Cell Cycle Regulation of NF- B-Binding Activity in Cells from Human Glioblastomas Sameer A. Ansari, 1 Mahmut Safak, 1 Luis Del Valle, Sahnila Enam, Shohreh Amini, and Kamel Khalili 2 Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 North 12th Street, 015-96, Philadelphia, Pennsylvania 19122 Glioblastoma multiforme is a highly malignant and anaplastic tumor of the central nervous system repre- senting more than 50% of all malignant gliomas. The cell origin of this highly undifferentiated tumor re- mains obscure, although it is postulated that glioblas- tomas are developed from astrocytes. The rapid growth of the glioma and the state of its undifferen- tiation are attributed to the deregulation of several signal transduction pathways and cell cycle events. Recent studies showed diverse functions for the NF- B/Rel family of inducible transcription factors in- cluding differentiation, apoptosis, oncogenesis, and cell cycle regulation. We sought to examine the level of NF-B activity throughout the glioma’s cell cycle. Re- sults from band-shift studies indicated a biphasic NF-B DNA-binding activity in the nuclei of cycling glioblastoma cells. We showed that NF-B-binding ac- tivity maximizes in nuclear extracts at specific cell cycle stages including G0/G1, mid-late G1, and S phase. Results from Northern blotting studies revealed that the differential expression of the NF-B subunits, p50 and p65, may not be responsible for cell cycle stage- specific association of NF-B subunits with DNA. How- ever, results from Western blotting analysis utilizing nuclear extracts from glioma cells throughout the cell cycle demonstrated that the nuclear accumulation of p50 and p65 perfectly correlates with their DNA-bind- ing activity. These observations suggest that the nu- clear translocation of the p50/p65 subunit of NF-B in glioma cells is cell cycle stage-dependent and that is distinct from the differential mRNA expression of these genes during glioma cell cycling. The possible role of NF-B in glioma cell formation and regulation of cellular genes by NF-B in these tumor cells is dis- cussed. © 2001 Academic Press INTRODUCTION The NF-B/Rel family of transcription factors is a critical component of signal transduction pathways that regulate genes involved in inflammatory and im- mune responses. Recent evidence implicates NF-B/ Rel proteins in diverse functions mediating antiapop- totic pathways, differentiation, and cell cycle regulation. Interestingly, the small multigene NF-B/ Rel family shares homology with the v-rel oncogene from the avian transforming retrovirus, reticuloendo- theliosis virus strain T, suggesting that NF-B/Rel members may function in growth control or oncogene- sis via mitogenic pathways [1]. Prototypical NF-B, the best-characterized complex of the NF-B family, consists of the p50 and p65 het- erodimer and is regulated by inhibitor proteins (IBs) [2–5]. A family of IB proteins, which are present in the cytoplasm, bind to NF-B heterodimers consisting of p50 and p65 subunits in a ternary complex and mask their nuclear localization signals [6]. Upon induction by a stimulus, IBs are phosphorylated for proteolysis [7, 8] by the ubiquitin-26S proteasome pathway [9] or by a proteolytic-independent mechanism [10], leading to nuclear translocation of the p50/p65 heterodimer complex. In nuclei, NF-B heterodimers bind to well- defined DNA sequences, 5'-GGGACTTTCC-3', found within the enhancers of many regulatory and structur- ally important cellular genes to stimulate their levels of transcription. NF-B-binding activity has been shown to be regulated by several environmental factors such as exposure to cytokines, UV, and viral infection. In addition, NF-B-binding activity can be modulated during the cell cycle and previous studies have shown induction of NF-B at the G0/G1 transition in fibro- blasts in response to serum [11]. Rel-related proteins localize the nucleus at specific phases of the cell cycle [12, 13] and RelA may act as an effector for the p21BCR-ABL oncogene. Interestingly, RelA interacts with the cyclin E:CDK2 complex through the p300 adapter protein and its cooperative transcriptional ef- fect with the CDKI inhibitor p21 poses provocative questions as to the function of NF-B in the cell cycle [14]. Recent evidence suggests that NF-B is involved in G0/G1 to S-phase transition in fibroblast cell lines and inhibits myogenic differentiation through kinetic and transcriptional regulation of cyclin D1 [15–17]. 1 These authors equally contributed to this work. 2 To whom correspondence and reprint requests should be ad- dressed. Fax: (215) 204-0679. E-mail: kkhalili@astro.temple.edu. 0014-4827/01 $35.00 221 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 265, 221–233 (2001) doi:10.1006/excr.2001.5168, available online at http://www.idealibrary.com on