Thomas P. Condon

Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2′-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nm) that target the 5′ cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5′ cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5′ cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation. Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2′-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nm) that target the 5′ cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5′ cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5′ cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation. Antisense oligonucleotides have been shown to be effective agents for inhibition of gene expression at the mRNA level (1Crooke S.T. Med. Res. Rev. 1996; 16: 319-344Google Scholar, 2Wagner R.W. Nature. 1994; 372: 333-335Google Scholar, 3Mirabelli C.K. Crooke S.T. Crooke S.T. Lebleu B. Antisense Research and Applications. CRC Press, Boca Raton, FL1993: 7-35Google Scholar). They may be described as exogenous regulators of mRNA metabolism intended to sterically interfere with one or more metabolic processes upon hybridization, such as initiation of translation, or to promote enzyme-mediated mRNA degradation by formation or exposure of a region for nuclease activity, such as RNase H. The mode of action of an antisense oligonucleotide in cells is dependent upon its composition (sugar, backbone, and base residues) and mRNA binding site location (5′-UTR, coding region, 3′-UTR). 1The abbreviations used are: UTR, untranslated region; ICAM-1, intercellular adhesion molecule 1; HUVECs, human umbilical vein endothelial cells; TNF-α, tumor necrosis factor α; PE, phycoerythrin; DTT, dithiothreitol; PBS, phosphate-buffered saline; G3PDH, glycerol-3-phosphate dehydrogenase. 1The abbreviations used are: UTR, untranslated region; ICAM-1, intercellular adhesion molecule 1; HUVECs, human umbilical vein endothelial cells; TNF-α, tumor necrosis factor α; PE, phycoerythrin; DTT, dithiothreitol; PBS, phosphate-buffered saline; G3PDH, glycerol-3-phosphate dehydrogenase. Although several types of antisense oligonucleotides, which differ in composition and target site, have been found to be effective agents for sequence-specific inhibition of gene expression in mammalian cells, direct or detailed evidence of their mode(s) of action remains limited (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar, 6Bonham M.A. Brown S. Boyd A.L. Brown P.H. Bruckenstein D.A. Hanvey J.C. Thomson S.A. Pipe A. Hassman F. Bisi J.E. Froehler B.C. Matteucci M.D. Wagner R.W. Noble S.A. Babiss L.E. Nucleic Acids Res. 1995; 23: 1197-1203Google Scholar, 7Wagner R.W. Matteucci M.D. Lewis J.G. Gutierrez A.J. Moulds C. Froehler B.C. Science. 1993; 260: 1510-1513Google Scholar, 8Giles R.V. Spiller D.G. Tidd D.M. Antisense Res. Dev. 1995; 5: 23-31Google Scholar, 9Condon T.P. Bennett F.C. J. Biol. Chem. 1996; 271: 30398-30403Google Scholar).Intercellular adhesion molecule 1 (ICAM-1) is one of several cell adhesion molecules expressed on the cell surface of vascular endothelium that participates in a broad range of immune and inflammatory responses (10Hogg, N. (ed) (1991) Integrins and ICAM-1 in Immune Responses: ChemicalImmunology, Vol. 50, pp. 98-158, S. Karger, BaselGoogle Scholar). ICAM-1 is also expressed on nonendothelial cells, such as keratinocytes, monocytes, and fibroblasts in response to inflammatory mediators. Elevated levels of ICAM-1 expression have been observed in a number of immune-related human diseases (11Albelda S.M. Smith C.W. Ward P.A. FASEB J. 1994; 8: 504-512Google Scholar, 12Pilewski J.M. Albelda S.M. Am. J. Respir. Cell Mol. Biol. 1995; 12: 1-3Google Scholar),e.g. rheumatoid arthritis, psoriasis, and asthma. Thus, regulation of ICAM-1 gene expression is of therapeutic interest (13Brady H.R. Curr. Opin. Nephrol. Hypertens. 1993; 2: 171-182Google Scholar, 14Bennett C.F. Crooke S.T. Henderson B. Bodmer M.W. Therapeutic Modulation of Cytokines. CRC Press, New York1996: 171-193Google Scholar, 15Bennett C.F. Crooke S.T. Adv. 1994; Scholar). The ICAM-1 gene been and the initiation site been for several cell by a of C. J. Immunol. 