Transformation of Infinite Life Span Human Fibroblasts

描述（由申请人提供）：已证明培养中的人成纤维细胞以及其他类型的人细胞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。我们从正常的人皮肤成纤维细胞开始，通过连续的克隆选择，现在已经获得了100多个完全恶性的细胞株，使用癌基因转染或致癌物处理和选择。一个涉及端粒酶阳性、二倍体、无限寿命菌株（称为MSU-1.0）自发转化为端粒酶阳性、染色体稳定、无限寿命菌株（称为MSU-1.1）的步骤仍然是一个黑匣子。这一步骤非常重要，因为MSU-1.1细胞可以容易地转化为恶性细胞，但是我们还不能恶性转化MSU-1.0细胞。为了鉴定这些菌株之间的基因表达差异，我们进行了Affyssin基因芯片分析。总共46个基因在MSU-1.1细胞中以比MSU-1.0细胞中高至少3倍的水平表达; 21个基因在MSU-1.1细胞中以比MSU-1.0细胞中低至少3倍的水平表达。为了检验MSU-1.1细胞中基因表达的关键变化是导致其恶性转化的原因这一假设，我们将：1）通过北方印迹法确认MSU-I.0细胞和MSU-I.1细胞之间在所鉴定的每种基因的表达方面的差异，并且如果可能，通过蛋白质印迹法确认，2）通过感染将目的基因的cDNA导入适当的菌株（即，通过检测细胞在生长因子受限的培养基中生长和/或在琼脂糖中形成集落的能力，将在MSU-1.1细胞（组1）中以较高水平表达的基因转化为MSU-1.0细胞，将在MSU-1.1细胞（组2）中以较低水平表达的基因转化为MSU-1.1细胞。将检测感染的MSU-1.0细胞在这些条件下生长的能力，检测感染的MSU-1.1细胞细胞生长的减少或阻断。3)确定发现在生长因子反应性中表现出适当关键变化的细胞株是否可以通过转染H-RAS癌基因而恶性转化。

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.