Abstract
Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.
Highlights
Expression of ICAM-1 on the surfaceof human umbilical vein endothelial celalsnd a human lung carcinoma The bindingof circulating leukocytes to vascular endothecell line (A549) was increased by interleukin-16, tu- lium is an obligatory step in the emigrationof leukocytes out mor necrosis factorCYa,nd interferony in a concentra- of the vasculature to the soifteinfection or injury (1).Several tion-dependentmanner.Phosphorothioateantisense endothelial proteins have recently been identified that meoligonucleotides designedto hybridize t1o0target sites diate the adherenceof leukocytes to inflamedvascular endoon the human ICAM-1 mRNA were tested for inhibi- thelium and subsequent emigration oouftthe vasculature(2
Effect of DOTMA on ICAM-1Expression-Preliminary experiments demonstrated that the cationic lipid DOTMA enhanced the potency of antisense oligonucleotideinhibition of ICAM-1 at least 100-foldand cellular uptake of oligonucleotide 10-fold.' The differences between enhanced biological activity and enhanced uptake can be partially explained by changes in the subcellular distribution of the oligonucleotide in the presence of DOTMA-containing vesicles.' To demonstrate that DOTMA does not directly modify cytokine-induced ICAM-1 expression, HUVEC and A549 cells were stimulated with increasing concentrations of IL-lp, TNF-a, and IFN-y in the presence or absence of DOTMA
It was possiblethat ISIS 1939 decreased ICAM-1expression by modulating the levels of ICAM-1 mRNA.To address this possibility, total cellular RNA was analyzed for the presence of ICAM-1mRNAby Northern blot hybridization in cells that had been treated with oligonucleotides
Summary
Effect of DOTMA on ICAM-1Expression-Preliminary experiments demonstrated that the cationic lipid DOTMA enhanced the potency of antisense oligonucleotideinhibition of ICAM-1 at least 100-foldand cellular uptake of oligonucleotide 10-fold.' The differences between enhanced biological activity and enhanced uptake can be partially explained by changes in the subcellular distribution of the oligonucleotide in the presence of DOTMA-containing vesicles.' To demonstrate that DOTMA does not directly modify cytokine-induced ICAM-1 expression, HUVEC and A549 cells were stimulated with increasing concentrations of IL-lp, TNF-a, and IFN-y in the presence or absence of DOTMA. ISIS 1570, which hybridized to the AUG translation initiation codon, was one of the most active antisense oligonucleotides, completely inhibiting IL-ID-induced ICAM-1 expression at concentrations as low as 70 nM in HUVEC and effecting 70% inhibition in A549 cells a t 0.7 PM. The control oligonucleotide ISIS 1821 did inhibit ICAM-1 expression in HUVEC with activity comparable to ISIS 1934; in A549 cells, ISIS 1821was less effective than ISIS 1934 The weak activity exhibited by ISIS 1821 may be due, in part, to hybridization to the ICAM-1 mRNA, 12 out of 13 bases at a position 15 bases 3‘ to the AUG translation initiationcodon These studies demonstrate that the AUG translation initiation codon and specific 3’untranslated sequences in theICAM-1 mRNA werethe most susceptible to antisense oligonucleotide inhibition of ICAM1 expression.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
