Transformation of Infinite Life Span Human Fibroblasts

描述(申请人提供)：培养中的人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，我们发展了MSU-1谱系。我们从正常人皮肤成纤维细胞开始，通过连续、克隆的选择，现在已经获得了100多个完全恶性的细胞株，使用癌基因转染法或致癌物处理，并进行筛选。其中一个步骤涉及将端粒酶阳性的二倍体无限寿命菌株MSU-1.0自发转化为染色体稳定的端粒酶阳性无限寿命菌株MSU-1.1，这一步骤仍然是一个黑匣子。这一步非常重要，因为MSU-1.1细胞很容易转化为恶性细胞，但我们还无法将MSU-1.0细胞恶性转化。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。共有46个基因在MSU-I.1细胞中的表达水平比MSU-1.0细胞高至少3倍；21个基因在MSU-1.1细胞中的表达水平至少比MSU-1.0细胞低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这一事实的假设，我们将：1)通过Northern blotting确认MSU-1.0细胞和MSU-I.1细胞之间在所确定的每个基因的表达上的差异，如果可能，通过Western blotting，2)确定其在1中确认表达的基因是否通过通过感染将感兴趣的基因的cDNA导入适当的菌株而在使MSU-I.1细胞可转化中起关键作用(即，通过检测MSU-1.1细胞(组1)对MSU-1.0细胞表达水平较高的基因，以及MSU-1.1细胞(组2)对MSU-I.1细胞低水平表达的基因，检测细胞在生长因子受限的培养基中的生长能力和/或在琼脂糖集落中形成集落的能力。被感染的MSU-1.0细胞将被测试它们在这些条件下的生长能力，被感染的MSU-1.1细胞是否减少或阻止细胞的生长。3)确定被发现在生长因子反应性方面出现适当临界变化的细胞株是否可以通过H-RAS癌基因的转染而恶性转化。

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.