Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RIalpha subunit.
The primary mediator of cAMP action in mammalian cells is cAMP-dependent protein kinase (PKA). There are two types of PKA, type I (PKA-I) and type II (PKA-II), which share a common catalytic subunit but contain distinct regulatory subunits, RI and RII, respectively. Evidence suggests that increased expression of RIalpha/PKA-I correlates with neoplastic cell growth. Here, we show that sequence-specific oligonucleotide inhibition of RIalpha expression results in inhibition of growth and modulation of cAMP signaling in cancer cells. The antisense promoted growth inhibition in a time-dependent, concentration-dependent, and sequence-dependent manner in human cancer cells in monolayer culture, and it inhibited colony formation in soft agar and tumor growth in nude mice. Among the cancer cells are LS-174T, HCT-15, and Colo-205 colon carcinoma cells; A-549 lung carcinoma cells; LNCaP prostate adenocarcinoma cells; Molt-4 leukemia cells; and Jurkat T lymphoma cells. Northern blot and immunoprecipitation analyses revealed that the growth inhibitory effect of the antisense correlated with a decrease in RIalpha expression at both the mRNA and protein levels. Pulse-chase experiments revealed that the antisense-directed inhibition of RIalpha expression resulted in compensatory changes in expression of the isoforms of R and C subunits and cAMP signaling in a cell type-specific manner. These results demonstrate that cAMP is ubiquitous in the regulation of cell growth and that the antisense oligonucleotide, which inhibits the synthesis of the RIalpha subunit of PKA, can be targeted to a single gene for treatment of cancer in a variety of cell types.
Nesterova
M
M
Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1750, USA.
Cho-Chung
Y S
YS
D020413:3' Untranslated Regions / Q000187:drug effects / Q000235:genetics; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000187:drug effects / Q000378:metabolism; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D000242:Cyclic AMP / Q000502:physiology; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D016373:DNA, Antisense / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression / Q000187:drug effects*; D018390:Gene Targeting; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology*; D016372:RNA, Antisense / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D016373:DNA, Antisense; D016376:Oligonucleotides, Antisense; C517834:PRKAR1A protein, human; D016372:RNA, Antisense; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases