Research at Harlow Laboratory
Research in the Harlow laboratory relies on new approaches for functional analysis in mammalian cells. Cells use signaling pathways to respond to extracellular and intracellular cues and regulate important physiological events. It is clear that many regulatory processes integrate biochemical readouts from multiple pathways to control important cellular events. In simple genetic model systems, synergism and antagonism between different pathways can be identified and studied by use of mutation screens that can ask, in an unbiased way, which pathways contribute to key decision-making steps. Until recently, similar approaches have been unavailable in mammalian species, particularly in humans. The growing availability of comprehensive repositories of all coding regions and inhibitory RNAs, such as siRNA or shRNA, for each gene provides a technical approach to studies the role of each protein individually.
We use high throughput screens to identify proteins that affect key phenotypes of cancer biology. Screens rely on increasing or decreasing the levels of a protein of interest by expression of a cDNA or shRNA specific for the protein under study. cDNA and shRNA expression constructs are typically used in retroviral vectors. Constructs are transduced into cells in separate wells of 96- or 384-well plates, and changes in cell phenotypes are measured. For mammalian studies we have isolated full-length sequence verified cDNAs for 6500 human proteins and have a growing collection of shRNAs available for functional studies. Our goal is to collect and use repositories of the entire human coding potential.
Currently we are using these types of functional screens to perform three types of screens. cDNA and shRNA screens are being used to identify proteins that play a role in particular biological phenotypes. These screens include identification of proteins that are essential for cell viability and that affect cancer phenotypes such as genome instability and epigenetic changes in promoter regulation. The essential protein screens compare two related cell types such as tumor and normal cells or cells that have been engineered to have only a single change. The essential protein screens have a long-term goal of identifying new potential drug targets. A second class of screens examines regulatory interactions between different cellular pathways. Specific reagents that affect the function of a protein, such as treatment with a chemotherapeutic drug or expression of an shRNA specific for the protein under study, are used to perturb the cell in a measured way. Then cDNAs or shRNAs are screened to find other events that modify the cells response to the first agent and thus identify other components that affect the role of the first protein. A third class of on-going screens uses related strategies to perform epistasis experiments and map new regulatory pathways within cells. All of these methods are being used to learn how cancer cells are regulated, and we anticipate that these methods will be useful in answering many questions of modern mammalian cell biology.