POSTTRANSLATIONAL MODULATION OF P GLYCOPROTEIN

描述：(申请人摘要)本申请的目的是 研究翻译后磷酸化的调节作用 药物外排泵P-糖蛋白(Pgp)在多药耐药中的作用 (MDR)人乳腺癌细胞及体外重组 杆状病毒的表达。PGP是一种依赖于ATP的质膜 负责传递抗性的转运体 结构多样的天然产物抗癌药物。PGP是 MDR1基因的产物，是一个高度保守的多基因家族，包括 在人类的两个基因中，只有MDR1具有MDR表型 转染组为对照组。PGP作为蛋白质丝氨酸的底物 蛋白激酶C(PKC)家族的激酶。最近的研究来自 申请人的实验室已经证明MDR1可以增加MDR- 用PKCA转染人乳腺癌细胞后表达， 与药物滞留减少和佛波醇增加有关 酯刺激的Pgp磷酸化。这种影响可以部分地 被PKCA反义表达逆转。此外，现场指导 Ser671在Pgp中的突变降低药物结合和 PKCA激活药物诱导的PGP-ATPase在杆状病毒中的表达 系统。因此，此应用程序的目标是确定 不同亚型的PKC在调节Pgp活性中的作用 在此背景下，探索通过以下方式下调PGP活性的方法 选择性地抑制特定的PKC亚型。根据申请人的 乳腺癌细胞中PKC亚型的先前特征， 这个应用程序的第一个目标将是确定Wild的效果 乳腺癌多药耐药中PKCA的类型或活性形式 癌细胞稳定表达Pgp。在MDR的情况下 表型显示一个特定的PKC亚型减少或缺失，例如。 将PKC-beta-2、Delta和epsilon等低分子基因导入细胞 PKC形式丰富。第二个目标将是选择性地抑制 在多药耐药细胞中过度表达的PKC亚型，例如。PKC-Alpha，由 稳定表达反义c DNA。第三个目标将是 确定突变一个或多个PKC共识的效果 Pgp中的磷酸化位点与其转运药物的能力有关。这个 突变的Pgp(药物蓄积、药物结合和ATPase)的功能 活性)将在MCF-7细胞中通过稳定表达和在 杆状病毒感染后的昆虫细胞。

DESCRIPTION: (Applicant's Abstract) The objective of this application is to study the regulatory effects of post-translational phosphorylation of the drug efflux pump, P-glycoprotein (PGP), in multidrug-resistant (MDR) human breast cancer cells and in vitro using recombinant baculovirus expression. PGP is an ATP-dependent plasma membrane transporter that is responsible for conferring resistance to structurally diverse natural product anticancer drugs. PGP is the product of the MDR1 gene, a highly conserved multigene family consisting of two genes in man, and of which only MDR1 confers the MDR phenotype after transfection. PGP serves as a substrate for protein-serine kinases of the protein kinase C (PKC) family. Recent studies from the applicant's laboratory have shown that MDR can be increased in MDR1- expressing human breast cancer cells following transfection with PKCa, and is associated with decreased drug retention and increased phorbol ester-stimulated PGP phosphorylation. This effect can be partially reversed by antisense expression of PKCa. Moreover, site directed mutagenesis of Ser671 in PGP attenuates drug binding and the ability of PKCa to activate drug-induced PGP ATPase in a baculovirus expression system. Therefore, the goals of this application are to determine the roles of different isoforms of PKC in modulating PGP activity, and in this context, to explore ways in which to down-regulate PGP activity by selectively inhibiting specific PKC isoforms. Based on the applicant's previous characterization of PKC isoforms in breast cancer cells, the first goal of this application will be to determine the effect of wild type or constitutively active forms of PKCa on MDR in human breast carcinoma cells stably expressing PGP. In instances where the MDR phenotype shows a reduction or absence of a particular PKC isoform, eg. PKC-beta-2, delta and epsilon, cells will be transfected with the low abundance form of PKC. The second goal will be to selectively inhibit the PKC isoform which is over-expressed in MDR cells, eg. PKC-alpha, by stably expressing the antisense cDNA. The third goal will be to determine the effect of mutating one or more PKC consensus phosphorylation sites in PGP on its ability to transport drugs. The function of mutated PGP (drug accumulation, drug binding and ATPase activity) will be assessed in MCF-7 cells by stable expression and in insect cells after baculovirus infection.