1991; Scholar, J. Biol. Chem. 1991; 266: human umbilical vein endothelial cells with by T.P. J. Biol. Chem. 1995; that expression of ICAM-1 may be in cells by antisense oligonucleotides (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar). that the effective oligonucleotides that with RNase H and the of the in have a number of oligonucleotide at the which in and nuclease but not RNase H Chem. Res. 1995; Scholar). Antisense oligonucleotides that more to the target mRNA to be more effective at with the processes of metabolism at at this also have been shown to a of nuclease and of a of these oligonucleotides S. A. S. A. 2′-O-(2-methoxy)ethyl 1995; and M.D. Freier S.M. Zounes C. J. Med. Chem. 1993; that target the 5′ of the ICAM-1 transcript to of the oligonucleotides, and for an of their mode of action in 1 and The 5′ cap of mRNA been shown to be a that functions throughout mRNA metabolism Rev. Scholar, B. S. 1996; 2: Scholar, S. 1996; 2: Scholar, N. S. A. Scholar, P.A. Scholar, 1995; Scholar, J. J. Cell Biol. Scholar, J. Scholar, N. N. Press, Scholar, Scholar, H. in CRC Press, Boca Raton, Scholar, Nucleic Acids Res. 1991; Scholar, A. A.J. Nature. 266: Scholar). antisense oligonucleotides which target the 5′ cap region of a transcript have the potential to modulate one or more metabolic stages of the message inside the cell Crooke S.T. Lebleu B. Antisense Research and Applications. CRC Press, Boca Raton, FL1993: Scholar). In this study the antisense mode of action was by of the target metabolic processes translation, and antisense and of gene and of antisense was by of cell surface expression of ICAM-1 protein with oligonucleotide at in the range of as described the of of that was or observed in the was as described C. Lima W.F. Freier S.M. J. Biol. Chem. 1993; = and = as of the and in a oligonucleotides in a to that inhibition of ICAM-1 protein expression by the oligonucleotides, and was The oligonucleotides, and effects on ICAM-1 protein expression in the and the oligonucleotides more potent of ICAM-1 expression in the RNase oligonucleotides effective at ICAM-1 protein expression in to the RNase for and in to and the and The is a of the antisense in base is the is the 2′-O-(2-methoxy)ethyl and is the 2′-O-(2-methoxy)ethyl of the oligonucleotides was by oligonucleotides, to and the of their antisense with to the C.F. Chiang M-Y. Chan H. J.E. C.K. Mol. of with the in the of the in a of the oligonucleotide in the cell as as in of with the a of in the to degradation of the by J. Scholar, Freier S. Nucleic Acids Res. 1995; 23: Scholar). In the oligonucleotide a to the with a of in the in to the the a to its to the of D.A. S. S.M. S. J. Biol. Chem. 1994; Scholar, B. Nucleic Acids Res. 1995; 23: and more with the for and in of oligonucleotides observed with of cells with and These that the and oligonucleotides the cell in the of C.F. Chiang M-Y. Chan H. J.E. C.K. Mol. was and to determine inhibition of ICAM-1 protein expression by and degradation of the target transcript, an observed with RNase antisense oligonucleotides (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar). was at and a for and HUVECs. Interestingly, a increase in abundance of the ICAM-1 transcript in cells with the oligonucleotides at that this increase in transcript abundance was not due to an increase in the of of the ICAM-1 gene not in target transcript abundance in cells with antisense oligonucleotides which the 5′ cap region of of for ICAM-1 and abundance of ICAM-1 transcript, to the mRNA oligonucleotide was at a of at and by is a oligonucleotide used as a in this determine the antisense on transcript abundance was to ICAM-1, for the transcript expression is also by in HUVECs. with ICAM-1 an increase in abundance of the transcript was observed in cells with the that the region of the transcript The increase in target transcript abundance antisense may be an of these expressed in with the antisense mode of in target transcript abundance also in cells with antisense oligonucleotides which target the 5′ cap region of of for and abundance of transcript, to the oligonucleotide was at a of at and by is to of the transcript A. J. M.A. J. Biol. Chem. 1991; 266: 5′ cap structure of mRNA been shown to splicing of the of in several H. Dev. Scholar, H. S. A. Scholar). The ICAM-1 gene of by with 1 in C. J. Immunol. 