Gretchen Stein - Senior Instructor

Normal human diploid fibroblasts (HDF) have a finite proliferative lifespan at the end of which they are unable to enter S phase in response to mitogenic stimuli. Senescent HDF are also enlarged, flattened, and synthesize an altered repertoire of cell type specific proteins, suggesting that they have differentiated as well as aged. We are studying the molecular mechanism for the senescent cell cycle arrest and testing the hypothesis that these cells also become terminally differentiated at the end of lifespan.

Previously, we showed that senescent HDF fail to phosphorylate the retinoblastoma protein (pRb), an event necessary for the release of E2F transcription factors that promote the expression of late G1 genes whose products are required for S phase initiation and progression. Because phosphorylation of pRb is carried out by cyclin D-Cdk4/6 andcyclin E-Cdk2 complexes, we investigated the ability of senescent HDF to form and activate these complexes. Our recent results have shown that senescent HDF have abundant cyclin E-Cdk2 complexes that are inactive, and that inactivation of these complexes can be accounted for by their binding to the p21Sdi1, Cip1,Waf1 inhibitor of cyclin-dependent kinases. Senescent HDF also have abundant cyclin D-Cdk4/6 complexes that are bound to an increased amount of p21, but it has not yet been shown that this is sufficient to inhibit cyclin D-associated kinase activity. On the other hand, we have shown that the increased amount of the cyclin-dependent kinase inhibitor p16Ink4a, which sequesters Cdk4/6, does not increase significantly until after the cells become senescent owing to high p21. Interestingly, p16 increases in parallel with several markers of the differentiation of senescent cells, suggesting that the increase in p16 may be part of a program of differentiation that is initiated in senescent cells. Concurrently, p21 decreases from its maximum at the initiation of the senescent cell cycle arrest, suggesting that it may be downregulated as part of the same differentiation program.
Based on the work described above, we are currently pursuing two main avenues of research. The first is to determine the mechanism for senescence in HDF that are deficient in p21Sdi1, Cip1, Waf1 owing to expression of the HPV16 E6 oncogene, which leads to the rapid degradation of p53, a positive transactivator of p21. We have found that these cells eventually become senescent, albeit inefficiently, even though they have a reduced amount of p21 throughout their lifespan. Thus, these cells have the potential to reveal mechanisms for senescence that may be cryptic in wild-type cells owing to the dominant effect of p21. Our second avenue of research is to study the mechanism for the differentiation of the senescent cells, and determine how this relates to the upregulation of p16 and downregulation of p21. Because aging cells enter senescence very heterogeneously over a long period of time, it is difficult to study this transition in a naturally aging population. Consequently, we have worked on a model system whereby young HDF treated with DNA damaging agents become irreversibly arrested and differentiated like senescent cells. They mimic the changes seen in p21, p16, and a variety of differentiated characteristics in senescent cells, making it possible for us to study the mechanisms for these changes in a semi-synchronous population.