Antisense oligonucleotides to proliferating cell nuclear antigen and Ki-67 inhibit human mesangial cell proliferation.

Abstract

Proliferating cell nuclear antigen (PCNA) and Ki-67 are cell cycle-associated nuclear proteins and are used as markers for proliferating cells. This study attempted to inhibit glomerular mesangial cell (MC) proliferation, which is the hallmark of many forms of glomerular disease, by inhibiting these nuclear proteins with antisense oligodeoxynucleotides. The antisense and sense phosphorothioate oligodeoxynucleotides complementary to PCNA and Ki-67 mRNA, including the initiation codon, were synthesized. Human MC were cultured in growth medium in the presence of sense or antisense oligodeoxynucleotides, and the effects of these oligodeoxynucleotides on mesangial cell proliferation were evaluated by direct cell count. Both PCNA and Ki-67 antisense oligodeoxynucleotides significantly inhibited mesangial cell proliferation as compared with sense oligodeoxynucleotides. Antisense oligodeoxynucleotides (10 microM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50%. The effect of antisense oligodeoxynucleotides on target protein expression was examined by immunocytochemistry using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction also was performed to evaluate the effect of antisense oligodeoxynucleotides on PCNA and Ki-67 mRNA expression. Studies of target protein and mRNA expression revealed that the inhibitory effects of the antisense oligonucleotides were mediated through decreases in the expression of both mRNA and protein. Sense oligodeoxynucleotides produced little effect. These results indicate that antisense oligodeoxynucleotides targeting PCNA and Ki-67 mRNA reduce the expression of these gene products and inhibit mesangial cell proliferation. Moreover, these results suggest the feasibility of antisense strategies designed to inhibit PCNA and Ki-67 expression for the inhibition of mesangial cell proliferation in vivo.