Delivery of a DNAzyme targeting c-myc to HT29 colon carcinoma cells using a gold nanoparticulate approach.

The objective of the current study was to develop cellular delivery approaches for catalytic DNA enzymes (DNAzymes) which cleave targeted messenger RNA, using vectors based on colloidal gold. The model DNAzyme was a 32mer oligonucleotide designed to specifically interact with and cleave c-myc mRNA. Colloidal gold particles were prepared by reduction of tetrachlororauric [III] acid with sodium citrate. Particles could be produced in the 1-90 nm range. A cationic substrate linked to transferrin was electrostatically/hydrophobically bound to the gold particle. These vectors were then treated with the DNAzyme to yield the condensed DNA-cationic polymer-particulate product. The pH (4-11.5), the quantity of the DNAzymes (0.079-0.567 microg/probe), the cationic polymer (polylysine (PL) or polyethylenimine (PEI)) as well as the surfactant (PVP) concentration (0-0.5%) were varied to give stable constructs which decomplexed under the desired conditions (i.e., in lysosomes and at lower pH values). Cellular uptake of the FITC-labelled c-myc DNAzyme incorporated in this vector was measured using FACS analysis in human HT29 colon carcinoma cells. Data suggested that PEI gave better delivery efficiencies than PL. The use of PVP to stabilize the formed dispersions was detrimental to DNAzyme delivery when PL was used but had little effect in the PEI systems. In the best cases, delivery to 77% of the cells was possible using PEI with the PVP stabilizer and completing the DNA condensation at pH 5.5 with 0.118 microg of DNAzyme/probe. In contrast, the best conditions for PL gave only transfection to 43% of the cells (no PVP, condensed at pH 5.7 and with a loading of 0.079 microg DNAzyme/probe). The PL probe tended to be more toxic than the PEI-based systems (65% cell death in PL transfected cells compared to 22% for PEI). These results suggest that cellular targeting using colloidal gold appears feasible for DNAzyme delivery.

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity.

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Retinoic acid receptor alpha 1 isoform is induced by estradiol and confers retinoic acid sensitivity in human breast cancer cells.

Retinoic acid (RA) inhibits proliferation of estrogen receptor (ER)-positive human breast cancer cells, but not the growth of ER-negative cells. We have shown previously that ER-positive cells express higher levels of retinoic acid receptor (RAR) alpha, suggesting that RAR alpha gene expression may be regulated in breast cancer cells by estrogens. We here report that estradiol (E2) increases RAR alpha mRNA in a time- and concentration-dependent manner resulting in a marked increase in RAR alpha protein expression, and present evidence that RAR alpha 1 is the only known isoform of RAR alpha regulated by E2 in breast cancer cells. In parallel we demonstrate that ER-positive cells exhibit greater RA sensitivity in the presence of E2, suggesting that E2-induced expression of RAR alpha 1 is involved in growth inhibition by RA. To directly investigate the role of RAR alpha 1 in RA-mediated growth inhibition, we introduced RAR alpha 1 expression vectors into RA-resistant and ER-negative MDA-MB-231 cells. The RAR alpha 1-transfected cells were growth inhibited by RA, while mock- and untransfected cells were unresponsive. Together, our data indicate that adequate levels of RAR alpha 1, either generated by introduction of expression vectors or endogenously induced by estrogens, are required for growth inhibition of breast cancer cells by RA.

E1A functions as a coactivator of retinoic acid-dependent retinoic acid receptor-beta 2 promoter activation.

The retinoic acid (RA) receptor (RAR) beta 2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RAR beta 2 promoter activation in the E1A-expressing cells, which was not observed in the parental cell line, indicating a possible role for E1A in RAR beta 2 activation. In transient transfection assays, E1A functioned as a coactivator of RA-dependent RAR beta 2 promoter activation and, moreover, was able to restore this activation in cells lacking RAR beta 2 activation. By deletion analysis, two regions in the RAR beta 2 promoter were identified that mediate the stimulatory effect of E1A: the RA response element and TATA box-containing region and a more up-stream region between -180 and -63, in which a cAMP response element-related motif was identified as a target element for E1A. In addition, determination of endogenous E1A-like activity by measuring E2A promoter activity in transient transfection assays in EC and differentiated cells revealed a correlation between RA-dependent RAR beta 2 promoter activation and the presence of this activity, suggesting an important role for the cellular equivalent of E1A in regulation of the RAR beta 2 promoter.

