Ehrlich G, Patinkin D, Ginzberg D, Zakut H, Eckstein F, Soreq H
Department of Biological Chemistry, Life Sciences Institute, Hebrew University of Jerusalem, Israel.
Antisense Res Dev. 1994 Fall;4(3):173-83. doi: 10.1089/ard.1994.4.173.
To distinguish between sequence-dependent effects and non-specific cytotoxicity of phosphorothioate antisense oligonucleotides (AS-oligos), we introduced AS-oligos blocking expression of 2Hs, the Homo sapiens cell division controller cdc2 kinase, its hematopoietically expressed homolog CHED, and the acetylcholine-hydrolyzing enzyme butyrylcholinesterase (BCHE) into primary murine bone marrow (BM) culture. Antisense oligonucleotides were fully phosphorothioated (Ts) or prepared with three phosphorothioate groups at their 3' termini (S3). Each of these oligos could cause reductions in colony counts either as a result of its sequence-dependent biological capacity or due to sequence-independent cytotoxicity. The Ts and S3 forms of the matching sense oligo, S-BCHE, served for comparison. The S3 forms of AS-2Hs, AS-BCHE, and S-BCHE caused more limited drops in colony counts than their Ts counterparts, reflecting lower cytotoxicity. When incubated with electroblotted BM proteins, Ts but not S3 oligos intensively labeled two protein bands. Moreover, 5'-end 32P-labeled (Ts) S-BCHE labeled nuclear proteins in situ in small, mitotic cells, suggesting correlation between oligo-protein interactions and the sequence-independent cytotoxicity of Ts AS-oligos. Extension of the apparently nontoxic AS-CHED by two adenosine residues at the 3' end, creating a potential for intramolecular hydrogen bond formation, resulted in increased toxicity. These findings recommend the use of nonlooped, partially phosphorothioated oligos for the modulation of hematopoiesis.
为了区分硫代磷酸酯反义寡核苷酸（AS-oligos）的序列依赖性效应和非特异性细胞毒性，我们将阻断人类细胞分裂控制因子cdc2激酶、其在造血细胞中表达的同源物CHED以及乙酰胆碱水解酶丁酰胆碱酯酶（BCHE）表达的AS-oligos引入原代小鼠骨髓（BM）培养物中。反义寡核苷酸完全硫代磷酸化（Ts）或在其3'末端制备有三个硫代磷酸酯基团（S3）。这些寡核苷酸中的每一种都可能由于其序列依赖性生物学能力或由于序列非依赖性细胞毒性而导致集落计数减少。匹配的正义寡核苷酸S-BCHE的Ts和S3形式用于比较。AS-2Hs、AS-BCHE和S-BCHE的S3形式导致的集落计数下降比其Ts对应物更有限，反映出较低的细胞毒性。当与电印迹的BM蛋白一起孵育时，Ts寡核苷酸而非S3寡核苷酸强烈标记了两条蛋白带。此外，5'-末端32P标记的（Ts）S-BCHE在原位标记了小的有丝分裂细胞中的核蛋白，表明寡核苷酸-蛋白相互作用与Ts AS-寡核苷酸的序列非依赖性细胞毒性之间存在相关性。在3'末端将明显无毒的AS-CHED延伸两个腺苷残基，产生分子内氢键形成的可能性，导致毒性增加。这些发现建议使用无环的、部分硫代磷酸化的寡核苷酸来调节造血作用。
