The principal direction of research in our lab is the use of new technologies to couple rapid identification of interesting genes with methods to study the consequences of their expression in an organismic context. Toward this end we have developed a very efficient type of expression cloning of signal transduction intermediates that allows us to rapidly identify cDNAs encoding genes that engage a number of known transduction pathways. In addition, we continue to work on methods for creating mutant cell lines that have lesions in signal transduction pathways, and appropriate ways to uncover the genetic basis of those lesions. We have also begun to develop systems for rapid generation of mice bearing targeted disruptions of specific candidate genes. The specific pathways on which we have focused most heavily are those arising from the T cell antigen receptor and other activating receptors, such as those for tumor necrosis factor alpha and lipopolysaccharide.
However recently we have found that the nuclear receptor PPAR-gamma, known to play a key role in the development of adipocytes, acts on human macrophages to inhibit inflammatory cytokine production. A number of human nonsteroidal anti-inflammatory drugs have PPAR-gamma activity at high doses, comparable to the serum concentration attained during treatment of rheumatoid arthritis. Hence an unrecognized component of the action of these drugs may be to inhibit inflammatory cytokine production.
To study gene activation in vivo we have begun to explore the use of engineered forms of Green Fluorescent Protein (GFP). One subject we have begun to study is stromal-tumor interaction in solid neoplasms. Tumor angiogenesis is known to be a critical element of the growth and metastasis of solid tumors. The angiogenic process requires normal tissue (blood vessels and stromal cells) to infiltrate neoplastic tissue. To help identify which normal cells are involved in angiogenesis, we created transgenic mice in which the promoter for Vascular Endothelial Growth Factor (VEGF) was placed upstream of GFP. The transgenic mice show green fluorescence about the margins and in the granulation tissue of healing wounds. In collaboration with the laboratory of Dr. Rakesh Jain at this institution, we implanted tumors in dorsal skin chambers that allow intravital microscopy of the tumors as they develop. These studies showed that the neoplastic cells strongly activate the VEGF promoter activity of surrounding tissue and induce the migration of bright green cells into the tumor itself. The fluorescent cells in both wound and tumor models are fibroblasts. By crossing the VEGF-GFP transgene into mice that are genetically susceptible to mammary tumors, we were also able to examine the promoter activation of endogenous tumors. These studies showed that the stromal cells surrounding the tumor nodules are highly induced for the GFP transgene, whereas the tumor itself shows no reactivity. Hence stromal fibroblasts show potent activation of the VEGF promoter. Although it has widely been supposed that the tumor itself is responsible for angiogenesis, the results of this work suggest that tumor-stromal collaboration is complex and further study is needed to identify which aspects of angiogenesis are regulated by tumor and which are regulated by the untransformed tissues of the tumor bed.
A second broad area of study relates to gene therapy and its potential utility both for the treatment of human diseases and for the generation of tools to facilitate the study of genes in an organismic context. This work is directed at creating better vectors for gene therapy, better tools for turning genes on and off, and better ways to regulate tissue specific expression. We have developed novel vectors for gene therapy that combine some of the best features of viral vectors with the strengths of nonviral expression platforms. Specific objectives include treatments for HIV infection and inborn errors of metabolism.
Selected Publications:
Jiang, C. Ting, A.T. and Seed, B. PPAR-_ agonists inhibit production
of monocyte inflammatory cytokines. Nature 391, 82-86 (1998).
Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. Membrane compartmentation is required for efficient T cell activation. Immunity 8, 723-732 (1998).
Fukumura, D., Xavier, R., Sugiura, T., Chen, Y., Park, E.C., Lu, N.,
Selig, M., Nielsen, G., Taksir, T., Jain, R.K., and Seed, B. Tumor Induction
of VEGF Promoter activity in stromal cells. Cell 94, 715-725
(1998).