Investigation of biological significance of neutral amino acid transporter expression in human brain tumors and development of novel therapeutic strategies

目标.恶性肿瘤细胞显示出升高的增殖活性，这需要增加细胞内代谢。LAT 1在其辅因子4F 2 hc的存在下，构成负责大多数必需氨基酸的细胞转运的系统L神经质氨基酸转运蛋白。最近的研究表明LAT 1在肿瘤细胞中的表达增加，这促使我们研究其在人脑胶质瘤细胞中的表达和生物学作用。采用Western blot和RT-PCR方法检测了13株人脑胶质瘤细胞系中LAT 1和4F 2 hc的表达。用L系统抑制剂BCH处理LAT 1阳性胶质瘤细胞，分别用BrdU染色和TUNEL法检测其增殖和凋亡率。用编码人LAT 1 cDNA的质粒转染LAT 1阴性胶质瘤细胞，并对表达高水平LAT 1的细胞进行体外生长测定，并将其皮下注射到裸鼠中。AMI ...更多信息 使用14 C-Leu没有测量酸摄取活性<14>。大多数胶质瘤细胞系表达LAT 1蛋白。所有细胞系4F 2 hc mRNA表达均为阳性。BCH处理导致LAT 1阳性LNZ 308细胞的增殖显著降低，而在LAT 1阴性LN 319细胞中无效。BCH处理后LNZ 308细胞增殖下降，凋亡增加，细胞周期调控因子表达水平和caspase活性发生变化。LAT 1的引入并不影响LN 319细胞在体外的生长速率。然而，LAT 1过表达导致U87 MG细胞中氨基酸摄取活性增加3.4倍，导致体内致瘤性增强。LAT 1及其配偶体4F 2 hc在大多数人脑胶质瘤细胞中表达。我们的研究结果表明，LAT 1，氨基酸转运系统的主要组成部分，可能在胶质瘤的发生中发挥重要作用，并可能是一个新的潜在的恶性胶质瘤的治疗靶点。少

Objectives. Malignant tumor cells show an elevated proliferative activity which would require increased intracellular metabolism. LAT1, in the presence of its co-factor 4F2hc, constitutes the system L neurtral amino acid transporter which is responsible for cellular transport of most essential amino acids. Recent reports demonstrating increased expression of LAT1 in cancer cells prompted us to investigate its expression and biological roles in human glioma cells.Methods. Expression of LAT1 and 4F2hc was determined in 13 human glioma cell lines using Western blot and RT-PCR analyses. LAT1-positive glioma cells were treated with a system L inhibitor BCH, and their proliferation and apoptosis rates were examined by BrdU staining and TUNEL assay, respectively. LAT1-negative glioma cells were transfected with the plasmid encoding human LAT1 cDNA and the cells expressing high levels of LAT1 were subjected to in vitro growth assays, and were injected into nude mice subcutaneously as well. Ami … More no acid uptake activity was measured using ^<14>C-Leu.Results. Majority of glioma cell lines expressed LAT1 protein. All cell lines were positive for 4F2hc mRNA expression. BCH treatment resulted in significant reduction of proliferation in LAT1-positive LNZ308 cells, whereas it was ineffective in LAT1-negative LN319 cells. LNZ308 cells showed reduced proliferation and increased apoptosis upon BCH treatment, which were accompanied with changes of expression levels of cell-cycle regulators and caspase activation. Introduction of LAT1 did not affect growth rates in LN319 cells in vitro. However, LAT1 overexpression leading to 3.4-fold increase in amino acid uptake activity in U87MG cells resulted in enhanced tumorigenicity in vivo.Conclusions. LAT1 and its partner 4F2hc are expressed in majority of human glioma cells. Our results suggest that LAT1, the major component of the amino acid transport system, may play an important role in gliomagenesis and may be a novel potential therapeutic target for malignant gliomas. Less