1991; Scholar). of the that this of antisense oligonucleotides on splicing of the ICAM-1 to the transcript, as by of ICAM-1 mRNA or of not and was to determine antisense inhibition of ICAM-1 protein expression inhibition of transport of the transcript of the to the cytoplasm. mRNA was by 2 for 2′-O-(2-methoxy)ethyl and antisense and at nm) and cells this in the abundance of the ICAM-1 transcript was observed in the of antisense cells and In a increase in the abundance of the ICAM-1 transcript was observed in the the antisense cells, and in to the and and cells abundance of the ICAM-1 transcript in was also these the abundance of the ICAM-1 transcript was and in the and and in the of the antisense cells, and to the The increase in abundance of the transcript in the of the antisense cells a in the at which the transcript is The of a in ICAM-1 mRNA abundance in the that the antisense oligonucleotides not the transport of the ICAM-1 antisense oligonucleotides have on of the ICAM-1 The increase in ICAM-1 mRNA abundance in the cytoplasm. of and 2 for ICAM-1 and abundance of ICAM-1 transcript, to oligonucleotide was at a of to determine the of antisense oligonucleotide upon the translation of the target ICAM-1 transcript ICAM-1 protein and mRNA a cells with antisense oligonucleotides and and their and by The of the a of the and of the by a in the of the ICAM-1 transcript in cells with and in to of the and The for the and cells the and of the target transcript in the and the and the and of the target transcript in the and of translation initiation to be mode of action for oligonucleotides that target the 5′ cap region of the ICAM-1 transcript in HUVECs. for cells with and at cells as was on for to for and that inhibition of ICAM-1 protein expression interference with translation initiation and as by the of transcript the The formation of a in the 5′ cap region is the of this The increase in abundance of the ICAM-1 mRNA in the of the antisense cells in with the in the that one of the target is coupled to translation. These with of that in the coding region, and N. Mol. Biol. 1995; J. Mol. Biol. 1994; of gene expression may at one or more stages of mRNA The known of regulation mRNA metabolism have been found at the stages of translation N. Press, and degradation J. Rev. 1995; of the transcript, mRNA and have been found to be been shown that and in the region may initiation of translation J. Biol. Chem. 1991; 266: Scholar, S. A. Scholar, J. N. Scholar). The antisense oligonucleotides, to the region of the target transcript this mode of regulation in cells by formation of the 80 S translation initiation complex. that this in the turnover of the expressed ICAM-1 Antisense oligonucleotides have been shown to be effective agents for inhibition of gene expression at the mRNA level (1Crooke S.T. Med. Res. Rev. 1996; 16: 319-344Google Scholar, 2Wagner R.W. Nature. 1994; 372: 333-335Google Scholar, 3Mirabelli C.K. Crooke S.T. Crooke S.T. Lebleu B. Antisense Research and Applications. CRC Press, Boca Raton, FL1993: 7-35Google Scholar). They may be described as exogenous regulators of mRNA metabolism intended to sterically interfere with one or more metabolic processes upon hybridization, such as initiation of translation, or to promote enzyme-mediated mRNA degradation by formation or exposure of a region for nuclease activity, such as RNase H. The mode of action of an antisense oligonucleotide in cells is dependent upon its composition (sugar, backbone, and base residues) and mRNA binding site location (5′-UTR, coding region, 3′-UTR). 1The abbreviations used are: UTR, untranslated region; ICAM-1, intercellular adhesion molecule 1; HUVECs, human umbilical vein endothelial cells; TNF-α, tumor necrosis factor α; PE, phycoerythrin; DTT, dithiothreitol; PBS, phosphate-buffered saline; G3PDH, glycerol-3-phosphate dehydrogenase. 1The abbreviations used are: UTR, untranslated region; ICAM-1, intercellular adhesion molecule 1; HUVECs, human umbilical vein endothelial cells; TNF-α, tumor necrosis factor α; PE, phycoerythrin; DTT, dithiothreitol; PBS, phosphate-buffered saline; G3PDH, glycerol-3-phosphate dehydrogenase. Although several types of antisense oligonucleotides, which differ in composition and target site, have been found to be effective agents for sequence-specific inhibition of gene expression in mammalian cells, direct or detailed evidence of their mode(s) of action remains limited (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar, 6Bonham M.A. Brown S. Boyd A.L. Brown P.H. Bruckenstein D.A. Hanvey J.C. Thomson S.A. Pipe A. Hassman F. Bisi J.E. Froehler B.C. Matteucci M.D. Wagner R.W. Noble S.A. Babiss L.E. Nucleic Acids Res. 1995; 23: 1197-1203Google Scholar, 7Wagner R.W. Matteucci M.D. Lewis J.G. Gutierrez A.J. Moulds C. Froehler B.C. Science. 