CELL GROWTH DEPENDS ON MITOCHONDRIALLY-DERIVED CITRATE

在多阶段实验设计中， 调查了有据可查的 去调节的（连续操作的）胆固醇生成途径， 以及诱导新的DNA合成（作为细胞的前奏 增殖）。 重点将集中在：（1）作用 线粒体三羧酸（柠檬酸-苹果酸）交换 （2）上述现象的解释; 实验室最近发现线粒体的能力， 柠檬酸盐转运阻断剂1，2，3-苯三甲酸 (BTC)显著抑制DNA合成和增殖， 培养的肿瘤细胞;和（3）检测、分离和部分 甲羟戊酸衍生的异戊二烯化（和 可能是磷酸化的）蛋白质加合物 快速生长的细胞可能需要DNA合成 以及肿瘤发生过程中的细胞周期。 (1)Morris肝癌3924 A (with正常大鼠肝脏作为对照），以及 鼠B细胞淋巴瘤70 Z/3，（2）T细胞白血病L5178 Y和 (3)腹水肝癌MH 129（最近适应于生长在 悬浮培养物），其一起包含不同的 胚层来源，应作为繁殖材料+/- BTC和/或线粒体后上清液（PMS）的来源 无细胞脂质合成系统。 我们将研究先驱 通过甾醇生物合成的碳通量作为作用的函数 通过14 C标记的中间体分离以及通过 在通过CMR的13 C富集的前体基质通量之后， 利用肿瘤PMS制剂。 随着新推出的3 H-BTC， 我们将试图描述细胞内分布的特征， 这种抑制剂。 BTC对DNA、RNA和蛋白质的影响 同步化细胞中细胞周期各阶段的合成 人口将被探索，以更好地了解如何 线粒体柠檬酸载体抑制剂减缓了 细胞 检测和部分表征的独特 衍生的蛋白质物种将采用我们已经改进的 成功的二维NEPHGE分析和放射自显影 由O 'Farrell凝胶制成。 显然， 在细胞内检测到的独特异戊二烯化蛋白衍生物 扩散条件将是这些国家的主要努力， 问题研究 一旦分离和纯化，这些独特的抗体， 衍生物应提高寻找其生产和细胞 在细胞周期中的部分位置。

In a multi-stage experimental design, relationships will be investigated between selected aspects of the well-documented deregulated (continuously operating) cholesterogenesis pathway, and the induction of new DNA synthesis (as a prelude to cell proliferation) in tumors. Emphasis will be focused on: (1) the role of the mitochondrial tricarboxylate (citrate-malate) exchange carrier in the above phenomena; (2) the clarification of this laboratory's recent discovery of the ability of the mitochondrial citrate transport blocking agent 1,2,3-benzenetricarboxylate (BTC) to dramatically inhibit DNA synthesis and proliferation in cultured tumor cells; and (3) the detection, isolation and partial characterization of a mevalonate-derived, isoprenylated (and possibly phosphorylated) protein adduct from the cytosol of rapidly growing cells which may be required for DNA synthesis and cell cycling during tumorigenesis. (1) Morris hepatoma 3924A (with normal rat liver as control), as well as suspension cultures of murine B-cell lymphoma 70Z/3, (2) T-cell leukemia L5178Y and (3) ascites hepatoma MH129 (recently adapted to growth in suspension culture), which together comprise cancers of different germ layer origins, shall serve as material for propagation +/- BTC, and/or the source of post-mitochondrial supernatant (PMS) cell-free, lipid synthesizing systems. We shall study precursor carbon flux through sterol biosynthesis as a function of the action of BTC via 14C labelled intermediate isolation as well as by following 13C enriched precursor substrate flux via CMR, utilizing tumor PMS preparations. With newly available 3H-BTC, we will attempt to characterize the intracellular distribution of this inhibitor. The effects of BTC on DNA, RNA and protein synthesis during stages of the cell cycle in synchronized cell populations will be explored, in an effort to better understand how the mitochondrial citrate carrier inhibitor slows the growth of cells. Detection and partial characterization of the unique derivatized protein species will employ refinements of our already successful 2-D NEPHGE analysis and the autoradiograms generated from O'Farrell gels. The isolation of the apparently unique isoprenylated protein derivatives detected under cell proliferation conditions shall be a principal effort of these studies. Once isolated and purified, antibodies to these unique derivatives shall be raised to search for their production and cell fraction location during the cell cycle.