BADERC-Brian Seed

Brian Seed, Ph.D.

Genetic Analysis of Signal Transduction

1. Genetic analysis of signal transduction. Following on our earlier development of methods for expression cloning of cell surface receptors, we have developed tools to accelerate the identification of the intracellular paths that underly cellular communication and pathogenic responses in the immune system. As part of that program we have created automated approaches that allow the rapid identification of genes that control transcriptional activation, subcellular localization and receptor deactivation. We have also developed systems for rapid generation of mice bearing targeted disruptions of specific candidate genes. We are integrating the two approaches with an emphasis on scaffold proteins in T cells and their role in activation.

2. Stromal contributions to tumorigenesis. In collaboration with the Jain lab at MGH we have begun to explore 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 whic 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 wound, and studies of tumors implanted in dorsal skin chambers (which allow intravital microscopy of the tumors as they develop) 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 which 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. Because this study has highlighted the importance of stromal cells in tumor growth we have begun to study the factors that are responsible for eliciting the induction of the VEGF promoter in vivo. Surprisingly, the developmental state of the fibroblast contributes in a major way to VEGF activation potential, suggesting that stromal influences are both complex and hierarchical.

3. Gene Therapy. A third 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. Specific objectives include treatments for HIV infection and inborn errors of metabolism.

References:

1. Jiang, C. Ting, A.T. and Seed, B. PPARg agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391, 82-86.

2. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. Membrane compartmentation is required for efficient T cell activation. Immunity 1998; 8, 723-732.

3. 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 1998; 94, 715-725.

4. Pickl, W.F., Pimentel, F.X.P., Seed, B. Lipid rafts and pseudotyping. J. Virol. 2000 75; 7175-83.

5. Randow, F., Seed B., Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat. Cell Biol. 2001; 10, 891-6.

6. Yang, Y., Seed, B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat Biotechnol. 2003; 21, 447-51.

7. Wang X, Seed B. Selection of oligonucleotide probes for protein coding sequences.Bioinformatics. 2003;19:796-802.

8. Lichtenthaler SF, Dominguez DI, Westmeyer GG, Reiss K, Haass C, Saftig P, De Strooper B, Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem. 2003;278:48713-9.

9. Rabizadeh S, Xavier RJ, Ishiguro K, Bernabeortiz J, Lopez-Ilasaca M, Khokhlatchev A, Mollahan P, Pfeifer GP, Avruch J, Seed B. The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.J Biol Chem. 2004(JBC, in press).






			Brian Seed, Ph.D. Genetic Analysis of Signal Transduction 1. Genetic analysis of signal transduction. Following on our earlier development of methods for expression cloning of cell surface receptors, we have developed tools to accelerate the identification of the intracellular paths that underly cellular communication and pathogenic responses in the immune system. As part of that program we have created automated approaches that allow the rapid identification of genes that control transcriptional activation, subcellular localization and receptor deactivation. We have also developed systems for rapid generation of mice bearing targeted disruptions of specific candidate genes. We are integrating the two approaches with an emphasis on scaffold proteins in T cells and their role in activation. 2. Stromal contributions to tumorigenesis. In collaboration with the Jain lab at MGH we have begun to explore 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 whic 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 wound, and studies of tumors implanted in dorsal skin chambers (which allow intravital microscopy of the tumors as they develop) 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 which 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. Because this study has highlighted the importance of stromal cells in tumor growth we have begun to study the factors that are responsible for eliciting the induction of the VEGF promoter in vivo. Surprisingly, the developmental state of the fibroblast contributes in a major way to VEGF activation potential, suggesting that stromal influences are both complex and hierarchical. 3. Gene Therapy. A third 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. Specific objectives include treatments for HIV infection and inborn errors of metabolism. References: 1. Jiang, C. Ting, A.T. and Seed, B. PPARg agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391, 82-86. 2. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. Membrane compartmentation is required for efficient T cell activation. Immunity 1998; 8, 723-732. 3. 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 1998; 94, 715-725. 4. Pickl, W.F., Pimentel, F.X.P., Seed, B. Lipid rafts and pseudotyping. J. Virol. 2000 75; 7175-83. 5. Randow, F., Seed B., Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat. Cell Biol. 2001; 10, 891-6. 6. Yang, Y., Seed, B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat Biotechnol. 2003; 21, 447-51. 7. Wang X, Seed B. Selection of oligonucleotide probes for protein coding sequences.Bioinformatics. 2003;19:796-802. 8. Lichtenthaler SF, Dominguez DI, Westmeyer GG, Reiss K, Haass C, Saftig P, De Strooper B, Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem. 2003;278:48713-9. 9. Rabizadeh S, Xavier RJ, Ishiguro K, Bernabeortiz J, Lopez-Ilasaca M, Khokhlatchev A, Mollahan P, Pfeifer GP, Avruch J, Seed B. The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.J Biol Chem. 2004(JBC, in press).