Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.
We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.
Dean
N M
NM
Department of Molecular and Cellular Biology, ISIS Pharmaceuticals, Carlsbad, California 92008.
McKay
R
R
Condon
T P
TP
Bennett
C F
CF
D001483:Base Sequence; D002277:Carcinoma; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010703:Phorbol Esters; D012333:RNA, Messenger; D013873:Thionucleotides; D018799:Intercellular Adhesion Molecule-1; D011493:Protein Kinase C