The retinoic acid receptor-beta 2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand-binding domain.

In contrast to other members of the steroid/thyroid hormone superfamily, not much is known about the regions involved in transactivation of the receptors for retinoic acid. To determine the transactivation function of RARs, fusion proteins between the DNA-binding domain of the yeast transcription factor GAL4 and retinoic acid receptor-alpha (RAR alpha) or RAR beta were made. Transfection of these constructs resulted in RA-induced activation of a GAL4-responsive element-containing promoter. Deletion analysis revealed that RAR beta-2 has two transcription activation functions (TAFs). TAF-1 activates transcription constitutively and was mapped to the first 32 amino acids of the A-region. TAF-2 is located in the ligand-binding domain between amino acids 137 and 410 and activated transcription only in the presence of RA. The presence of two TAFs was confirmed by cotransfection of RAR beta deletion constructs with the human RAR beta-2 promoter as reporter, showing that the absence of RAR beta TAF-1 causes a decrease in transactivation, whereas truncation of TAF-2 completely blocks this function. Internal deletions in the ligand-binding domain in both GAL-RAR beta and RAR beta expression constructs resulted in a nonfunctional receptor, indicating that the complete ligand-binding domain is required for its transactivation function. Furthermore, we have shown that the contribution of the two TAFs in transcription activation varies among different cell lines, suggesting that they act in a cell-specific manner.

Genomic organization of the human retinoic acid receptor beta 2.

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.

Differentiation-dependent expression of provirus-activated int-1 oncogene in clonal cell lines derived from a mouse mammary tumor.

The int-1 mammary oncogene is frequently activated by proviral insertion in mouse mammary tumors. To characterize the target cell for the oncogenic action of int-1, we have isolated permanent cell lines with distinct morphologies and differentiation characteristics, starting from a tumor with a rearranged int-1 gene. Polygonal cells had retained many differentiation markers of epithelial cells and produced adenocarcinomas upon transplantation in syngenic mice. Sphere-forming-cuboidal cells are poorly differentiated and produced anaplastic tumors. Cuboidal and elongated cells were negative for epithelial markers. Cuboidal cells were poorly tumorigenic, but elongated cells produced highly malignant sarcoma-like tumors. In all lines, the int-1 gene was identically rearranged due to insertion of proviral DNA of the Mouse Mammary Tumor Virus, but the expression of int-1 varied with the state of differentiation of the cells. Polygonal cells contained relatively high levels of int-1 RNA, which were not influenced by steroid hormones. In the sphere-forming-cuboidal cells, expression of int-1 was low but inducible by dexamethasone. In the cuboidal and elongated cells no expression of int-1 was detectable, showing that the continued expression of int-1 was not required for progression to more malignant cells. By immunoprecipitation, two int-1 protein species, of 42 and 40 kD were identified in polygonal and in sphere-forming-cells but not in the culture media.

A method for the determination of antibody affinity using a direct ELISA.

Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.

The transport of reactive intermediates in a co-cultivation system: the role of intercellular communication.

The transport of reactive intermediates through gap junctions was studied in a co-cultivation system consisting of primary chick-embryo liver cells and V79 Chinese hamster cells. The formation of gap junctions was studied by measurement of the incorporation of [3H]hypoxanthine in HGPRT deficient V79 mutant cells after co-cultivation with the hepatocytes. Under control conditions the heterologous gap junctions allowed for the transfer of [3H]hypoxanthine resulting in an average value of 13 grains/cell. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited this transfer in a dose related way to background values. The transfer of reactive intermediates was measured as the number of sister chromatid exchanges (SCEs) induced in the V79 cells. For both compounds tested, benzo[a]pyrene and dimethylnitrosamine, transport of reactive intermediates through gap junctions was observed. When the inhibitory effects of TPA were investigated at different time points after start of the co-cultivation, inhibition was measurable after 6 h and increased to a maximal inhibition of 50%, observed after 24 h. No effect of TPA was observed on the number of SCEs. When the metabolic cooperation deficient V79 mutant cell line MC--27, was used the effects were completely comparable to those of TPA for both compounds. The lower mutagenic effects in the MC--27 cells cannot be attributed to a lower intrinsic sensitivity for mutagens of these cells.