The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (-123 to -108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.

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The gene encoding the human pregnancy-specific glycoprotein (PSG) belongs to a gene subfamily, comprised of the carcinoembryonic antigen (CEA) and PSG subgroups, within the immunoglobulin superfamily. To study the functional roles of PSG during development in an animal model, we isolated and characterized a near full-length cDNA (rnCGM6) encoding a PSG-related protein from a rat placental cDNA library. rnCGM6 is 2,068 bp in length and contains an open reading frame that encodes a 475-amino-acid polypeptide with a predicted molecular mass of 53 kD. The 5' noncoding sequence is 173 nucleotides, and primer-extension experiments demonstrate that the transcriptional initiation site is located 22-24 nucleotides further upstream. The 3' noncoding sequence contains 470 nucleotides which is followed by a poly(A) tail. In contrast to human PSGs, which contain one immunoglobulin variable-like and two to three immunoglobulin constant-like protein domains, rnCGM6 contains three immunoglobulin variable-like domains and one immunoglobulin constant-like domain. rnCGM6 contains six potential N-linked glycosylation sites and, in its carboxyl-terminal domain, a tyrosine protein kinase phosphorylation site. The tyrosine phosphorylation site is conserved among all rat and human PSG members. rnCGM6 hybridized with a major 2.5-kb and two minor 3.0- and 3.5-kb mRNAs, all primarily expressed in the rat placenta. Ribonuclease protection analysis, using probes specific to the 5', middle, and 3' regions of rnCGM6, and the 5' region of a previously identified cDNA, rnCGM1, mainly yielded fully-protected fragments indicating relatively low sequence similarity among rat PSG-related proteins. Northern hybridization and ribonuclease protection assays also suggest that rnCGM6 may be the major PSG member in rat.