title: Design of nuclease resistant protein kinase calpha DNA enzymes with potential therapeutic application.
abstract: For the therapeutic application of catalytic nucleic acids it is desirable to have small, stable and inexpensive compounds that are active at physiological Mg(2+) concentrations. We have explored the possibility of using the versatile 10-23 DNA catalytic core to suppress the expression of the protein kinase Calpha (PKCalpha) isoform in malignant cells. By introducing either a 3'-3'-inverted thymidine nucleotide or site-specific phosphorothioate modification into a PKCalpha DNA enzyme, we have designed stable catalysts that retained a significant in vitro cleavage activity. In particular, a DNA enzyme containing phosphorothioate analogues in the antisense arms and in the pyrimidine residues of the catalytic core was found to be remarkably stable in 50 % human serum (t(1/2)>90 hours) and inhibited in vitro cell growth by up to 90 % at nanomolar concentrations. The inhibition of PKCalpha gene expression is sequence-specific, as a DNA enzyme with reversed antisense arms was found to be ineffective. Epifluorescence microscopic analysis of cells transfected with a 5' fluorescein isothiocyanate-conjugated DNA enzyme showed that the DNA enzyme molecules are mainly localised in the nuclei. Most of the DNA enzyme-treated cells were killed by apoptosis. The ability of the described PKCalpha DNA enzymes to trigger apoptosis (apoptozymes) in malignant cells illustrates their therapeutic potential. Furthermore, such agents can be a valuable tool for probing gene function.
authors.affiliation: Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, N-0310, Norway. mosioud@@embnet.uio.no
authors.forename: M
authors.initials: M
authors.lastname: Sioud
authors.forename: M
authors.initials: M
authors.lastname: Leirdal
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001692:Biological Transport; D002384:Catalysis; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000201:enzymology; D048429:Cell Size / Q000187:drug effects; D016373:DNA, Antisense / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use*; D003851:Deoxyribonucleases / Q000097:blood / Q000378:metabolism*; D015195:Drug Design; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015316:Genetic Therapy; D006207:Half-Life; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics*; D008081:Liposomes / Q000008:administration & dosage; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics*; D051571:Protein Kinase C-alpha; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D016373:DNA, Antisense; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D007527:Isoenzymes; D008081:Liposomes; C402582:RNA-cleaving DNA 10-23; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003851:Deoxyribonucleases