The applicant s aim is to gain understanding of how oligodendrocyte development and myelination are regulated in the CNS, with a view to design therapies for demyelinating disorders. Dr. Miskimins presents preliminary data indicating that the differentiation of oligodendrocyte progenitor cells is accompanied by a marked increase in the cyclin dependent kinase inhibitor p27kip1 (and a decrease in cyclin E). She proposes that the increase in p27kip1 is necessary for development and differentiation of oligodendrocytes. Specifically, the increase in p27kip1 would account for the cessation of proliferation prior to differentiation and could also play a role in the activation of the differentiation program. An initial goal is to define the specific stage of oligodendrocytes development at which p27kip1 levels rise. For this study Dr. Miskimins will utilize the oligodendrocyte progenitor (O-2A cell), as well as the CG-4 cell line which she reports retains many of the properties of the O-2A cell. Another set of studies is to define the effects of growth factors and signaling molecules (bFGF, TGFB and cAMP) known to moderate oligodendrocyte development on the expression of p27kip1. The applicant predicts that basic fibroblasts growth factor (bFGF) will interfere with the elevation of p27kip1 while transforming growth factor b (TGF b) and cAMP should increase p27kip1 levels. For these studies she will utilize O-2A cells and monitor p27kip1 by immunofluorescence in Western blots and she will quantitate mRNA by Northern blotting.
The third aim i s to determine if expression of p27kip1 is sufficient to promote differentiation of O-2A cells. Constructs for expression of p27kip1 under the control of an inducible promoter will be transfected into either CG-4 or proliferating O-2A cells. The applicant predicts that induction of p27kip1 should bring proliferation to a halt and promote differentiation. Antisense technology will be utilized to determine if p27kip1 is required for oligodendrocyte development. An addendum provides evidence suggesting that cyclin E levels should also be monitored and this is proposed.
| Miskimins, Robin; Srinivasan, Rekha; Marin-Husstege, Mireya et al. (2002) p27(Kip1) enhances myelin basic protein gene promoter activity. J Neurosci Res 67:100-5 |
| Miskimins, W K; Wang, G; Hawkinson, M et al. (2001) Control of cyclin-dependent kinase inhibitor p27 expression by cap-independent translation. Mol Cell Biol 21:4960-7 |
| Wang, G; Miskimins, R; Miskimins, W K (2000) Mimosine arrests cells in G1 by enhancing the levels of p27(Kip1). Exp Cell Res 254:64-71 |
| Wang, G; Miskimins, R; Miskimins, W K (1999) The cyclin-dependent kinase inhibitor p27Kip1 is localized to the cytosol in Swiss/3T3 cells. Oncogene 18:5204-10 |
