Translational inhibition of messenger RNA of the human pi class glutathione S-transferase by antisense oligodeoxyribonucleotides.
In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.
Department of Experimental Pediatrics, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.
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Keller
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Ali-Osman
D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase