Studies on Expression-Mechanism (s) of Malignant Phenotypes Using Growth-Factor Gene-Transfer Methods

使用生长因子基因转移方法研究恶性表型的表达机制

基本信息

批准号：
02454521
负责人：
SAITO Masaki
金额：
$ 4.35万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The growth and development of a murine myeloid leukemia cell line, NFS-60, is dependent upon several hemopoietic growth factors such as interleukin-3 (IL-3), GM-CSF, G-CIF, and erythropoietin. We established a subclone (1-7) which was exclusively responsive to IL-3 and introduced into it a murine IL-3 gene. Ten IL-3-independent cell lines with autonomous proliferation were established from the transfected parental 1-7 subclone. The IL-3 activity, secreted from the IL3-independent cell lines into conditioned media, varied from less than 10 to 280 U/mL. Two representative transfectants, clone 1-7/7 (#7) producing the highest IL-3 activity and clone I-7/5 (#5) producing the lowest activity, were mainly investigated. Only a single copy of the IL-3 gene was introduced into each cell line, but. the level of its transdescription was different, i. e. the #7 cells expressed the IL-3 gene approximately ten times more than the #5 cells. A remarkable difference in extracellular IL-3 activity was found between the two cell lines while the intracellular IL-3 activity was not so different. The growth curve of the #5 cells in the presence of exogenous IL-3 was similar to that seen in the absence of IL-3. Anti-IL-3 antiserum did not inhibit the proliferation of the transfected cells. These results suggested that IL-3 secreted outside the transfected cells might not be obligatory for autonomous growth and that intracellular IL-3 might play an important role in autocrine growth. Simultaneously, the IL-3 gene-transfected cells exhibited remarkable changes in membrane glycosphingolipids (GSLs), especially in acidic sialoGSLs (gangliosides). The IL-3 gene transfection characteristically induced a new appearance of ganglioside GD3, which is known to be intimately related to malignant transformation. In addition, considerable changes of neutral GSLs were observed in the transfectants, i. e. the total amount and some constituents significantly increased.

小鼠骨髓性白血病细胞系 NFS-60 的生长和发育依赖于多种造血生长因子，例如白细胞介素 3 (IL-3)、GM-CSF、G-CIF 和促红细胞生成素。我们建立了一个仅对 IL-3 敏感的亚克隆 (1-7)，并向其中引入了鼠 IL-3 基因。从转染的亲代1-7亚克隆建立了10个具有自主增殖能力的不依赖于IL-3的细胞系。从不依赖 IL3 的细胞系分泌到条件培养基中的 IL-3 活性从小于 10 到 280 U/mL 不等。主要研究了两种代表性转染子，即产生最高IL-3活性的克隆1-7/7 (#7)和产生最低活性的克隆I-7/5 (#5)。仅将 IL-3 基因的单个拷贝引入每个细胞系中，但是。其转述水平不同，i。 e. #7 细胞表达的 IL-3 基因大约是 #5 细胞的十倍。两种细胞系之间的细胞外 IL-3 活性存在显着差异，而细胞内 IL-3 活性差异不大。在存在外源性IL-3的情况下#5细胞的生长曲线与在不存在IL-3的情况下观察到的相似。抗IL-3抗血清不抑制转染细胞的增殖。这些结果表明，转染细胞外部分泌的IL-3可能不是自主生长所必需的，细胞内IL-3可能在自分泌生长中发挥重要作用。同时，IL-3基因转染细胞的膜鞘糖脂（GSL），特别是酸性唾液酸GSL（神经节苷脂）表现出显着变化。 IL-3基因转染特征性地诱导神经节苷脂GD3的新出现，已知其与恶性转化密切相关。此外，在转染子中观察到中性 GSL 的显着变化，即。 e.总量及部分成分显着增加。