Kitada S, Miyashita T, Tanaka S, Reed J C
La Jolla Cancer Research Foundation, Cancer Research Center, California.
Antisense Res Dev. 1993 Summer;3(2):157-69. doi: 10.1089/ard.1993.3.157.
Expression of the bcl-2 gene becomes deregulated in many non-Hodgkin lymphomas as the result of t(14;18) chromosomal translocations. Because bcl-2 regulates the survival of cells, and because its over-expression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation, this gene and its mRNA and protein products represent ideal targets for designing novel therapeutic strategies for the treatment of cancer. Here we describe the effects of an 18-mer phosphodiester oligonucleotide that is complementary to the first 6 codons of the bcl-2 mRNA's open reading frame. When tested for inhibition of in vitro protein synthesis using RNAse-H-supplemented reticulocyte lysates and RNA prepared by in vitro transcription of a human bcl-2 cDNA, the bcl-2 antisense (AS) oligomer completely abolished Bcl-2 protein production at 10 microM, but had no effect on the in vitro translation of a chicken bcl-2 RNA that contained three mismatches relative to the oligomer binding site on the human bcl-2 RNA. A control 18-mer having the same base composition as the AS oligomer but with scrambled order (SC) was not inhibitory. Addition of AS and SC oligomers to cultures of a NIH-3T3 fibroblast cell line that had been stably infected with a recombinant retrovirus containing the same human bcl-2 cDNA used for in vitro transcription/translation experiments revealed concentration-dependent reductions in the relative levels of the 26-kD human Bcl-2 protein (as determined by immunoblotting) by the AS but not by the SC oligomer. Similar results were obtained when AS and SC oligomers were applied to a t(14;18)-containing lymphoma cell line SU-DHL-4 that was cultured in low-serum media. When used at 200 microM, the bcl-2 AS oligomer produced 84-95% reductions in Bcl-2 protein levels in SU-DHL-4 cells but had relatively little effect on the levels of other mitochondrial control proteins, suggesting that the inhibitory effects were specific. Treatment of SU-DHL-4 cells with AS oligomer lead to essentially complete loss of bcl-2 mRNA from cells within 1 day of addition to cultures, but presumably because of the long half-life of the Bcl-2 protein (approximately 14 h), commensurate reductions in Bcl-2 protein levels did not occur until 3 days.(ABSTRACT TRUNCATED AT 400 WORDS)
由于t(14;18)染色体易位，bcl-2基因的表达在许多非霍奇金淋巴瘤中变得失调。因为bcl-2调节细胞的存活，并且其过度表达与细胞对化疗药物和γ射线杀伤的抗性相关，所以该基因及其mRNA和蛋白质产物是设计癌症治疗新策略的理想靶点。在此我们描述了一种18聚体磷酸二酯寡核苷酸的作用，它与bcl-2 mRNA开放阅读框的前6个密码子互补。当使用补充了RNA酶H的网织红细胞裂解物和通过人bcl-2 cDNA体外转录制备的RNA来测试其对体外蛋白质合成的抑制作用时，bcl-2反义（AS）寡聚物在10μM时完全消除了Bcl-2蛋白的产生，但对含有相对于人bcl-2 RNA上寡聚物结合位点三个错配的鸡bcl-2 RNA的体外翻译没有影响。与AS寡聚物碱基组成相同但顺序打乱的对照18聚体（SC）没有抑制作用。将AS和SC寡聚物添加到稳定感染了含有用于体外转录/翻译实验的相同人bcl-2 cDNA的重组逆转录病毒的NIH-3T3成纤维细胞系培养物中，结果显示AS寡聚物使26-kD人Bcl-2蛋白的相对水平（通过免疫印迹测定）呈浓度依赖性降低，而SC寡聚物则无此作用。当将AS和SC寡聚物应用于在低血清培养基中培养的含t(14;18)的淋巴瘤细胞系SU-DHL-4时，也得到了类似的结果。当以200μM使用时，bcl-2 AS寡聚物使SU-DHL-4细胞中Bcl-2蛋白水平降低了84% - 95%，但对其他线粒体控制蛋白的水平影响相对较小，这表明抑制作用是特异性的。用AS寡聚物处理SU-DHL-4细胞，在添加到培养物后1天内细胞内bcl-2 mRNA基本完全丧失，但可能由于Bcl-2蛋白的半衰期较长（约14小时），直到3天后Bcl-2蛋白水平才相应降低。（摘要截断于400字）
