Hammerhead ribozymes targeted against cyclin E and E2F1 cooperate to down-regulate coronary smooth muscle cell proliferation.
BACKGROUND
Anti-proliferative drugs released from endo-vascular stents have substantially contributed to reduce in-stent restenosis rates in coronary arteries bearing single primary lesions by down-regulating coronary smooth muscle cell (CSMC) growth. However, the considerably lower drug efficacy shown in treatment of more complex coronary lesions suggests that alternative anti-proliferative approaches can be beneficial. Thus, we explored the use of hammerhead ribozymes as tools to knock down cyclin E and E2F1, two potent activators of cell proliferation which cooperate to promote the G1 to S phase transition.


METHODS
Two ribozymes, one directed against cyclin E and the other against E2F1 mRNAs, were delivered by liposomes to cultured human CSMCs. The influences on cell proliferation were measured evaluating BrdU incorporation into newly synthesised DNA. The effects on cell cycle phase distribution were determined by BrdU and 7-aminoactinomycin D incorporation into DNA.


RESULTS
Both ribozymes exhibited a sequence-specific and dose-dependent reduction in BrdU incorporation, which, at a concentration of 280 nM, persisted up to 4 days after transfection of CSMCs. A combined administration of the two ribozymes (210+210 nM) resulted in a more pronounced decrease in BrdU incorporation compared to the administration of an equimolar amount (420 nM) of each of them. Finally, both ribozymes induced a significant (P<0.05) reduction in S phase cells with a concomitant increase of G1/G0 and G2-M phase cells, compared to controls.


CONCLUSIONS
The ribozymes selected represent potent tools to prevent CSMC proliferation, especially when administered together, and thus are ideal candidates for in vivo application.
Grassi
G
G
Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstr. 8, 72076 Tübingen, Germany. ggrassi@units.it
Schneider
A
A
Engel
S
S
Racchi
G
G
Kandolf
R
R
Kuhn
A
A
D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D023921:Coronary Stenosis / Q000188:drug therapy; D019927:Cyclin E / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D015536:Down-Regulation; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006801:Humans; D008081:Liposomes; D009206:Myocardium / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects*; D016337:RNA, Catalytic / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D014162:Transfection
D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D008081:Liposomes; D016337:RNA, Catalytic; D014157:Transcription Factors; C427204:hammerhead ribozyme