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J Biol Chem, Vol. 273, Issue 31, 19587-19591, July 31, 1998

Antisense Oligonucleotides Targeted against Protein Kinase C and CII Block 1,25-(OH)₂D₃-induced Differentiation

Robert U. Simpson, Timothy D. O'Connell, Quintin Pan, Judy Newhouse, and Martha J. Somerman^**

From the  Department of Pharmacology and ^** Department of Periodontics/Prevention/Geriatrics, the University of Michigan, Ann Arbor, Michigan 48109-0632

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKC, PKC, PKCI, and PKCII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)₂D₃ (20 nM for 48 h) was inhibited similarly in cells treated with PKC antisense (30 µM) 24 h prior to or at the same time as hormone treatment (86 ± 9% inhibition; n = 4 versus 82 ± 8% inhibition; n = 4 (mean ± S.E.), respectively). In contrast, cells treated with PKC antisense 24 h after 1,25-(OH)₂D₃ were unaffected and fully differentiated. PKC antisense did not block 1,25-(OH)₂D₃ promotion of HL-60 cell differentiation. Next, the ability of PKCI- and PKCII-specific antisense oligonucleotides to block 1,25-(OH)₂D₃ promotion of cell differentiation was examined. PKCII antisense (30 µM) completely blocked CD14 expression induced by 1,25-(OH)₂D₃, whereas PKCI antisense had little effect. Interestingly, PKCII antisense blocked differentiation by 87 ± 7% (n = 2, mean ± S.D.) but had no effect on 1,25-(OH)₂D₃ inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)₂D₃ on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCII expression.

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