Molecular Neuro-Oncology Laboratory

Dr Reeves | Molecular Neuro-Oncology Laboratory Recent Publications | Links

Steven A. Reeves, Ph.D., Principal Investigator Tiziana Servidei, Ph.D., Post-Doc. Petra Schwartz, M.D., Post-Doc. Hirofuma Hiyama, M.D., Post-Doc. Holger Willenbring, M.D./Ph.D. candidate, Medical student. Bibrama Sinha, BS, Research Technician.

The role of the SHPTP2 tyrosine phosphatase in the control of cell signaling.

The ability of a cell to grow and divide is initiated with the binding of growth factors to specific receptors. The relaying of the growth factor signal from the receptor to the nucleus of the cell, where genes responsible for growth or differentiation are then turned on, is orchestrated by a network of cellular proteins which shuttle the signal from the cell surface to the nucleus. Many of the signaling proteins that shuttle the signal interact with each other in a modular fashion. The mechanics of which are analogous to the way telephones are connected. Snap-in plugs connect the hand held receiver with the body of the telephone, which in turn is connected to a wall plug through additional snap-in plugs. So called SH2 domains (Src homology 2 domains) are the equivalent snap-in plugs found within a variety of signaling proteins. SH2 domains are small motifs that recognize phosphorylated tyrosine residues. Specificity for any given SH2 domain is achieved through specific residues flanking the phosphorylated tyrosine. Similar to a phone conversation which is disrupted if any of the snap-in plugs are removed, the transmission of a signal to the nucleus of the cell is also disrupted if the SH2 domain connectors are unplugged. One way to disrupt the connection between the SH2 domain and the phosphorylated tyrosine is by removal of the phosphate group from the phosphorylated tyrosine. Candidate cellular enzymes likely responsible for this activity are the Tyrosine-Specific Phosphatases. One of the areas of research of this laboratory is in defining what specific role the SHPTP2 (PTP1D or Syp) tyrosine phosphatase has in mediating growth factor simulation of cells. Work in this laboratory has shown that SHPTP2 associates with several different signaling proteins including the mitogen activated proteins kinases (MAPK), ERK1 and ERK2. Because the activities of the MAPKs are essential for the ability of a cell to respond to a growth or differentiation signal, we are investigating whether SHPTP2 is important in regulating the activities of MAPK.

Retroviral Vector to Deliver & Regulate the Production of Therapeutic Genes

Another major focus of the laboratory is in the development of retroviral vectors that potentially can be used for specific killing of glioma tumor cells. We are currently designing retroviral vectors that deliver a toxin gene to the tumor cells. The major hurdle in the design of these vectors, however, is in controlling the expression, and thus the activities, of the toxin gene. In order to control the expression of the toxin we are constructing composite promoters that confer high level regulation to expression of the toxin gene.

For example, we have designed a tetracycline-regulatable retroviral vector that can deliver genes encoding therapeutic proteins to diseased cells. This unique retroviral vector allows the production of these therapeutic proteins in infected cells to be under the control of tetracycline, a compound with well known pharmacology in humans and rodents. We have used this vector in several different applications. In one study, we introduced the programmed cell death gene, ICE (which induces apoptosis) into the tetracycline-regulatable retroviral vector and utilized the intrinsic cell death program of ICE as a means for tumoricidal therapy in a rat brain tumor model. In this study, suppression of ICE expression was extremely tight in the presence of tetracycline, both in cultured cells and in a rat brain tumor model, as determined by cell viability and morphological criteria. However, when tetracycline was withdrawn, ICE gene expression was rapidly turned on and apoptosis-mediated cell death occurred in essentially all tumor cells.

In another study, we are using the tetracycline-regulatable retroviral vector in a rodent Parkinson's disease model. In clinical practice the concept would be to genetically modify non-neuronal cells from the Parkinsonian patient with the capacity to produce proteins that potentially could restore normal dopamine levels. In the rodent model, we have genetically modified rat fibroblasts by infecting the cells with different retroviral vectors containing genes the encode either tyrosine hydroxylase (TH), which is a rate-limiting enzyme necessary for the biosynthesis of dopamine or glial-derived neurotrophic factor (GDNF), which encodes a protein that increases cellular dopamine uptake and neuronal survival in dopaminergic and motor neurons. We are currently implanting these cells into the substantia nigra of 6-hydroxydopamine-treated rats and will measure, in a tetracycline-dependent manner, whether these proteins can benefit animals in a unilateral rotation model. Importantly, these tetracycline-regulated retroviral vectors not only provide a means of regulating expression of the TH and GDNF genes in an off or on manner, but also allow intermediate levels of expression. Varying the levels of expression of the TH and GDNF genes by increasing or decreasing the pharmacological levels of tetracycline should be of considerable benefit in situations of greater or lesser dopamine need.

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