A "stealth" approach to inhibition of lymphocyte activation by oligonucleotide complementary to the putative G0/G1 switch regulatory gene G0S30/EGR1/NGFI-A.
The putative G0/G1 switch regulatory gene G0S30/EGR1/NFGI-A show increased expression shortly after adding concanavalin-A (ConA) to cultured T lymphocytes. However, it is reported that lymphocytes from mice in which the gene has been deleted proliferate normally in response to ConA. This suggests that G0S30 expression is not critical for the response. Paradoxically, others report that proliferation of ConA-stimulated rat lymphocytes is inhibited by an antisense oligonucleotide complementary to G0S30. Because the G0S30 sequence is highly conserved between species, we used a similar oligonucleotide (differing by 1 base) to show for humans that the response to ConA is also inhibited. However, no oligonucleotide-induced changes in the concentrations of G0S30 protein or mRNA are detectable. This suggests that the oligonucleotide is not acting by influencing the expression of G0S30, and may be targeting another gene. The phosphorothioated oligonucleotide was maximally inhibitory at a 50 nM concentration, which is near to the "physiological" concentration found with CpG-containing oligonucleotides to activate mouse B lymphocytes. In the present work, increasing the concentration above 50 nM, or adding further quantities of control oligonucleotides, decreased the inhibition. It is suggested that by using low oligonucleotide concentrations (the "stealth" approach), one may avoid "tripping" an endogenous defense system directed against exogenous oligonucleotides, yet still get sufficient uptake to inhibit lymphocyte activation.
Cristillo
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Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Heximer
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Forsdyke
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D001483:Base Sequence; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D051766:Early Growth Response Protein 1; D016193:G1 Phase / Q000235:genetics; D005812:Genes, Switch; D006801:Humans; D017874:Immediate-Early Proteins; D007399:Interphase / Q000235:genetics*; D008213:Lymphocyte Activation / Q000235:genetics*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D016192:Resting Phase, Cell Cycle / Q000235:genetics; D013873:Thionucleotides / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D014157:Transcription Factors; D003208:Concanavalin A