Design of nuclease resistant protein kinase calpha DNA enzymes with potential therapeutic application.
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.
Sioud
M
M
Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, N-0310, Norway. mosioud@@embnet.uio.no
Leirdal
M
M
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
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