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2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Sheri L. Holmen, Ph.D. Laboratory of Molecular Medicine and Virology 38 Dr. Holmen received her M.S. in biomedical science from Western Michigan University in 1995 and her Ph.D. in tumor biology from the Mayo Clinic College of Medicine in 2000. She did her postdoctoral work at VARI from 2000 to 2003 and became a Junior Investigator at VARI in December 2003. Staff Laboratory Staff Todd Whitwam, Ph.D. Marleah Russo Sarah Warlick Students Visiting Scientists

VARI | 2006 Research Interests My laboratory is focused on defining critical targets in the signaling pathways of cancer cells that can become the focus for therapeutic intervention. Because of the high cost of developing new therapies, it is essential that we first identify which genetic alterations can be productively targeted. We are concentrating our initial efforts on melanoma and glioblastoma, tumors which demonstrate constitutive activation of Ras and Akt signaling. We plan to further validate the roles of these pathways using pharmacological inhibitors of clinical importance. The RCAS system We use a series of replication-competent retroviral vectors based on Rous sarcoma virus (RSV), a member of the avian leukosis virus family, to study the roles of various genes in tumor initiation and progression. RSV is the only known naturally occurring, replication-competent retrovirus that carries an oncogene, src. In RCAS vectors, the region encoding src (which is dispensable for viral replication) has been replaced by a synthetic DNA linker. Foreign genes inserted into this linker are expressed from the viral LTR promoter via a subgenomic splice site (just as src is in RSV). RCAN vectors differ from RCAS vectors in that they lack the src splice acceptor, so the gene of interest is inserted along with an internal promoter. Higher-titer viruses subsequently have been generated by replacing the RSV pol gene with the pol gene of the Bryan strain of RSV; these vectors are termed RCASBP or RCANBP. The ability of these vectors to infect non-avian cells relies on expression of the corresponding receptor on the cell surface. The viral receptor is typically introduced into mammalian cells (or mice) via an inducible and/or tissue-specific transgene. Therefore, this system allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. Alternatively, when targeted infection of mammalian cells is not required (e.g., in cell culture), infection can be achieved through the use of non-avian envelopes, such as the amphotropic envelope from murine leukemia virus. The receptor for this envelope is endogenously expressed on almost all mammalian cells. 39 We have used the RCASBP/RCANBP family of retroviral vectors extensively in both cultured cells and live animals for studies of viral replication and for cancer modeling in mice. Most of these studies have analyzed gain-of-function phenotypes by delivering and overexpressing a particular gene of interest. Recently we engineered the RCANBP vector to reduce the expression of specific genes through the delivery of short hairpin RNA sequences. We also engineered this vector to control the expression of the inserted gene using the tetracycline (tet)-regulated system. Sequences inserted into this region are transcribed from a tet-responsive element and not the viral LTR. This virus allows inserted genes to be turned on and off, in order to determine if expression of the gene is required for tumor initiation, maintenance, or progression. The ability to turn off gene expression will help determine if that gene is a good target for therapy.

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