An oligonucleotide decoy for the transcription factor e2f inhibits
mesangial cell proliferation in vitro.
Tomita, Naruya, Masatsugu Horiuchi, Sawako Tomita, Gary H Gibbons,
John Ys Kim, Dana Baran, Victor J Dzau.
Department of Medicine, Harvard Medical School, Brigham and Women_s
Hospital, 75 Francis Street, Boston MA 02115, Division of Nephrology,
McGill University, 3775 University St., Montreal, Canada H3A 2B4
APStracts 5:0093F, 1998.
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, increased
specific binding of E2F was detected in MC following serum
stimulation. This binding was completely inhibited by pre-incubation
of MC nuclear extracts with the double stranded ODN with high
affinity for E2F, but not by pre-incubation 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 co-transfection 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.
Received 6 January 1997; accepted in final form 4 May 1998.
APS Manuscript Number F5-7.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 8 May 1998