New insights into the metastasis-associated 67 kD laminin receptor

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated by different cell surface molecules, including the monomeric 67 kD laminin receptor. This molecule appears to be very peculiar since so far only a full-length gene encoding a 37 kD precursor protein has been isolated and the mechanism by which the precursor reaches the mature form is not understood. Based on clinical data, which clearly demonstrate the importance of the receptor in tumor progression, studies were conducted to define the structure, expression, and function of this laminin receptor as a step toward developing therapeutic strategies that target this molecule. The data suggest that acylation of the precursor is the key mechanism in maturation of the 67 kD form. The function of the membrane receptor is to stabilize the binding of laminin to cell surface integrins, acting as an integrin-accessory molecule, although homology of the gene encoding the receptor precursor with other genes suggests additional functions. Downregulation of the receptor expression on tumor cells might open new therapeutic approaches to decrease tumor aggressiveness. J. Cell. Biochem. 67:155–165, 1997. © 1997 Wiley-Liss, Inc.     

Relative role of CSF-1, MCP-1/JE,and RANTES in macrophage recruitment during successful pregnancy

It has been previously demonstrated that macrophage colony stimulating factor (CSF-1) is produced by uterine epithelial cells in response to estrogen and progesterone. Studies in normal and op/op mice demonstrated that accumulation of a portion of the uterine macrophage population could be attributed to the chemotactic properties of CSF-1. Op/op mice exhibit greatly reduced rates of fertility, but successful pregnancy is not completely blocked. Also, uteri from op/op mice are not completely macrophage deficient. There are two possible explanations for this. One is that not all tissue macrophages are recruited from the bone marrow pool; some may be derived from primitive mesenchyme. Alternatively, tissue macrophages may be recruited from the bone marrow pool through expression of other type I chemokines such as JE, RANTES, MIP-1α, MIP-1β, IP-10, and KC. Both RANTES and JE are expressed at higher levels than CSF-1 during early pregnancy. The variable expression and relative role of these various chemokines in pregnancy was addressed by measuring mRNA expression during the first 8 days of pregnancy and in a pseudopregnant model. The expression of these various genes relative to macrophage numbers and macrophage distribution will be discussed. The relative role of these various factors in preparing the uterus for blastocyst implantation will be discussed. Mol Reprod Dev 46:62–70, 1997. © 1997 Wiley-Liss, Inc.     

Formation of the 67-kDa laminin receptor by acylation of the precursor

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. J. Cell. Biochem. 69:244–251, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Development of a high-throughput assay to measure histidine decarboxylase activity

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.     

A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-α in metastatic melanoma patients

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage®) given with GM-CSF and IFN-α in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days –1, 0 and +1, while IFN-α (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-α is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.L.P. and R.P. have equally contributed to the work.