1993; 260: 1510-1513Google Scholar, 8Giles R.V. Spiller D.G. Tidd D.M. Antisense Res. Dev. 1995; 5: 23-31Google Scholar, 9Condon T.P. Bennett F.C. J. Biol. Chem. 1996; 271: 30398-30403Google Scholar). adhesion molecule 1 (ICAM-1) is one of several cell adhesion molecules expressed on the cell surface of vascular endothelium that participates in a broad range of immune and inflammatory responses (10Hogg, N. (ed) (1991) Integrins and ICAM-1 in Immune Responses: ChemicalImmunology, Vol. 50, pp. 98-158, S. Karger, BaselGoogle Scholar). ICAM-1 is also expressed on nonendothelial cells, such as keratinocytes, monocytes, and fibroblasts in response to inflammatory mediators. Elevated levels of ICAM-1 expression have been observed in a number of immune-related human diseases (11Albelda S.M. Smith C.W. Ward P.A. FASEB J. 1994; 8: 504-512Google Scholar, 12Pilewski J.M. Albelda S.M. Am. J. Respir. Cell Mol. Biol. 1995; 12: 1-3Google Scholar),e.g. rheumatoid arthritis, psoriasis, and asthma. Thus, regulation of ICAM-1 gene expression is of therapeutic interest (13Brady H.R. Curr. Opin. Nephrol. Hypertens. 1993; 2: 171-182Google Scholar, 14Bennett C.F. Crooke S.T. Henderson B. Bodmer M.W. Therapeutic Modulation of Cytokines. CRC Press, New York1996: 171-193Google Scholar, 15Bennett C.F. Crooke S.T. Adv. 1994; Scholar). The ICAM-1 gene been and the initiation site been for several cell by a of C. J. Immunol. 1991; Scholar, J. Biol. Chem. 1991; 266: human umbilical vein endothelial cells with by T.P. J. Biol. Chem. 1995; Scholar). that expression of ICAM-1 may be in cells by antisense oligonucleotides (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar). that the effective oligonucleotides that with RNase H and the of the in have a number of oligonucleotide at the which in and nuclease but not RNase H Chem. Res. 1995; Scholar). Antisense oligonucleotides that more to the target mRNA to be more effective at with the processes of metabolism at at this also have been shown to a of nuclease and of a of these oligonucleotides S. A. S. A. 2′-O-(2-methoxy)ethyl 1995; and M.D. Freier S.M. Zounes C. J. Med. Chem. 1993; that target the 5′ of the ICAM-1 transcript to of the oligonucleotides, and for an of their mode of action in 1 and The 5′ cap of mRNA been shown to be a that functions throughout mRNA metabolism Rev. Scholar, B. S. 1996; 2: Scholar, S. 1996; 2: Scholar, N. S. A. Scholar, P.A. Scholar, 1995; Scholar, J. J. Cell Biol. Scholar, J. Scholar, N. N. Press, Scholar, Scholar, H. in CRC Press, Boca Raton, Scholar, Nucleic Acids Res. 1991; Scholar, A. A.J. Nature. 266: Scholar). antisense oligonucleotides which target the 5′ cap region of a transcript have the potential to modulate one or more metabolic stages of the message inside the cell Crooke S.T. Lebleu B. Antisense Research and Applications. CRC Press, Boca Raton, FL1993: Scholar). In this study the antisense mode of action was by of the target metabolic processes translation, and antisense and of gene Antisense was by of cell surface expression of ICAM-1 protein with oligonucleotide at in the range of as described the of of that was or observed in the was as described C. Lima W.F. Freier S.M. J. Biol. Chem. 1993; = and = as of the and oligonucleotides in a to that inhibition of ICAM-1 protein expression by the oligonucleotides, and was The oligonucleotides, and effects on ICAM-1 protein expression in the and the oligonucleotides more potent of ICAM-1 expression in the RNase of the oligonucleotides was by oligonucleotides, to and the of their antisense with to the C.F. Chiang M-Y. Chan H. J.E. C.K. Mol. of with the in the of the in a of the oligonucleotide in the cell as as in of with the a of in the to degradation of the by J. Scholar, Freier S. Nucleic Acids Res. 1995; 23: Scholar). In the oligonucleotide a to the with a of in the in to the the a to its to the of D.A. S. S.M. S. J. Biol. Chem. 