REGULATION OF DIHYDROFOLATE-REDUCTASE GENE-EXPRESSION AND E2F COMPONENTS IN HUMAN-DIPLOID FIBROBLASTS DURING GROWTH AND SENESCENCE

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA b iosynthesis that is induced just before the onset of S phase, is marke dly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp pro moter region of the human DHFR gene (-381 to -17) indicated that nucle ar proteins bind to a cluster of cis-elements, including two overlappi ng E2F binding sequences, two SP1 sites, and one Yi sequence. Gel mobi lity shift assays were performed to assess the role of each cis-elemen t in the regulation of DHFR gene expression. We found that 1) Sp1 bind ing activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetec table in both early passage and senescent cells; and 3) E2F binding ac tivity was serum-inducible, senescence-dependent, and prominent in pre senescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed t hat the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-depend ent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and sen escence-independent. In contrast, E2F-2 mRNA was not detectable in IMR -90 or WI-38 human fibroblasts. Western blot analysis showed that amon g the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nucl ear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells. (C) 1996 Wiley -Liss, Inc.