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JOHN SEDIVY Professor Department of Molecular and Cell Biology and Biochemistry Ph.D., Harvard University, 1985 (401) 863-7631 |
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Research Summary The Sedivy laboratory is funded by two major grants: "Genetic Studies of c-Myc Gene Function in the Cell Cycle" (NIH GM41690), and "Mechanisms of Replicative Senescence in Human Cells" (NIH AG16694). The key objectives of each project are outlined below. Genetic Studies of c-Myc Gene Function in the Cell Cycle The goal of this project is to answer the question: How does the c-myc protooncogene regulate cellular proliferation? The c-Myc protein is a transcription factor, and a large number of genes have been proposed to be targets of its regulation. c-Myc has been implicated in the control of cellular proliferation, differentiation, and programmed cell death, but the mechanisms by which it exerts its activity on the cellular machinery are complex and not well understood. Our fascination with c-Myc stems not only from our interest in understanding basic molecular regulatory mechanisms, but also because the deregulation of c-Myc activity is associated with a wide range of human cancers. A genetic analysis can often lead to profound insights into molecular mechanisms, and the construction of knockout mice can be especially powerful. Unfortunately, the c-myc knockout mouse, because of its early embryonic lethality, did not yield insights into either cellular or molecular phenotypes. All attempts to recover c-myc -/- cells from homozygous embryos have been frustrated by the outgrowth of cells that express one of the other Myc family members. To overcome this problem, we used gene targeting to eliminate c-myc expression in a fibroblast cell line shown not to express the other family members. These knockout cells are the first experimental system in which c-myc loss-of-function phenotypes can be thoroughly investigated. The c-myc -/- cells are viable but display profound cell cycle defects. We have initiated a systematic molecular analysis of known cell cycle regulators in the c-myc -/- cells. Our new insights to date are: 1) loss of c-myc affects progression through both the G1 and G2 phases of the cell cycle; 2) the activity of all cyclin-Cdk complexes is affected; 3) the earliest and most prominent defect is an impairment in the activation of cyclin D-Ddk4/6 complexes; 4) c-Myc affects multiple points in the cell cycle, both before and after the restriction point; 5) loss of c-Myc has profound effects on the accumulation of cellular mass (cell growth). Our objective is to build on these results to fully elucidate how c-Myc regulates transition through the cell cycle. We currently working towards constructing homozygous c-myc knockouts in human cells, isolating new c-Myc target genes by a combination of microarray expression profiling and chromatin immunoprecipitation, defining the biochemical lesions in G1 and G2 cell cycle mechanisms that result from loss of c-Myc activity, and genetically testing the physiological relevance of the observed biochemical lesions. Mechanisms of Replicative Senescence in Human Cells Two fundamentally different aging phenomena have been described at the cellular level: 1) the gradual decline of life processes in postmitotic cells, and, 2) the decline and eventual complete cessation of cell division observed in most replicating cell lineages. The former is measured in simple chronological time, and comes into play during the aging of postmitotic adult organisms, such as the nematode, or during the aging of largely postmitotic tissues such as the brain or muscle in more complex organisms. In contrast, finite replicative lifespan, often referred to as 'cellular replicative senescence', is measured in terms of cell divisions rather than chronological time. The current consensus is that both postmitotic and replicative aging processes are causally related to the aging of humans. The topic of this research project is the molecular mechanism of replicative aging processes, specifically, the molecular machine that actually executes and maintains senescence. The major tool is targeted homologous recombination (gene targeting), which is being performed in normal (nonimmortalized) fibroblastic human cell strains. The targets of gene targeting are the following genes: the tumor suppressors p53 and retinoblastoma (Rb), and the Cdk kinase inhibitors p16INK4A, p19ARF, and p21CIP1/WAF1. The objective is to ablate gene action and subsequently investigate the resultant senescence phenotypes on both the cellular and molecular levels. This direct interventive approach is expected to reveal the functionally relevant components of the molecular senescence machine. The project is based on a model which predicts that the molecular machine that establishes senescence is composed of components that also play roles in cell cycle control during the normal proliferative lifespan of the cell, specifically, the p53-p21 and p16-Rb pathways. All experiments are being performed in normal human cells grown in in vitro cell culture. This is because a large body of evidence indicates that the regulation of replicative senescence mechanisms is significantly different in humans and in rodents. Therefore, due to the ethical unacceptability of experimentally altering the human germ line, and the limited utility of the rodent model to address the specific issues under investigation, the whole-organism transgenic route is not appropriate and the experimental model has been confined to human somatic cell culture. |
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Selected Publications:
Sedivy, J.M. and Joyner, A. (1992). Gene Targeting. W.H. Freeman Press,
NY |
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