POSTTRANSLATIONAL MODULATION OF P GLYCOPROTEIN

说明：（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 (MDR)人乳腺癌细胞和体外使用重组 杆状病毒表达。PGP是一种ATP依赖的质膜 一种负责赋予耐药性的转运蛋白， 结构多样的天然产物抗癌药物。 PGP是 MDR 1基因是一个高度保守的多基因家族， 其中只有MDR 1赋予MDR表型 转染后。 PGP作为蛋白丝氨酸的底物 蛋白激酶C（PKC）家族的激酶。最近的研究， 申请人的实验室已经表明，MDR可以在MDR 1- 在用PKCa转染后表达人乳腺癌细胞， 并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 通过PKCa的反义表达逆转。此外，现场指导 PGP中Ser 671的突变减弱了药物结合和药物结合的能力。 PKCa激活药物诱导的PGP ATP酶在杆状病毒表达中的作用 系统因此，本应用程序的目标是确定 PKC的不同亚型在调节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： 选择性抑制特定的PKC亚型。 根据申请人的 以前在乳腺癌细胞中PKC亚型的特征， 本申请的第一个目标将是确定野生 型或组成性激活形式的PKCa对人乳腺MDR的影响 稳定表达PGP的癌细胞。在MDR 表型显示特定PKC亚型的减少或缺失，例如， PKC-β-2、δ和PKC-β 2细胞将用低浓度的 PKC的丰富形式。第二个目标是选择性地抑制 在MDR细胞中过度表达的PKC亚型，例如：PKC-α，通过 稳定表达反义cDNA。第三个目标是 确定突变一个或多个PKC共有序列的影响 PGP中的磷酸化位点对其转运药物的能力的影响。的 突变的PGP的功能（药物蓄积、药物结合和ATP酶 活性）将在MCF-7细胞中通过稳定表达进行评估， 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.