1994; Scholar, B. Nucleic Acids Res. 1995; 23: and more with the for and in of oligonucleotides observed with of cells with and These that the and oligonucleotides the cell in the of C.F. Chiang M-Y. Chan H. J.E. C.K. Mol. was and to determine inhibition of ICAM-1 protein expression by and degradation of the target transcript, an observed with RNase antisense oligonucleotides (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar). was at and a for and HUVECs. Interestingly, a increase in abundance of the ICAM-1 transcript in cells with the oligonucleotides at that this increase in transcript abundance was not due to an increase in the of of the ICAM-1 gene not in target transcript abundance in cells with antisense oligonucleotides which the 5′ cap region of of for ICAM-1 and abundance of ICAM-1 transcript, to the mRNA oligonucleotide was at a of at and by is a oligonucleotide used as a in this determine the antisense on transcript abundance was to ICAM-1, for the transcript expression is also by in HUVECs. with ICAM-1 an increase in abundance of the transcript was observed in cells with the that the region of the transcript The increase in target transcript abundance antisense may be an of these expressed in with the antisense mode of in target transcript abundance also in cells with antisense oligonucleotides which target the 5′ cap region of of for and abundance of transcript, to the oligonucleotide was at a of at and by is to of the transcript A. J. M.A. J. Biol. Chem. 1991; 266: 5′ cap structure of mRNA been shown to splicing of the of in several H. Dev. Scholar, H. S. A. Scholar). The ICAM-1 gene of by with 1 in C. J. Immunol. 1991; Scholar). of the that this of antisense oligonucleotides on splicing of the ICAM-1 to the transcript, as by of ICAM-1 mRNA or of not and was to determine antisense inhibition of ICAM-1 protein expression inhibition of transport of the transcript of the to the cytoplasm. mRNA was by 2 for 2′-O-(2-methoxy)ethyl and antisense and at nm) and cells this in the abundance of the ICAM-1 transcript was observed in the of antisense cells and In a increase in the abundance of the ICAM-1 transcript was observed in the the antisense cells, and in to the and and cells abundance of the ICAM-1 transcript in was also these the abundance of the ICAM-1 transcript was and in the and and in the of the antisense cells, and to the The increase in abundance of the transcript in the of the antisense cells a in the at which the transcript is The of a in ICAM-1 mRNA abundance in the that the antisense oligonucleotides not the transport of the ICAM-1 antisense oligonucleotides have on of the ICAM-1 The increase in ICAM-1 mRNA abundance in the cytoplasm. of and 2 for ICAM-1 and abundance of ICAM-1 transcript, to oligonucleotide was at a of to determine the of antisense oligonucleotide upon the translation of the target ICAM-1 transcript ICAM-1 protein and mRNA a cells with antisense oligonucleotides and and their and by The of the a of the and of the by a in the of the ICAM-1 transcript in cells with and in to of the and The for the and cells the and of the target transcript in the and the and the and of the target transcript in the and of translation initiation to be mode of action for oligonucleotides that target the 5′ cap region of the ICAM-1 transcript in HUVECs. for cells with and at cells as was on for to for and that inhibition of ICAM-1 protein expression interference with translation initiation and as by the of transcript the The formation of a in the 5′ cap region is the of this The increase in abundance of the ICAM-1 mRNA in the of the antisense cells in with the in the that one of the target is coupled to translation. These with of that in the coding region, and N. Mol. Biol. 1995; J. Mol. Biol. 1994; of gene expression may at one or more stages of mRNA The known of regulation mRNA metabolism have been found at the stages of translation N. Press, and degradation J. Rev. 1995; of the transcript, mRNA and have been found to be been shown that and in the region may initiation of translation J. Biol. Chem. 1991; 266: Scholar, S. A. Scholar, J. N. Scholar). The antisense oligonucleotides, to the region of the target transcript this mode of regulation in cells by formation of the 80 S translation initiation complex. that this in