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, assessment of appropriate promoter/enhancer elements to drive these molecules and methods for the delivery of genes or combinations of genes to primary 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 that are required for immortalizing CNS neurons. This mutant oncogene, called T155, is capable of overcoming cell cycle arrest and immortalizing primary rodent mesencephalic neurons and various other primary cultured rodent somatic cells. T155 appears to interfere with the expression of differentiated phenotypes to a much smaller degree than wild-type SV40 large T antigen, and moreover, avoids the problem of interference with the normal activity of p53. Primary rodent mesencephalic cell cultures immortalized with T155 express differentiated neuronal markers (e.g., neurofilaments, b-III-tubulin), neural precursor markers (e.g., nestin), glial markers (e.g., GFAP), and neuronal phenotypic markers such as indicators of GABAergic function. In contrast, 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 were developed that produce high levels of GABA, and these cells are effective in transplantation studies in animals. Cell lines, both from kidney epithelium and from rodent mesencephalon, that produce high levels of growth factors have also been produced. The efficacy of these cells when used for neural transplantation in an animal models of stroke has also been examined. Several additional variants of T155 have been produced including T155g (genomic), T155c (cDNA), and E107K, which includes a mutation of the Rb binding site for T155. We have recently found that plasmid-based gene transfer results in unreliable and variable transfer of various functional components of the plasmids which are used;thus, in the future it will be necessary to employ viral vectors for gene transfer, a project which is currently being developed. In addition, a series of alternative promoter systems, including a nestin-derived promoter, RSV, EF1, and CMV-based promoters has been cloned into expression vectors in combination with T155 oncogene fragments. A series of cell lines has been produced from rat cultures to test and compare these promoters and oncogene variants, and a number of cell lines with neural progenitor properties have been developed using several of the combinations of oncogene and promoter. For use in human cells, a series of vectors to co-express one of the oncogene fragments and the human telomerase catalytic unit, either via an IRES sequence or as a fusion protein, have been developed. One of these fusion proteins has been found to be capable of immortalizing primary human astrocytes. We have developed an improved version of the T155c oncogene fragment with codons optimized for mammalian expression. Lentiviral vectors for delivery of these oncogene variants in various forms are presently being developed. Currently, these oncogene fragments and various mammalian promoters are being evaluated for their efficacy in producing CNS derived cell lines, especially from cultured human cells and from specific cell types differentiated from human embryonic stem cells. Systems in use include rodent and human cell cultures, mouse and human 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 drugss of abuse. We have also developed a lentiviral vector for delivery of v-myc to human embryonic stem cells and to neural progenitor cells derived from embryonic stem cells. This system has been used to produce a series of neural progenitor cell lines, some of which undergo differentiation to mesencephalic-type dopaminergic neurons at moderate frequency under appropriate conditions. These cells are easily propagated in vitro and but lose their differentiation capacity during long-term culture. We have tested a number of human medulloblastoma and astrocytoma cell lines for differential sensitivity to growth regulation by cocaine and related compounds, and are testing these in comparison to immortalized neural progenitor cells. It might be possible to exploit differences between the sensitivity of tumor cell lines and neural progenitor cells to growth regulation by compounds similar to cocaine in order to protect normal dividing neural progenitor cells from radiation-induced damage. Additional experiments involve the use of immortalized cell lines to investigate cellular mechanisms of drugs of abuse. In this context, a particularly notable property of rodent cells immortalized by T155 is that these cells retain normal p53 function, and thus can be employed to study mechanisms of neurotoxicity and cell cycle arrest. We have employed the AF5 neural progenitor cell line, in addition to human primary cell cultures, to show that proliferation of both neural progenitor cells and oligodendrocyte progenitor cells is inhibited by cocaine, and that this inhibition is mediated by decreased expression of cyclin A. Moreover, proliferation inhibition via cyclin A occurs only for progenitor cells, and is not seen of other cell types such as astrocytes or mature cells. In another series of experiments, a cell line was employed to show that tetrahydrocannabinol produces a neuroprotective effective which is mediated in part by changes in expression of 14-3-3 proteins. Thus, cell lines produced by these techniques have enabled us to examine certain cellular mechanisms involved in the effects of drugs of abuse;in particular, studies of cocaine-induced inhibition of neural progenitor cell proliferation would not have been possible without the AF5 cell line.
