title: Toxicity of cationic lipid-ribozyme complexes in human prostate tumor cells can mimic ribozyme activity.
abstract: 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.
authors.affiliation: Department of Medical Pathology, University of California, Davis, California, 95616, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Freedland
authors.forename: R W
authors.initials: RW
authors.lastname: Malone
authors.forename: H M
authors.initials: HM
authors.lastname: Borchers
authors.forename: Z
authors.initials: Z
authors.lastname: Zadourian
authors.forename: J G
authors.initials: JG
authors.lastname: Malone
authors.forename: M J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: M H
authors.initials: MH
authors.lastname: Nantz
authors.forename: J H
authors.initials: JH
authors.lastname: Li
authors.forename: P H
authors.initials: PH
authors.lastname: Gumerlock
authors.forename: K L
authors.initials: KL
authors.lastname: Erickson
mesh_terms: 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
chemical_list: D002412:Cations; D015850:Interleukin-6; D016337:RNA, Catalytic; D012333:RNA, Messenger; D008156:Luciferases