Increased expression of H/T-cadherin in tumor-penetrating blood vessels.

Neovascularization is a prerequisite for tumor growth. Thus, selective destruction of the tumor vasculature should prevent tumor expansion. We have established a method to identify proteins that are specifically expressed on the surface of endothelial cells in tumors. CD31-positive endothelial cells were isolated from Lewis lung carcinoma lung metastases as well as from normal lung tissue. cDNAs derived from these cells were subjected to a subtractive hybridization procedure, and cDNAs overrepresented in tumor-derived endothelial cells were isolated; those encoding surface proteins were selected using a signal sequence trap assay. One isolated cDNA encoded H/T-cadherin. In this report, we show that mouse H/T-cadherin is overexpressed on endothelial cells of several tumors, whereas it is expressed only on a subset of endothelial cells in healthy organs. On the basis of the expression of H/T-cadherin in lung metastases of different tumors, we suggest that different tumors can have a differential influence on the expression of endothelial cell surface proteins.

The IL-1 receptor-related T1 antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors.

Expression of the T1 gene, also known as ST2, DER4, and Fit-1, has been shown to be associated with cell proliferation. It gives rise to two different mRNAs that encode a receptor-like protein and a soluble molecule representing the ectodomain of the receptor form. Although T1 is a member of the IL-1R family, its biologic function is currently unknown. In this study, we have analyzed the expression of the T1 surface Ag in murine hemopoietic organs. Mast cells (MCs) were shown to be the only identifiable cell lineage that expressed T1 at high levels. T1 expression was found on cultured bone marrow-derived immature MCs. Similarly, freshly isolated connective tissue-type MCs from the i.p. cavity were also shown to express high levels of T1. Interestingly, the earliest detectable committed MC precursor isolated from fetal blood (FB) at day 15.5 of gestation, but not circulating hemopoietic stem cells in FB, also expresses high level of T1. Since FB promastocytes lack expression of the high affinity IgE receptor (Fc epsilonRI), T1 expression precedes expression of Fc epsilonRI in MC ontogeny. The finding that the T1 Ag is selectively expressed at several stages during development of the MC lineage suggests that this cell surface molecule, in combination with the well-established markers c-Kit and Fc epsilonRI, should be valuable for studying the MC lineage.

GATA-Dependent expression of the interleukin-1 receptor-related T1 gene in mast cells.

The murine delayed-early serum-responsive gene T1 encodes glycoproteins of the interleukin-1 receptor family. Transcriptional initiation in fibroblasts is regulated by c-Fos and gives rise to a rare 5-kb mRNA and an abundant 2.7-kb mRNA. These transcripts are translated into a receptor-like membrane-anchored protein and a secreted protein consisting only of the ectodomain. In mast cells, T1 gene transcription is initiated 10.5 kb further upstream than in fibroblasts and gives rise predominantly to the 5-kb transcript under normal growth conditions. Here we demonstrate that calcium ionophore stimulation of mast cells resulted in an upregulation of T1 gene expression and a switch from the long to the short T1 transcript. This was paralleled by the disappearance of the receptor-type T1 protein on the mast cell surface and the secretion of large amounts of the truncated T1 protein. c-Fos and a T1 enhancer, which have previously been identified to be essential for T1 expression in fibroblasts, were not required for calcium ionophore-mediated T1 gene upregulation. Overexpression of the transcription factor GATA-1 in mast cells caused elevated T1 synthesis. Three GATA elements were identified in the minimal GATA-responsive mast cell promoter. Mutational analysis revealed that all three GATA elements are involved in T1 gene expression. Point mutations within the middle GATA element eliminated promoter activity completely, while mutations of the distal and proximal GATA binding sites reduced promoter strength by factors of 2 and 5, respectively. Exogenous expression of GATA-1 was not sufficient to activate the mast cell-specific promoter in NIH 3T3 fibroblasts.

Expression analysis of the soluble and membrane-associated forms of the interleukin-1 receptor-related T1 protein in primary mast cells and fibroblasts.

The murine T1 gene encodes a membrane-bound glycoprotein (T1M) and a soluble variant (T1S) which represents the ectodomain of the receptor-type form. T1 is an orphan receptor belonging to the interleukin-1 receptor family. Its biological function is currently unknown. We analyze the expression of the two T1 proteins in mast cells and fibroblasts by using a set of monoclonal antibodies (MAb) that specifically recognize the extracellular portion of the T1 receptor. To generate anti-T1 MAbs, we immunized Lewis rats with a eukaryotically expressed chimeric protein consisting of the T1-receptor ectodomain fused to a human immunoglobulin domain. The two MAbs DJ4 and DJ8 were shown to specifically detect the murine T1M protein on the surface of primary IL-3-dependent bone marrow-derived mast cells as shown by flow cytometry and immunohistochemistry. Both antibodies were also capable of immunoprecipitating the membrane-associated 110-120 kDa T1M protein from mast cell lysates. In serum-stimulated but not in quiescent NIH3T3 fibroblasts, DJ4 and DJ8 MAbs detected both the soluble T1S protein as a 45-65 kDa band on SDS polyacrylamide gels as well as the membrane-bound 95 kDa T1M protein. The T1M protein in fibroblasts was less abundantly expressed and exhibited a lower molecular weight than the mast cell-produced T1M, probably as a consequence of different protein glycosylation. The MAbs described here represent highly specific reagents and valuable tools that should facilitate the establishment of the murine T1 protein expression pattern thus contributing to the solution of the question of its function.