Altered Profile of Transcription Factor-Binding Activities in Senescent Human Fibroblasts

Abstract

Normal eukaryotic cells divide a limited number of times, after which they enter a state of irreversible growth arrest and altered function termed senescence. Cell senescence entails changes in the expression of growth- and differentiation-specific genes, suggesting that senescent cells express an altered profile of transcription factors. Nuclear extracts were prepared from presenescent (quiescent and growing) and senescent human fibroblasts (WI-38) and from SV40-immortalized WI-38 cells and Y79 human retinoblastoma tumor cells—both of which have escaped senescence. The extracts were assayed for ability to form specific protein-DNA complexes with oligonucleotides containing binding sites for the general transcription factors CTF (CAAT-binding transcription factor), SP1 (promotor-specific transcription factor-1), and TFIID (transcription factor-IID) and the more gene-specific factors AP1 (activator protein factor-1), CREBP (cAMP response element-binding protein), GREBP (glucocorticoid response element-binding protein), NF-κB (nuclear factor κB) and OctBP (octamer-binding protein). Two TFIID complexes and the GREBP, NF-κB, and SP1 complexes were similar in presenescent and senescent cells. AP1, CREBP, and CTF complexes were reduced in senscent cells. Two activities were more abundant in senescent cells: OctBP and one TFIID complex. This TFIID complex was present in quiescent cells, but absent from four human cell lines that lack a functional retinoblastoma protein (pRb); both pRb-specific and TFIID-specific antibodies selectively disrupted it. The data suggest that an altered profile of transcription factors may specify the senescent phenotype and that pRb may interact with TFIID or a TFIID-associated protein(s).