David Kwiatkowski , MD, Ph.D.
Division of Hematology
Brigham and Women's Hospital
221 Longwood Avenue, EBRC 301
Boston, MA 02115

A chemical library screen for inhibitors of growth in Tsc1/Tsc2 null cells:

Tuberous sclerosis is an autosomal dominant human genetic disorder characterized by the development of hamartomas in multiple organ systems, with involvement of the brain causing the most serious clinical problems in many patients. We have recently developed several mouse models of TSC, including null and conditional,Tsc1 and Tsc2 alleles. We have cultured null embryos of each of Tsc1 and Tsc2, to establish cell lines of two distinct types, murine embryo fibroblasts, cultured in conventional media with 10% serum, and neuroepithelial progenitor (NEP) cells, cultured in media with bFGF and EGF but no serum. We have also established fibroblast cultures that are p53 null and Tsc2 null. We have identified abnormalities of the PI3-kinase/Akt/mTOR/p70S6kinasesignalling pathway in these cells, following up insights provided by the studies in Drosophila and suggestions from colleagues.

We will use the cell lines we have generated to do a chemical library screen for growth inhibitors that are specific for theTsc2 null genotype in the Harvard Institute of Chemistry and Cell Biology.. Six fibroblast cell lines, all p53 null, 3 Tsc2 null and 3 Tsc2 wild type, will be plated in 384 well plates. The cells will be treated with 60,000 different chemical compounds under carefully controlled growth conditions to permit identification of compounds that have a differential effect on the growth ofTsc2 null compared with wild type fibroblasts. Compounds that have a differential effect on growth in the Tsc2 null fibroblasts, will be retested to assess dose response, and then tested on the other cell lines we have available. Compounds that make it through this aggregate screening will then be tested in more detail to assess dose response. Studies will proceed to characterize the precise chemical structure required for the inhibitory activity ,and identify the intracellular targets to which compounds bind. We will also see if such compounds reverse the abnormal phosphorylation of p70S6kinase and other proteins we have observed in these cells.

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