A Genetic Approach to Identification of Genes Involved in Cellular Senescence and Immortalization

2000 new Scholar Award in aging

One major advance in the study of mammalian aging was the discovery made by Hayflick in the 1960's. He observed that normal human cells had a finite lifespan in vitro and could execute only a limited number of cell divisions. Beyond this limit, cells undergo an irreversible growth arrest known as replicative senescence, or M1 (mortality-1). It was further demonstrated that the lifespan of normal cells in vitro was proportional to the longevity of the organism in vivo. Thus, cellular senescence may reflect the aging process of an organism.

The onset of senescence can also be activated prematurely by oncogenic stimulus, such as the oncogenic form of Ras. Therefore, oncogenic transformation requires immortalizing genetic changes that overcome both replicative senescence and premature senescence elicited by oncogenes.

So far, little is known about the molecular mechanisms underlying replicative senescence and oncogene-mediated premature senescence, or how these control points are bypassed in transformed cells. We have developed a novel retrovirus-based expressional cloning system (the MaRX system), which enables large scale, high throughput phenotypic screening in cultured mammalian cells, even when the desired phenotype has a high background. I propose to use the MaRX system to systematically search for genes that can regulate replicative senescence and premature senescence induced by activated Ras. Through these studies, we hope to gain insights into the molecular pathways leading to immortalization and tumorigenesis in normal human cells.

Researchers
Peiqing Sun Ph.D.
Scripps Research Institute

While the ras oncogenes play a major role in the development of human tumors, in primary, normal cells, oncogenic ras activates the Raf-MEK-ERK MAPK pathway, which in turn triggers a tumor-suppression defense response known as premature senescence. As a result, additional genetic alterations are required to bypass the senescence response in order for ras to induce transformation. Although the downstream effectors mediating the oncogenic activity of ras have been extensively investigated, little is known about the cellular pathway that is essential for ras to induce senescence and the mechanisms by which ras-induced senescence is bypassed in human tumors. Our studies revealed that ras induces senescence through sequential activation of the Raf-MEK-ERK pathway and the stress-induced MKK3/6-p38 MAPK pathway. Thus, the Raf-MEK-ERK cascade, once activated by ras, triggers two distinct downstream signaling branches, with one mediating the mitogenic and transforming potential of ras and the other inducing the anti-oncogenic response via p38. These findings have identified a signaling pathway that mediates the ras-induced senescence response in primary cells, and demonstrated for the first time that the p38 pathway has a tumor-suppressing function, in addition to its known role in stress and inflammatory responses. Further analysis of the components of the ERK and p38 pathways has allowed us to identify proteins that mediate ras-induced senescence and the tumor suppressing function of p38. Furthermore, our analysis of the senescence-bypassing activities of a viral oncoprotein E1 A demonstrated that inactivation of Rb family proteins and inactivation of p300/CBP proteins both contribute to the bypass of ras-induced premature senescence. These findings have provided insights into the mechanism of senescence bypass in human tumors. Overall, our studies have furthered our understandings on tumorigenesis and tumor suppressing pathways, and have revealed potential targets for the development of novel cancer therapeutics.