The ability of cancer cells to proliferate inappropriately is due to the loss of tumor suppressor genes and the gain of function of oncogenes. Two classes of tumor suppressor genes exist, mutators and growth regulators. Mutators are genes that when mutant cause an increase in the rate of genomic instability and hence the rapid accumulation of mutations of all classes. Genes of the mutator classes include mis-match repair genes involved in HPCC, DNA repair genes such as X.P., and cell cycle checkpoint genes such as ATM and p53. Recently two breast cancer genes BRCA1 and BRCA2 have been implicated in DNA repair and fall into this class. The second class of tumor suppressors, growth regulators, are those directly involved in regulating cell proliferation or the ability of tumor cells to survive and metastasize. Oncogenes such as myc or ras are dominant and act in opposition to tumor suppressors. Many of the genes in these groups directly or indirectly regulate transcription, and the identification of target genes is important for elucidation of these regulatory pathways. Many oncogenes and tumor suppressors are involved in signaling pathways that activate the expression of a particular subset of relevant genes. Furthermore, the transcriptional induction of genes in response to DNA damage is a critical component of the cellular response to DNA damage and the prevention of mutagenesis. The identification of transcriptional target genes is critical to understanding how these regulatory pathways function. In this grant, we propose to develop novel methods for the identification of target genes that are differentially transcribed in response to oncogenic stimuli or DNA damage to better our understanding of these processes and to provide tools for future analysis.

The identification of genes regulated by particular stimuli has been a central strategy employed for elucidation of signaling pathways in many organisms. The original techniques were developed in prokaryotes using lacZ transcriptional fusions and in combination with transposable elements. Later, similar techniques were developed to identify developmentally regulated genes in Drosophila melanogaster, once again using lacZ and the transposable P-elements. Similar methods have been exploited in ES cells and used to identify transgenic mice that have tissue specific patterns of lacZ expression marking the existence of additional developmentally regulated genes. Not only have genes identified by these methods led to the elucidation of many signaling pathways, the creation of reporter gene using the promoters of regulated genes found by other methods has been the basis of many important studies. However, the most typical methods for identification of regulated genes in mammals are very labor intensive and generally result in the identification of a probe that is of limited utility.

This Idea grant proposes to design a new method for gene identification that involves the use of retroviral vectors containing the green fluorescent protein (GFP) or cell surface proteins as reporters of gene expression that allow living cells to be monitored for gene expression pattern changes. We propose to create a collection of cells containing retrovirus insertions that will only express GFP or a cell surface marker if it is under the control of an endogenous gene. These cells can then be purified by fluorescence-activated cell sorter (FACS) analysis on the basis of their basal expression levels and separated into equivalent expression groups. They can then be treated with different stimuli and sorted for cells that have increased expression of the gene-GFP fusion. This will generate clones containing fusions to genes that respond to the stimuli that can be used for further analysis to identify the pathways involved in their regulation. For example, we plan to search for genes whose expression is increased or decreased when oncogenes such as erbB2, myc, or activated ras are introduced on a second retrovirus. Furthermore, these collections of initial insertions can be screened for induction by any of a number of stimuli including the transient stimuli such as DNA damage or growth arresting agents. This is a novel method of gene discovery with applications to many branches of study that are relevant to breast cancer.