the turnover of the expressed ICAM-1 oligonucleotides in a to that inhibition of ICAM-1 protein expression by the oligonucleotides, and was The oligonucleotides, and effects on ICAM-1 protein expression in the and the oligonucleotides more potent of ICAM-1 expression in the RNase of the oligonucleotides was by oligonucleotides, to and the of their antisense with to the C.F. Chiang M-Y. Chan H. J.E. C.K. Mol. of with the in the of the in a of the oligonucleotide in the cell as as in of with the a of in the to degradation of the by J. Scholar, Freier S. Nucleic Acids Res. 1995; 23: Scholar). In the oligonucleotide a to the with a of in the in to the the a to its to the of D.A. S. S.M. S. J. Biol. Chem. 1994; Scholar, B. Nucleic Acids Res. 1995; 23: and more with the for and was and to determine inhibition of ICAM-1 protein expression by and degradation of the target transcript, an observed with RNase antisense oligonucleotides (4Bennett C.F. Condon T.P. Grimm S. Chan H. Chiang M-Y. J. Immunol. 1994; 152: 3530-3540Google Scholar, 5Chiang M-Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Google Scholar). was at and a for and HUVECs. Interestingly, a increase in abundance of the ICAM-1 transcript in cells with the oligonucleotides at that this increase in transcript abundance was not due to an increase in the of of the ICAM-1 gene not determine the antisense on transcript abundance was to ICAM-1, for the transcript expression is also by in HUVECs. with ICAM-1 an increase in abundance of the transcript was observed in cells with the that the region of the transcript The increase in target transcript abundance antisense may be an of these expressed in with the antisense mode of The 5′ cap structure of mRNA been shown to splicing of the of in several H. Dev. Scholar, H. S. A. Scholar). The ICAM-1 gene of by with 1 in C. J. Immunol. 1991; Scholar). of the that this of antisense oligonucleotides on splicing of the ICAM-1 to the transcript, as by of ICAM-1 mRNA or of not and was to determine antisense inhibition of ICAM-1 protein expression inhibition of transport of the transcript of the to the cytoplasm. mRNA was by 2 for 2′-O-(2-methoxy)ethyl and antisense and at nm) and cells this in the abundance of the ICAM-1 transcript was observed in the of antisense cells and In a increase in the abundance of the ICAM-1 transcript was observed in the the antisense cells, and in to the and and cells abundance of the ICAM-1 transcript in was also these the abundance of the ICAM-1 transcript was and in the and and in the of the antisense cells, and to the The increase in abundance of the transcript in the of the antisense cells a in the at which the transcript is The of a in ICAM-1 mRNA abundance in the that the antisense oligonucleotides not the transport of the ICAM-1 to determine the of antisense oligonucleotide upon the translation of the target ICAM-1 transcript ICAM-1 protein and mRNA a cells with antisense oligonucleotides and and their and by The of the a of the and of the by a in the of the ICAM-1 transcript in cells with and in to of the and The for the and cells the and of the target transcript in the and the and the and of the target transcript in the and The for and that inhibition of ICAM-1 protein expression interference with translation initiation and as by the of transcript the The formation of a in the 5′ cap region is the of this The increase in abundance of the ICAM-1 mRNA in the of the antisense cells in with the in the that one of the target is coupled to translation. These with of that in the coding region, and N. Mol. Biol. 1995; J. Mol. Biol. 1994; Scholar). of gene expression may at one or more stages of mRNA The known of regulation mRNA metabolism have been found at the stages of translation N. Press, and degradation J. Rev. 1995; of the transcript, mRNA and have been found to be been shown that and in the region may initiation of translation J. Biol. Chem. 1991; 266: Scholar, S. A. Scholar, J. N. Scholar). The antisense oligonucleotides, to the region of the target transcript this mode of regulation in cells by formation of the 80 S translation initiation complex. that this in the turnover of the expressed ICAM-1 and for oligonucleotide for for and and Crooke for their on the

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.