Kepone (chlordecone) disrupts adherens junctions in human breast epithelial cells cultured on matrigel.

Perturbations in cell-extracellular matrix (ECM) interactions are a consistent feature of mammary tumors and cells in culture. We have utilized MCF-10ATG3B human breast epithelial cells to examine whether the organochlorine Kepone induces alterations in cell adhesion molecules important to cell-cell and cell-ECM interactions. Kepone effects on the levels and association of proteins involved in adherens junctions or desmosomes were examined using immunoblot analysis and immunoprecipitation. MCF-10ATG3B cells cultured on an ECM of Matrigel form lattice-like structures that are disrupted with 0.1 and 1 microM Kepone. E-cadherin protein levels decreased significantly by approximately 23% and approximately 69% following treatment with 0.1 and 1.0 microM Kepone, respectively, relative to solvent-treated cells. Desmoglein and alpha- and gamma-catenin levels did not vary significantly with Kepone. Beta-catenin protein levels decreased significantly by approximately 37%, 36% and 53% at 0.01, 0.1 and 1.0 microM Kepone, respectively. E-cadherin-gamma-catenin association was disrupted with 0.1 and 1.0 microM Kepone. Thus, Kepone disrupts cellular architecture, specifically E-cadherin-gamma-catenin containing adherens junctions, which may ultimately affect cellular phenotype.

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA.

A microtiter well-based quantitative reverse transcriptase-PCR assay for determination of BCR-ABL mRNA, which relies on coamplification of the target with an RNA internal standard (IS), was developed. The hapten digoxigenin (Dig) is incorporated during PCR. Target RNA and IS contain identical primer recognition sites and generate same-sized amplification products distinguishable by hybridization with probes specific to the molecules' central part. The hybrids are determined with an anti-Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as substrate. Fluorescent complexes of fluorosalicylate-Tb(III)-EDTA are measured by time-resolved fluorometry. The ratio of fluorescence values for target and IS is linearly related to initial target RNA in the range of 1000 to 200000 molecules. Samples containing K562 total RNA amidst 1 microgram of RNA from normal cells give fluorescence ratios that are linearly related to 30-10000 K562 cells. CVs for 30, 200, and 900 K562 cells are approximately 11%.

Quantitative polymerase chain reaction using a recombinant DNA internal standard and time-resolved fluorometry.

Using a 308 bp DNA fragment (target DNA) as a template, we have synthesized an internal standard (IS) that is of the same size and uses the same primers as the target but differs by a 26 bp centrally located sequence. We then designed quantitative polymerase chain reaction (PCR) assays in which the target DNA is coamplified with a constant amount of IS (20,000 molecules). The presence of IS compensates for the reaction-to reaction variability of the amplification efficiency. The PCR products are assayed by two distinct hybridization protocols. The first approach (QPCR-1) requires that specific probes be immobilized onto microtiter wells, followed by hybridization with digoxigenin-labeled PCR product. In the second protocol (QPCR-2), PCR product is captured onto the wells and hybridized with digoxigenin-tailed specific probes. In both assays, the hybrids are detected using an antidigoxigenin-alkaline phosphatase conjugate and 5'-fluorosalicylphosphate as substrate. The hydrolysis product forms a highly fluorescent complex with Tb(3+)-EDTA, as measured by time-resolved fluorometry. The ratio of the fluorescence values obtained for the amplified target DNA and IS is linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear ranges are 1000-200,000 molecules for QPCR-1 and 2000-200,000 molecules for QPCR-2. The CVs ranged from 3.4 to 9.7%.

Time-resolved fluorometric hybridization assays with RNA probes synthesized from polymerase chain reaction-generated DNA templates.

DNA templates suitable for direct synthesis of RNA probes are produced by the polymerase chain reaction. The nucleic acid sequence of interest is amplified using a downstream primer carrying the T7 RNA polymerase promoter sequence. The modified primer is incorporated into the amplified DNA, which is subsequently used for RNA probe synthesis in the presence of T7 RNA polymerase and a hapten-labeled ribonucleotide (digoxigenin-UTP). As a model, we prepared RNA probes specific for the BCR-ABL mRNA characteristic of chronic myelogenous leukemia. The probes are used in time-resolved fluorometric hybridization assays. Mixtures of BCR-ABL positive and negative cells, as well as whole blood samples, are analyzed. The sample mRNA is amplified using a biotinylated upstream primer. The amplified product (target DNA) is captured onto streptavidin-coated wells and hybridized to the RNA probe. The hybrids are detected with an alkaline phosphatase (ALP)-labeled antibody. ALP hydrolyzes the phosphate ester of fluorosalicylic acid, and the fluorosalicylate produced forms highly fluorescent ternary complexes with Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. As low as 0.4 fmol of target DNA can be detected. Also, a single leukemic cell may be detected in the presence of 0.5 million "normal" cells.

Detection of BCR-ABL transcripts from the Philadelphia translocation by hybridization in microtiter wells and time-resolved immunofluorometry.

Two hybridization assays have been developed to detect BCR-ABL mRNA transcripts arising from the Philadelphia translocation. Both assays use time-resolved immunofluorometric detection of polymerase chain reaction-amplified BCR-ABL mRNA sequences hybridized to specific probes. In configuration I, biotinylated amplified target is immobilized onto streptavidin-coated wells and hybridized to a probe labeled with the hapten digoxigenin. Hybrids are detected via an alkaline phosphatase-labeled antibody and fluorosalicylylphosphate as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb(3+)-EDTA. In configuration II, biotinylated probe is immobilized onto streptavidin-coated wells. PCR, performed in the presence of hapten-labeled deoxyribonucleotide, generates labeled product, which is hybridized to immobilized probe and quantified as above. BCR-ABL transcripts from one leukemic cell amidst mRNA from 500,000 normal granulocytes are detectable with signal/background ratios as high as 36.4 and 24.6 for configurations I and II, respectively. The respective CVs for the assays were 6.6-9.0% and 5.1-12.5%.

Time-resolved immunofluorometric determination of specific mRNA sequences amplified by the polymerase chain reaction.

We report a highly sensitive time-resolved immunofluorometric method for quantification of polymerase chain reaction (PCR)-amplified mRNA sequences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, during PCR, in the amplified product. The PCR product is captured, through its biotin moiety, to a streptavidin-coated solid phase and subsequently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specific mRNA as a model system. mRNA molecules corresponding to 0.1 leukemic cell in the presence of 0.5 million normal cells may be detected (signal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.