Toxicity of cationic lipid-ribozyme complexes in human prostate tumor cells can mimic ribozyme activity.
Prostate tumor cell lines have been shown to both produce interleukin-6 (IL-6) and express the IL-6 receptor, suggesting a potential autocrine growth regulatory role for IL-6. We explored the role of IL-6 in the proliferation of the human prostatic carcinoma cell line, DU145, using ribozymes to inhibit IL-6 expression. Hammerhead-type ribozymes targeted against IL-6 mRNA sequences were prepared, and in vitro analyses were used to demonstrate that these molecules catalyzed the cleavage of IL-6 mRNA poly- nucleotide fragments. To test in situ activity, these ribozymes were transfected into DU145 cells using cationic transfection lipids, cytofectins. Treatment of cultured cells with ribozyme/cationic lipid complexes resulted in a reduction of IL-6 protein levels in the supernatant and reduced numbers of DU145 cells 48 h after treatment. However, similar results were also seen following treatment with control RNA/lipid complexes. This reduction in IL-6 levels and cell numbers was a function of the RNA/lipid complexes and was not seen with either lipid or RNA alone. Therefore, the reductions in IL-6 levels and cell numbers observed were not due to ribozyme-mediated cleavage of IL-6 mRNA, but rather reflected a dose-dependent, nonspecific toxic effect of the treatment with ribozyme/cytofectin complexes. This effect can resemble functional ribozyme activity, complicating analysis of the activity of synthetic ribozymes after transfection into cultured cells.
Freedland
S J
SJ
Department of Medical Pathology, University of California, Davis, California, 95616, USA.
Malone
R W
RW
Borchers
H M
HM
Zadourian
Z
Z
Malone
J G
JG
Bennett
M J
MJ
Nantz
M H
MH
Li
J H
JH
Gumerlock
P H
PH
Erickson
K L
KL
D001483:Base Sequence; D002412:Cations; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics; D050356:Lipid Metabolism; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D011471:Prostatic Neoplasms / Q000473:pathology*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D002412:Cations; D015850:Interleukin-6; D016337:RNA, Catalytic; D012333:RNA, Messenger; D008156:Luciferases