Abstract
Cyclin-dependent kinase inhibitor p16(INK4a) is implicated in replicative senescence, cell immortalization, and tumor generation. However, the mechanism regulating its overexpression in senescent cells is unknown. We used the enhanced green fluorescent protein reporter system to scan regulatory elements in the upstream region of p16(INK4a). The results of 5'-deletion studies indicated that the transcription regulatory elements contributing to overexpression of p16(INK4a) in senescent cells were located in the region of the p16(INK4a) promoter from -622 to -280 bp. According to the results of in vitro DNase I footprinting, EMSA, and Southwestern blotting, we found a novel negative regulatory element, the INK4a transcription silence element (ITSE), at -491 to -485 bp of the p16(INK4a) promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16(INK4a) by interacting with the ITSE. The activity of the p16(INK4a) promoter increased significantly in young cells when the ITSE was deleted. The GC-rich region of the p16(INK4a) promoter from -466 to -451 was a positive transcription regulatory element. Deletion of this region showed 91.4% loss of p16(INK4a) promoter activity in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably.
Highlights
Cellular senescence consists of the loss of proliferative potential produced by the accumulation of cell doublings [1]
The results of the 5Јdeletion assay indicated that the activities of different length (Ϫ622 to Ϫ3017 bp) of the p16INK4a promoter were enhanced by 5–7-fold during the fibroblast aging process, which was much lower than the increment level of p16INK4a mRNA and protein in senescent cells
In many cultured cell lines, such as fibroblasts, keratinocytes, and urothelial cells, p16INK4a accumulates with increasing numbers of population doublings [2, 5,6,7,8]
Summary
Cellular senescence consists of the loss of proliferative potential produced by the accumulation of cell doublings [1]. In certain human cell types, such as human keratinocytes or mammary epithelial cells, in which telomerase expression alone is insufficient to bypass senescence, the additional inactivation of p16INK4a by genetic or epigenetic mechanisms is required to bypass senescence and render the cells immortal [13, 14]. Viral oncoproteins such as SV40 large T antigen, adenoviral E1a protein, or herpesvirus E7 protein inactivate the growth-suppressive functions of Rb and facilitate immortalization [15, 16]. A GC-abundant element located in the region from Ϫ466 to Ϫ451 bp was involved with high expression of p16INK4a in senescent fibroblasts
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