An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro.
The transcription factor E2F controls expression of several genes involved in cell proliferation including c-myc, c-myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.
Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
N
N
Tomita
M
M
Horiuchi
S
S
Tomita
G H
GH
Gibbons
J Y
JY
Kim
D
D
Baran
V J
VJ
Dzau
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D016384:Consensus Sequence; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D005786:Gene Expression Regulation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects; D007672:Kidney Cortex / Q000166:cytology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D056465:Retinoblastoma-Binding Protein 1; D013873:Thionucleotides; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016376:Oligonucleotides, Antisense; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D056465:Retinoblastoma-Binding Protein 1; D013873:Thionucleotides; D050680:Transcription Factor DP1; D014157:Transcription Factors; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases