased in the same patient population. When the two ratios (ratio of cancer to normal HLBP31 mRNA and ratio of cancer to normal 67 LR mRNA) were compared, HLBP31 mRNA/67 LR mRNA was significantly lower (P less than .05) in primary tumors with metastases (mean +/- SD, 0.3 +/- 0.2) than in primary cancers without metastases (mean +/- SD, 0.7 +/- 0.5). The steady-state level of HLBP31 mRNA was directly correlated with the amount of HLBP31 protein in both colon tissue samples and human cancer cell lines. CONCLUSION: HLBP31 mRNA expression in colon cancer tissues is modulated inversely to that of 67 LR mRNA expression. The down-regulation of HLBP31 appears to be associated with the metastatic capabilities of colon cancer cells. IMPLICATIONS: Prospective studies on a large cohort should determine if the systematic detection of HLBP31 and 67 LR protein and/or mRNA can be a valuable adjunct in the prognostic evaluation of primary colon cancers.

Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.

Biosynthesis of the 67 kDa high affinity laminin receptor.

High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified.

Increased expression of the laminin receptor in human colon cancer.

It has been proposed that among the various cell-surface proteins capable of interacting with laminin, the 67-kd high-affinity laminin receptor plays a crucial role during tumor invasion and metastasis. In this study, the expression of laminin-receptor-precursor messenger RNA (mRNA) and 67-kd protein was analyzed in human colon adenocarcinoma. In 22 of 23 patients with colon cancer, we found a 2- to 23-fold increase in levels of laminin-receptor-precursor mRNA in the cancer tissues compared with those in matched normal adjacent colonic mucosa. In 10 of 11 cases studied, the level of 67-kd laminin receptor, detected by affinity-purified anti-laminin-receptor synthetic peptide antibodies on immunoblots of matched tumor and normal tissue extracts, was higher in the colon carcinoma tissue. Immunodetection of laminin receptor in tissue sections using anti-laminin-receptor-peptide antibodies confirmed that the increased expression of laminin receptor was specifically associated with the cancer cells. In a series of 72 paraffin sections of colon lesions, we observed a correlation between the expression of the laminin receptor and the Dukes' classification. Our observations indicate that increased expression of laminin-receptor-precursor mRNA is associated with enhanced levels of the 67-kd laminin receptor as well as with the invasive phenotype of colon carcinoma. Detection of this metastasis-associated gene product may be a valuable adjunct in the evaluation of human colon cancer.

Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinoma.

Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.

Platelet contribution to cancer cell growth and migration: the role of platelet growth factors.

Blood platelets contain several growth factors and inhibitors. The better known among them are named platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta), active as modulators of growth of normal mesenchymal and epithelial cells. Cancer cell growth in vitro seems to be independent of the effects of exogenous peptides, but dependent on the cell ability to release autocrine growth factors, similar to PDGF or TGF beta. In addition, platelet-associated growth factors and inhibitors, which are able to induce a fibrotic response in connective tissue cells, might also play a role to modulate the desmoplastic reaction surrounding the tumor.

Behavioural and biochemical studies on 6-methylamino-4,5,6,7-tetrahydrobenzothiazole (14.839JL), a new potent dopaminergic agonist.

6-Methylamino-4,5,6,7-tetrahydrobenzothiazole monochlorhydrate (14.839JL) is a new, potent dopaminergic agonist. The stereotypy induced by this drug was greater than that induced by an equivalent dose of apomorphine, was antagonized by pretreatment with sulpiride and counteracted the hypomotility induced by reserpine. Striatal levels of the dopamine metabolites homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) were significantly lowered for up to 4-6 h by doses from 0.05 to 1 mg/kg. The drug was also very effective in lowering prolactine secretion. 14.839JL displaced [3H]N-n-propylnorapomorphine [3H]NPA from striatal binding sites with an IC50 similar to dopamine (DA). Conversely, the ability of 14.839JL to displace 3H spiperone from its binding sites was 100 and 10 times lower than that of haloperidol and sulpiride, and similar to that of SCH 23390. Differently from the latter, however, 14.839JL did not modify adenylate cyclase activity. All these data suggest that 14.839JL is a new, potent, long-lasting direct DA agonist, probably acting on D2 receptors.