CELLULAR PHARMACOLOGY OF OLIGODEOXYNUCLEOTIDES

人工合成的寡核苷酸已被证明是序列抑制物 基因的表达，因此可能在实验性的治疗中有用。 恶性疾病。然而，人们对细胞的了解很少 这些药剂的药理学。这样的知识是至关重要的，因为 要使这些药物在临床上发挥重要作用，它们的疗效必须 进行优化。目前几乎没有关于动力学和 细胞表面结合的平衡参数；2)速率和机理 内化；3)细胞内分布；4)速率和 寡聚外排的机制。我们假设HL60细胞，尽管有些 令人振奋的文学成果，表现出对新时代的“抵抗” 寡核苷酸的作用。我们假设细胞防御系统 机制包括1)寡聚诱导的PKC激酶活性降低， 它促进胞吐作用；2)在非 酸性环境和寡聚缺乏对深层细胞的渗透 隔室(例如，溶酶体)导致；3)FULL的快速外流 长度材料，以使胞质内寡核苷酸的浓度为 大多数，只有外部集中度的几个百分点。我们将测试我们的 HL60细胞的假设：我们将使用标准的磷酸二酯15-聚体 5‘标记有荧光素的胸腺嘧啶核苷均聚物 治疗这些细胞。然后我们将监测内吞和胞吐的比率。 使用流式细胞术。通过这种方法，我们将确定动力学 外流，确定外流隔室的数量，并计算 动力学常数A，α，B，β等。我们将确定 双标记法测定低聚物胞内浓度 (荧光素+32P)寡核苷酸。我们将进行类似的实验 用不带电荷的~3H标记的甲基膦酸齐聚物。我们将增加 使用地奎宁和/或丙磺舒的细胞内寡聚保留。我们 会将外流动力学的变化与反义的变化联系起来 功效。这将通过使用与c-myc互补的寡核苷酸来实现。 通过免疫沉淀法和免疫沉淀法检测c-myc蛋白表达水平。 凝胶电泳法。

Synthetic oligodeoxynucleotides have been shown to be sequence inhibitors of gene expression, and thus may be useful in the experimental therapy of malignant disease. However, very little is known about the cellular pharmacology of these agents. Such knowledge is vital because in order for these agents ever to become clinically important, their efficacy must be optimized. Little information is currently available on 1) kinetic and equilibrium parameters of cell surface binding; 2) rates and mechanisms of internalization; 3) intracellular distribution; and 4) rates and mechanisms of oligo efflux. We hypothesize that HL60 cells, despite some encouraging literature results, manifest de novo "resistance" to the effects of oligonucleotides. We hypothesize that cellular defense mechanisms include 1) oligo-induced diminution in PKC kinase activity, which promotes exocytosis; 2) compartmentalization of oligos in a non- acidic environment and lack of penetration of oligo into deep cellular compartments (e.g., the lysosome) which leads to; 3) rapid efflux of full length material so that the concentration of intracytoplasmic oligo is, at most, only a few percent of the external concentration. We will test our hypotheses in HL60 cells: We will use a standard, phosphodiester 15-mer homopolymer of thymidine which has been 5' labeled with fluorescein, to treat the cells. We will then monitor rates of endocytosis and exocytosis using flow cytometry. By this method, we will determine the kinetics of efflux, determine the number of efflux compartments, and calculate the kinetic constants A, alpha,B,beta etc. We will determine the intracellular concentration of oligomer by synthesizing a double labeled (fluorescein + 32p) oligonucleotide. We will perform similar experiments with uncharged, 3H-labeled methylphosphonate oligomers. We will increase intracellular oligo retention by using dequalinium and/or probenecid. We will correlate changes in efflux kinetics with changes in antisense efficacy. This will be done by using an oligo complementary to the c-myc mRNA, and by examining levels of c-myc protein by immunoprecipitation and gel electrophoresis.