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

Y. Maeshima, N. Kashihara, Hitoshi Sugiyama, Hirofumi Makino, Z. Ota

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25 Citations (Scopus)

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 anti-sense and sense phosphorothloate 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 μM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50%. The effect of anti-sense 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.

Original languageEnglish
Pages (from-to)2219-2229
Number of pages11
JournalJournal of the American Society of Nephrology
Volume7
Issue number10
Publication statusPublished - 1996

Fingerprint

Mesangial Cells
Antisense Oligonucleotides
Oligodeoxyribonucleotides
Proliferating Cell Nuclear Antigen
Cell Proliferation
Messenger RNA
Nuclear Proteins
Proteins
Initiator Codon
Growth
Reverse Transcription
Cell Cycle
Cell Count
Immunohistochemistry
Monoclonal Antibodies
Gene Expression

Keywords

  • Antisense therapy
  • Growth factor
  • Ki-67
  • Mesangial cell proliferation
  • PCNA

ASJC Scopus subject areas

  • Nephrology

Cite this

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title = "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 anti-sense and sense phosphorothloate 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 μM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50{\%}. The effect of anti-sense 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.",
keywords = "Antisense therapy, Growth factor, Ki-67, Mesangial cell proliferation, PCNA",
author = "Y. Maeshima and N. Kashihara and Hitoshi Sugiyama and Hirofumi Makino and Z. Ota",
year = "1996",
language = "English",
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T1 - Antisense oligonucleotides to proliferating cell nuclear antigen and Ki- 67 inhibit human mesangial cell proliferation

AU - Maeshima, Y.

AU - Kashihara, N.

AU - Sugiyama, Hitoshi

AU - Makino, Hirofumi

AU - Ota, Z.

PY - 1996

Y1 - 1996

N2 - 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 anti-sense and sense phosphorothloate 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 μM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50%. The effect of anti-sense 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.

AB - 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 anti-sense and sense phosphorothloate 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 μM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50%. The effect of anti-sense 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.

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