ANTISENSE AS AN APPROACH TO CANCER CHEMOTHERAPY

两种类型的反义化合物（即， 寡脱氧核糖核苷甲基膦酸酯，ODNMP和 寡脱氧核糖核苷酸-聚-L-赖氨酸缀合物，ODPLC） 它可以进入细胞，对耐药性的表达将是 考察 主要目的是评估 这些化合物抑制二氢叶酸的合成 在DHFR过量生产细胞中， 结果 将定义这些限制和潜力 作为抗肿瘤剂的化合物（单独或与 其他药物）。 DHFR mRNA对杂交抑制敏感的区域 已经在体外使用 寡脱氧核糖核苷酸（ODs）杂交技术， 翻译测定 ODNMP和ODPLC与 这些敏感区域将在体外合成和测试， 它们特异性抑制DHFR mRNA翻译的能力 在包括异源底物的背景下， 与内部控制相同的反应。 ODNMPs和ODPLCs调节DHFR表达的能力 随后将在细胞培养中使用MTX抗性 和敏感细胞。 影响 将在分子水平和细胞水平上进行监测 生存能力 顺序的相对重要性， 观察效应所需的暴露量和浓度将 测定 放射性标记的ODNMP、ODPLC和OD将在 制备并用于研究长度和成分的影响 对这些化合物的吸收。 以类似的方式，OD、ODNMP和ODPLC将被 合成人β-珠蛋白前体的互补区域， mRNA以确定前mRNA的区域敏感性抑制 体外剪接。 从这些研究中获得的数据将 用于研究特异性抑制剪接的可行性 在表达人β-珠蛋白基因的培养细胞中， 高水平表达人DHFR小基因的细胞。

The impact of two types of antisense compounds (i.e., oligodeoxyribonucleoside methyl phosphonates, ODNMPs and oligodeoxyribonucleotide-poly-L-lysine conjugates, ODPLCs) which can enter cells, on the expression of drug resistance will be examined. The primary objective is to evaluate the ability of these compounds to suppress the synthesis of dihydrofolate reductase (DHFR) in DHFR overproducing cells. The results obtained will define the limitations and potential of these compounds as anti-tumor agents (alone or in combination with other drugs). Regions of DHFR mRNA sensitive to inhibition by hybridization arrest have been determined in vitro using oligodeoxyribonucleotide (ODs) hybridization techniques in a translational assay. ODNMPs and ODPLCs complementary to these sensitive regions will be synthesized and tested in vitro for their ability to specifically inhibit translation of DHFR mRNA against a background of heterologous substrates included within the same reactions as internal controls. The ability of ODNMPs and ODPLCs to regulate DHFR expression will subsequently be tested in cell culture using MTX-resistant and sensitive cells, alone and in combination with MTX. Effects will be monitored both on a molecular level and in terms of cell viability. The relative importance of sequence, timing of exposure and concentrations required to observe effects will be determined. Radiolabelled ODNMPs, ODPLCs, and ODs will be prepared and utilized to study effects of length and composition on the uptake of these compounds. In a similar fashion, ODs, ODNMPs and ODPLCs will be synthesized to complementary regions of human beta-globin pre- mRNA to determine regions sensitive inhibition of pre-mRNA splicing in vitro. The data obtained from these studies will then be used to study the feasibility of specifically inhibiting splicing in cultured cells which express the human beta-globin gene and in cells which express a human DHFR minigene at high levels.