Regulation of cellular senescence in human fibroblasts

Abstract

Normal mammalian cells only divide a limited number of times in culture before they undergo a permanent cell cycle arrest termed cellular senescence. In addition, expression of oncogenes and exposure to oxidative stress can elicit a similar response. The pathways that regulate senescence are disrupted in all cancer cells, suggesting that senescence evolved as a mechanism of tumor supression that protects cells against potential oncogenic changes. In human fibroblasts, the proliferative lifespan is limited primarily by the length of telomeres at the ends of chromosomes. Progressive telomere shortening eventually triggers a p53-mediated DNA damage response that leads to cell cycle arrest. Although the pathway from telomere shortening to cell cycle arrest has been studied in great detail, it has remained unclear how phenotypic changes that accompany senescence are regulated. In this work, I identified a novel transcription factor, APA-1, that regulates one aspect of the functional changes in senescent fibroblasts. Studies on the regulation of APA-1 have led to the discovery that senescence-associated changes such as matrix-remodeling are regulated, at least in part, through a telomere length-independent pathway. APA-1 was originally identified as a protein that interacts with the tumor suppressor ARF, a gene involved in senescence in murine fibroblasts. I have examined the functional consequences of this interaction and found that in some settings, ARF antagonizes the transcriptional activation function of APA-1. Finally, the study of APA-1 regulation led to the discovery that freshly isolated human fibroblasts are resistant to senescence induced by oncogenic RAS. Moreover, I found that exposure of fibroblasts to extended passaging in culture senesitizes cells to RAS-induced senescence, through telomere-independent upregulation of the cyclin dependent kinase inhibitor p16Ink4a. These findings challenge the belief that all normal cells senesce in response to RAS expression, and suggest that oxidative stress is a necessary cofactor that sensitizes cells to oncogene-induced senescence.