Transformation of Infinite Life Span Human Fibroblasts

描述（由申请人提供）：培养的人类成纤维细胞以及其他类型的人类细胞已被证明非常难以转化为恶性细胞。为了研究细胞转化的机制，我们开发了MSU-1谱系。我们从正常的人类皮肤成纤维细胞开始，通过连续的克隆选择，现在已经通过癌基因转染或致癌物治疗和选择获得了100多个完全恶性的细胞株。端粒酶阳性，二倍体，无限寿命的菌株，称为MSU-1.0，自发转化为端粒酶阳性，染色体稳定，无限寿命的菌株，称为MSU-1.1，这一步仍然是一个黑盒子。这一步非常重要，因为MSU-1.1细胞可以很容易地转化为恶性细胞，但我们还没有能够恶性转化msu -1。0细胞。为了确定这些菌株之间的基因表达差异，我们进行了Affymetrix基因芯片分析。在msu - 1中，共有46个基因的表达水平至少高出3倍。比MSU-1.0细胞多1个细胞；其中21个基因在MSU-1.1细胞中的表达量至少低于MSU-1.0细胞的3倍。为了验证MSU-1.1细胞中基因表达的关键变化是导致它们可以恶性转化的假设，我们将：1)通过Northern blotting证实msu -1细胞之间的差异。0细胞和msu - 1。2)确定在1中被证实表达的基因是否在msu - 1的形成中起关键作用。1细胞通过感染将目标基因的cDNA导入合适的菌株(即MSU-1.1细胞中表达水平较高的基因（组1）导入MSU-1.0细胞，MSU-1.1细胞中表达水平较低的基因（组2）导入msu -1细胞。1细胞，通过检查细胞在生长因子限制的培养基中生长和/或在琼脂糖中形成菌落的能力。受感染的MSU-1.0细胞将测试其在这些条件下的生长能力，受感染的MSU-1.1细胞将测试细胞生长的减少或阻断。3)确定转染H-RAS癌基因后，发现生长因子反应性发生相应临界变化的细胞株是否可发生恶性转化。

DESCRIPTION (provided by applicant): Human fibroblasts in culture as well as other types of human cells have proven to be extraordinarily difficult to transform into malignant cells. To study the mechanisms of cell transformation, we have developed the MSU-1 lineage. We began with normal human skin fibroblasts and by sequential, clonal selection, have now obtained more than 100 fully malignant cell strains using oncogene transfection or carcinogen treatment, and selection. One step that involves the spontaneous conversion of a telomerase positive, diploid, infinite life span strain, called MSU-1.0, to a telomerase positive, chromosomally stable, infinite life span strain called MSU-1.1 remains a black box. This step is very important because the MSU-1.1 cells can be readily transformed into malignant cells, but we have not been able to malignantly transform the MSU-I.0 cells. To identify differences in gene expression between these strains, we have carried out Affymetrix Gene Chip analysis. A total of 46 genes are expressed at a level at least 3-fold higher in the MSU-I.1 cells than in the MSU-1.0 cells; 21 genes are expressed at a level at least 3-fold lower in the MSU-1.1 cells than in the MSU-1.0 cells. To test the hypothesis that critical changes in gene expression in the MSU-1.1 cells are responsible for the fact that they can be malignantly transformed, we will: 1) confirm, by Northern blotting the differences between MSU-I.0 cells and MSU-I.1 cells in expression of each gene identified and if possible, by Western blotting, 2) determine whether genes whose expression were confirmed in 1 play a critical role in making the MSU-I.1 cells transformable by introducing by infection the cDNA of the genes of interest into the appropriate strain (i.e., genes expressed at a higher level in MSU-1.1 cells (Group 1) into MSU-1.0 cells and genes expressed at a lower level in MSU-1.1 cells(Group 2)into MSU-I.1 cells, by examining the cells' ability to grow in medium where growth factors are limiting and/or form colonies in agarose. Infected MSU-1.0 cells will be tested for their ability to grow under these conditions, infected MSU-1.1 cells for a reduction or blockage in cell growth. 3) determine whether the cell strains found to exhibit the appropriate critical change in growth factor responsiveness can be malignantly transformed by transfection of an H-RAS oncogene.