Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.