Establishment of Antisense Therapy for Hematological Mailg-nancies Targeted Their Rearranged Genes.

在本研究项目中，我们阐明了反义寡脱氧核苷酸（AS ODN）对重排和过表达癌基因抑制人白血病细胞增殖的机制。1) BCR-ABL AS ODN可通过诱导慢性髓系白血病（CML）细胞凋亡，有效抑制CML细胞的增殖。c-myc AS ODN能抑制人白血病细胞hl60的增殖，使G_1/S交界区白血病细胞数量突变上调。c-myc AS ODN还通过Fas/Fas配体系统抑制类风湿关节炎滑膜细胞的增殖并诱导细胞凋亡。2)建立了具有硫代磷和二酯键嵌合骨架结构的AS ODN的合成和纯化方法。5′端和3′端的前三个键都是通过硫代化学合成的。我们已经证明这些嵌合类似物能够抵抗核酸酶的降解。3)上述嵌合类似物具有更少的非特异性效应或适体效应，而当使用具有全硫代键的AS odn时，通常会遇到这种效应。4)我们已经发现，具有嵌合骨架的AS odn可以有效地对抗Bcl-2、c-myc、促红细胞生成素受体和基质衍生因子1，并且我们发现了新的靶向反义序列来降低Bcl-2的表达。

In this research project, we have clarified followings concerning the mechanism of suppression of the proliferation of human leukemic cells by antisense oligodeoxynucleotides (AS ODN) for rearranged and over-expressed oncogenes.1) BCR-ABL AS ODN could effectively suppress the proliferation of chronic myeloid leukemia (CML) cells by the induction of apoptosis of these leukemia cells. c-myc AS ODN could inhibit the proliferation of human leukemia cells HL6O, and the abrupt up-regulation of number of leukemia cells in G_1/S boundary. c-myc AS ODN also inhibited the proliferation of synovial cells from patients with rheumatoid arthritits and induced the apoptosis through Fas/Fas ligand system.2) We have established the synthesis and purification methods of AS ODN with chimeric backbone structure of phosphorothioate and phosphodiester linkages. The first three linkages of both 5'- and 3'-end are synthesized by phosphorothioate chemistry. We have shown these chimeric analogues were resistant to the degradation by nuclease.3) The above chimeric analogue have much less non-specific effects or aptamer effects that are often encountered when AS ODNs with all-phosphorothioate linkages are used.4) We have shown that AS ODNs with chimeric backbones against Bcl-2, c-myc, erythropoietin receptor, and stromal-derived factor 1 effectively work as functional antisense molecules, and we have found the novel target antisense sequence to reduce the Bcl-2 expression.