This project involves the development of continuous cell lines, for use as models to investigate effects of drugs in vitro, in neural transplantation as an alternative to primary cells and tissues, and for basic studies of neural cell biology. Current efforts include development of mutant truncated forms of SV40 large T antigen and methods for the delivery of genes or combinations of genes to cells in order to modify the cell cycle. A mutant form of SV40 large T antigen, which lacks p53 binding activity, has been cloned to examine those properties of SV40 large T antigen which are required for immortalizing CNS neurons. This mutant oncogene, called T155, is capable of overcoming cell cycle arrest and immortalizing primary mesencephalic neurons. T155 appears to interfere with the expression of differentiated phenotypes to a much smaller degree than wild-type SV40 large T antigen, and, moreover avoid the problem of interference with the activity of p53 which could promote the survival of cells with DNA damage. Primary mesencephalic cell cultures immortalized with T155 express differentiated neuronal markers (e.g., neurofilament) and glial markers (e.g., GFAP), whereas cells immortalized with wild-type SV40 large T rarely express markers characteristic of mature neurons or glia. In one set of experiments, striatal cell lines have been obtained that produce high levels of GABA, and these cells are effective in transplantation studies in animals. Cell lines from kidney epithelium that produce high levels of growth factors and can be used in neural transplantation in animal models of stroke have also been produced. Several additional variants of T155 have been produced including T155g (genomic), T155c (cDNA), and E107K. Viral vectors for delivery of these oncogene variants in various forms are presently being developed. Currently, these oncogene fragments are being quantitatively evaluated for their efficacy in producing rodent CNS cell lines. Systems in use include rodent and human cell cultures, and mouse embryonic stem cell lines. These studies may lead to the production of human cell lines that could be used for therapeutic purposes and as in vitro models for studying effects of drugs of abuse.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Intramural Research (Z01)
Project #
1Z01DA000410-06
Application #
6830628
Study Section
(CNRB)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2003
Total Cost
Indirect Cost
Name
National Institute on Drug Abuse
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Lee, Chun-Ting; Bendriem, Raphael M; Freed, William J (2015) A new technique for modeling neuronal connectivity using human pluripotent stem cells. Restor Neurol Neurosci 33:347-56
Nolte, Marc W; Loscher, Wolfgang; Herden, Christiane et al. (2008) Benefits and risks of intranigral transplantation of GABA-producing cells subsequent to the establishment of kindling-induced seizures. Neurobiol Dis 31:342-54
Castillo, Claudia G; Mendoza-Trejo, Soledad; Aguilar, Manuel B et al. (2008) Intranigral transplants of a GABAergic cell line produce long-term alleviation of established motor seizures. Behav Brain Res 193:17-27
Anantharam, Vellareddy; Lehrmann, Elin; Kanthasamy, Arthi et al. (2007) Microarray analysis of oxidative stress regulated genes in mesencephalic dopaminergic neuronal cells: relevance to oxidative damage in Parkinson's disease. Neurochem Int 50:834-47
Harvey, B K; Chen, G J; Schoen, C J et al. (2007) An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke. Cell Transplant 16:483-91
Chen, Jia; Lee, Chun-Ting; Errico, Stacie L et al. (2007) Increases in expression of 14-3-3 eta and 14-3-3 zeta transcripts during neuroprotection induced by delta9-tetrahydrocannabinol in AF5 cells. J Neurosci Res 85:1724-33
Marchionini, Deanna M; Lehrmann, Elin; Chu, Yaping et al. (2007) Role of heparin binding growth factors in nigrostriatal dopamine system development and Parkinson's disease. Brain Res 1147:77-88
Sanchez, Joseph F; Crooks, Daniel R; Lee, Chun-Ting et al. (2006) GABAergic lineage differentiation of AF5 neural progenitor cells in vitro. Cell Tissue Res 324:1-8
Castillo, Claudia G; Mendoza, Soledad; Freed, William J et al. (2006) Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav Brain Res 171:109-15
McNeill-Blue, Charlesene; Wetmore, Barbara A; Sanchez, Joseph F et al. (2006) Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine. Brain Res 1112:1-15

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