Biomedical subjects

E M McKeegan

Publications and source records attributed to E M McKeegan.

3 recordsLinked to original sources

PubMed2000

Application of QuantiGene nucleic acid quantification technology for high throughput screening.

To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.

Branched DNA Signal Amplification Assay↗

PubMed1991

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.

Animals↗

PubMed1989

Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance.

Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance. Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds. We therefore initiated a study of PKC and MDR. We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity. The mechanism of the increase appears to be quite different in the two cell lines. The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein. Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin. We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level. The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different. The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