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

小鼠髓系白血病细胞系NFS-60的生长发育依赖于多种造血生长因子，如白介素3(IL-3)、粒细胞集落刺激因子(GM-CSF)、G-CIF和促红细胞生成素。我们建立了一个对IL-3特异反应的亚克隆(1-7)，并将小鼠IL-3基因导入其中。从转基因亲本1-7亚克隆中建立了10个IL-3非依赖的自主增殖细胞系。IL-3非依赖性细胞株分泌到条件培养液中的IL-3活性在10~280U/mL之间变化。主要考察了IL-3活性最高的克隆1-7/7(#7)和活性最低的克隆I-7/5(#5)这两个具有代表性的转染体。只有一份IL-3基因被导入每个细胞系，但是。其转录水平不同，即#7细胞表达的IL-3基因大约是#5细胞的10倍。细胞外IL-3活性差异显著，细胞内IL-3活性差异不大。在外源IL-3存在的情况下，#5细胞的生长曲线与在没有IL-3的情况下看到的相似。抗IL-3抗血清对细胞增殖无明显抑制作用。这些结果提示，细胞外分泌的IL-3可能不是自主生长所必需的，而细胞内的IL-3可能在自分泌生长中起重要作用。同时，IL-3基因转染组细胞胞膜糖鞘糖脂(GSLS)发生明显变化，尤其是酸性唾液酸鞘糖脂(GSLs)。IL-3基因的转染诱导了神经节苷脂GD3的新出现，它与恶性转化密切相关。此外，在转染体中观察到中性GSLS的显著变化，即总量和部分成分显著增加。

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.