Pe. Rakoczy et al., EXPRESSION OF CATHEPSIN S ANTISENSE TRANSCRIPTS BY ADENOVIRUS IN RETINAL-PIGMENT EPITHELIAL-CELLS, Investigative ophthalmology & visual science, 39(11), 1998, pp. 2095-2104
PURPOSE. TO show the production of sense or antisense transcripts by r
ecombinant adenoviruses, to investigate whether the transcripts produc
ed were suitable for downregulating the expression of the targeted gen
e, cathepsin S (CatS), and to examine the effect of antisense transcri
pt production on the biologic function of retinal pigment epithelial (
RPE) cells, including the regulation of endogenous aspartic protease e
xpression. METHODS. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS rec
ombinant viruses were produced by homologous recombination. The recomb
inant viruses were tested by restriction enzyme digestion to confirm t
he orientation of the inserts. The expression of antisense transcripts
was tested by not-them blot analysis. Western blot analysis was used
to study the regulation of the endogenous CatS protein in ARPE19 cells
. The biologic effect of CatS downregulation in ARPE19 cells was teste
d by proliferation and phagocytosis assays, de novo cathepsin D (CatD)
synthesis, and measurement of aspartic protease activity. RESULTS. Af
ter characterization of the recombinant adenovirus constructs, the pro
duction of antisense and sense CatS transcripts was shown in ARPE19 ce
lls. The transcripts appeared at approximately 1.9 kb 48 hours after t
ransduction, and the expression of the antisense transcripts was simil
ar in constructs carrying either the MLP or the RSV promoter. Western
blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS a
nd Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a stro
ng signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.C
atSS. ARPE19 cells were transduced to a high level. The transduction o
f ARPE19 cells with the recombinant adenoviruses did not affect the mo
rphologic appearance of the cells, their proliferation, or their phago
cytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS re
combinant adenoviruses showed a significant downregulation of de novo
CatD synthesis and a twofold decrease in aspartic protease activity. C
ONCLUSIONS. Recombinant adenoviruses were shown to be suitable for pro
ducing antisense CatS transcripts to modulate endogenous CatS expressi
on in RPE cells. It is proposed that CatS may play an important role,
directly or indirectly, in the lysosomal digestion of outer segments t
hrough the regulation of other lysosomal enzyme activity, such as the
expression of CatD.