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Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular teratoma with ovary-seeking properties in vivo also adhered preferentially to MOE cells, while the preference for MBE cells was shared by glioma cells with an endothelioma and a bladder tumor line. The endothelioma, interestingly, showed a marked preferential adhesion to 3T3 cells, thus distinguishing it from the glioma. The experiments demonstrate that capillary endothelial cells derived from different sources are not alike and that differences expressed at the cell surface of these cells can be distinguished by tumor cells.

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, a-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a >1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescenceactivated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation ofendothelial cells but also as a tool for the identification and pharmacological study of ACE.Angiotensin-converting enzyme (ACE), or kininase II, which cleaves the terminal dipeptide from angiotensin I to form vasoactive angiotensin II and which is active as a dipeptidyl hydrolase in its action on bradykinin and other small peptides, serves as a useful marker for the identification ofendothelial cells from capillaries, veins, and arteries (1-4). Because the enzyme is associated with the cell surface (5), antibodies directed against ACE can serve not only to identify endothelial cells but also can mark them for analysis and isolation without loss of viability.Our interest in the growth and development of murine endothelial cells prompted us to generate monoclonal reagents directed at strongly cross-species reactive ACE. Tests carried out with conventionally generated rabbit anti-rat ACE (6) pointed to rat lung ACE as a suitable immunogen; therefore, we used a rat lung ACE preparation to induce an immune response in mice. Spleens from these mice then were fused by hybridization to nonsecreting mouse myeloma cells to permit isolation and characterization of hybrids (hybridomas) that produced monoclonal antibodies to ACE.We now report on the properties of one such hybridomaits cell-specific and enzyme-directed binding properties and its use as a reagent for identifying and isolating murine, bovine, and human endothelial cells.MATERIALS AND METHODS ACE Preparation. ACE was obtained by following the methods of Lanzillo and Fanberg (6, 7). In brief, rat lungs were dissected after lavage, lightly homogenized in 0.02 M potassium phosphate buffer (pH 8.3), and centrifuged (250 x g; 10 min) to remove cells and debris. The supernatant was recentrifuged (54,000 X g; 60 min) and the pellets then were rehomogenized to yield the "crude enzyme preparation." For further purification, sodium deoxycholate was added; this was followed by centrifugation and dialysis, filtration through Whatman no. 1 paper, and fractionation on DEAE-cellulose columns ...

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.Since the discovery that certain naturally occurring RNA motifs were capable of catalytically cleaving other RNA molecules in a sequence-specific manner, extensive studies have defined the sequence and structural characteristics that control the in vitro specificity and kinetics of these RNA enzymes or ribozymes (1-4). Ribozymes have a broad range of potential in vivo applications. These include the use of ribozymes as research tools for probing molecular mechanisms, the use of ribozymes to genetically engineer crops, and the use of ribozymes as therapeutics for human or animal diseases. Each of these applications requires that a ribozyme function efficiently within the intracellular environment. The sequence and structural features that promote optimal intracellular activity of ribozymes are currently under study.Several factors are likely to contribute to the intracellular efficacy of a ribozyme. A ribozyme must colocalize with its molecular target in the appropriate cellular compartment and must be present at sufficiently high concentration to promote hybridization. In addition, its catalytic cleavage rate must be fast enough, and its half-life must be long enough to allow cleavage of a substantial fraction of the target mRNA population. Finally, the cleavage site in the target mRNA must be accessible to ribozyme binding. When the ribozyme is made synthetically, a variety of modifications can be introduced to increase its half-life within the cell, to change its target sequence binding affinity, and possibly also to alter its intracellular trafficking properties. In this study, we have used chemically synthesized hammerhead ribozymes t...

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