Amphiregulin (AR) and cripto are proteins that are structurally related to epidermal growth factor (EGF) and transforming growth factor a (TGF-a). AR is also functionally related to this family of growth regulatory molecules and is able to bind and activate the 170-kDa EGF receptor (EGFR). Human EGFR-3 (HER3)/ERBB3 is a recently identified protein related to the EGFR that is widely expressed in breast carcinomas and is a candidate receptor for EGF-like growth factors. Differential expression ofthese putative ligands and receptors in transformed cells suggests that they may function in an autocrine manner to regulate tumor cell growth.Specific mRNA transcripts for TGF-a [4.8 kilobases (kb)], AR (1.4 kb), cripto (2.2 kb), and HER3 (6.2 kb) were expressed in a majority of human colon cancer cell lines. HER3 mRNA was detected in 55% of primary or metastatic human colorectal carcinomas but in only 22% of normal colon mucosa and 32% of normal liver samples. In contrast, cripto and AR mRNA were expressed in 60-70% of primary or metastatic human colorectal cancers but in only 2-7% of normal human colonic mucosa. Immunostaining also detected AR protein in primary and metastatic colorectal tumors but not in normal colon or uninvolved liver. These fings suggest that cripto and AR may be useful markers to discriminate between normal and malignant colonic epithelium and may provide a selective growth advantage for colorectal carcinomas.

Objectives-Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.Design-Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0 to 100 μg/ml) and time course (0 to 120 minutes) assays were performed to determine the optimal conditions for live cell surface binding using immuno-fluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.Results-Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 minutes. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.Conclusions-The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gpl20 and gp4l. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gpl20-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development. * Corresponding author. ing of gpl60 in mammalian cells (16a). The HIV p55 protein is anchored in cellular membranes by a myristic acid moiety added to the N-terminal penultimate glycine residue (2). Similar to other retrovirus systems (33), anchoring of the HIV-1 core protein has been shown to be necessary for production of virions (14). Development of an in vitro recombinant expression system that mimics the HIV-1 virus synthesis and maturation pathways would be advantageous for studying the role of env and gag protein association in regulating virus formation and release. We (S.

Abstract. BR 96 is an internalizing antibody that binds to Lewis Y (LeY), a carbohydrate determinant expressed at high levels on many human carcinomas (Hellstr6m, I., H. J. Garrigues, U. Garfigues, and K. E. Hellstr6m. 1990. Cancer Res. 50:2183-2190. Breast carcinoma cell lines grown to confluence bind less BR96 than subconfluent cultures (Garrigues, J., U. Garrigues, I. Hellstr6m, and K. E. Hellstr6m. 1993. Am. J. Path. 142:607-622). However, when the confluent cells are induced to migrate by scratch wounding, they again bind BR96 suggesting that antigens bearing the Ley determinant may promote cell migration. In the present study, BR96 was found to be highly enriched on microspikes and ruffled membranes, cell surface structures involved in cell migration. In addition, BR96 was a potent inhibitor of cell migration in vitro. When stationary BR96 treated cells were exposed to fresh culture media, membrane ruffles and microspikes developed at the cell margin and migration resumed. Immunogold microscopy showed that BR96 antigens were enriched on these membrane protrusions.BR96 cell surface immunoprecipitation analysis of 3H-glucosamine labeled breast carcinoma cells identified antigens with approximate molecular weights of 135 kd (upper antigen) and 85 kd (lower antigen). A short amino terminal sequence (8 residues) of the upper antigen matched that of human lysosomal membrane glycoprotein 1 (LAMP-l). In addition, the upper antigen was detected on immunoblots probed with anti-LAMP-l, and within the intracellular compartment BR96 was found predominantly in endosomes and lysosomes.A soluble LAMP-1/immunoglobulin fusion protein (LAMP-1/Ig) was transiently expressed in both BR96 binding and nonbinding cell lines. Immunoblot analysis of LAMP-1/Ig's from the various cell lines showed that (a) acquisition of the BR96 epitope is probably controlled at the level of polylactosamine modification (e.g., fucosylation) rather than LAMP-1 gene expression; (b) alternate forms of LAMP-1/Ig comigrate with the lower BR96 antigen raising the possibility that it may be a degradation product of the upper antigen; and (c) LAMP-1/Ig expressed in 3396 breast carcinoma cells has approximately 30-fold more BR96 epitopes than LAMP-1/Ig from non-tumorigenic mammary epithelial cells. Together these data indicate that a major BR96 antigen, LAMP-l, is present on unique cell surface domains involved in cell locomotion as well as membranes of the endocytic compartment. Altered glycosylation of LAMP-1 expressed in transformed cells may contribute to their ability to disseminate. MANY ligands have been identified that can regulate cell locomotion (Turley, 1992). Some studies indicate that altered cell migration is associated with aberrant glycosylation. In a number of instances, highly metastatic tumor cells were found to express more polylactosamine oligosaccharides (Gal/31-4GlcNAc/31-3), than normal or poorly metastatic ones (Hubbard, 1987;Pierce and Arango, 1986;Yamashita et al., 1984;Yousefi et al., 1991). Poly N-acetyllactosamine chains are oft...