(281a) Using Inducible Gene Expression to Characterize Oncogene Cooperation in a Murine Pre-B Cell Line

Cancer is caused by multiple gene mutations that each contribute to the transformation of normal cells. There is more to be learned, however, about the sequence of this transformation and how the genes involved are cooperating to produce cancerous cells. Drastic changes in the expression of a single oncogene often initiate antiproliferative signals that lead to senescence or apoptosis. Presumably, therefore, mutated genes must be expressed at tolerable levels and occur in an order that allows them to circumvent the cells natural mechanisms of tumor suppression. In this study we focused on two main goals. First, we created a system that would allow us to quantitatively determine the effect of cancer-associated genes on cell proliferation at a range of expression levels. Second, we used this system to screen pairs of genes to find novel cooperation that may help elucidate how the underlying regulatory pathways interact.

Generally, in order to determine the function of a gene, most studies highly overexpress and/or knockdown/knockout gene expression. In fact, in mammalian cells there are few tools available to inducibly express genes in a predictable manner. In this study we used a new approach wherein the proteins of interest are fused to destabilized protein domains which are inducibly stabilized by the presence of specific small molecules. Since it operates at the protein level the clonal variability is reduced and other markers/drug resistance genes can be driven off the same promoter. Next, we looked at proliferation rates as an indicator of cell response to the induced gene expression.

We looked at the interactions of pairs of genes. To identify gene candidates, we started by looking at a large mouse lymphoma tumor screen that used murine leukemia retrovirus to initiate tumorigenesis. It was assumed that pairs of genes that were frequently found in conjunction in tumors may cooperate in the transformation of normal cells. Using the technique outlined above and the two orthogonal inducible protein control systems, we expressed pairs of genes in a v-Abl induced mouse pre-B cell line.