John Sedivy
Department of Molecular and Cell Biology and Biochemistry
Ph.D., Harvard University, 1985
(401) 863-7631

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.

Selected Publications:

Sedivy, J.M. and Joyner, A. (1992). Gene Targeting. W.H. Freeman Press, NY
Karantza, V., Maroo, A., Fay, D. and Sedivy, J.M. (1993). Overproduction of Rb protein after the G1/S boundary causes G2 arrest. Mol. Cell. Biol. 13: 6640-6652.

Hanson, K.D., Shichiri, M., Follansbee, M.R. and Sedivy, J.M. (1994). Effects of c-myc expression on cell cycle progression. Mol. Cell. Biol. 14: 5748-5755.

Hanson, K.D., and Sedivy, J.M. (1995). Analysis of biological selections for high efficiency gene targeting. Mol. Cell. Biol. 15: 45-51.

Li, S., Janosch, P., Tanji, M., Rosenfeld, G.C., Waymire, J.C., Mischak, H., Kolch, W. and Sedivy, J.M. (1995). Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins. EMBO J., 14: 685-696.

Brown, J.P., Wei, W. and Sedivy, J.M. (1997). Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277: 831-834.

Mateyak, M.K., Obaya, A.J., Adachi, S. and Sedivy, J.M. (1997). Phenotypes of c-Myc-deficient fibroblasts isolated by targeted homologous recombination. Cell. Growth Diff. 8: 1039-1048.

Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J.P., Sedivy, J.M., Kinzler, K.W. and Vogelstein, B. (1998). The induction of p21 by p53 is required for sustained G2 arrest following DNA damage. Science 282: 1497-1501.

Bush, A., Mateyak, M.K., Dugan, K., Obaya, A., Adachi, S., Sedivy, J.M. and Cole, M.D. (1998). c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets. Genes Dev. 12: 3797-3802.

Xiao, Q., Claassen, G., Shi, J., Adachi, S., Sedivy, J.M. and Hann, S.R. (1998). Transactivation-defective c-MycS retains the ability to regulate growth and apoptosis. Genes Dev. 12: 3803-3808.

Sedivy, J.M., Vogelstein, B., Liber, H.L., Hendrickson, E. and Rosmarin, A. (1999). Gene targeting in human cells without isogenic DNA. Science 283: 9-9a
Wei, S., Wei, W. and Sedivy, J.M. (1999). Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts. Cancer Res. 59: 1539-1543.

Mateyak, M.K., Obaya, A.J. and Sedivy, J.M. (1999). c-Myc regulates cyclin D/Cdk4/6 activity but affects cell cycle progression at multiple independent steps. Mol. Cell. Biol. 19: 4672-4683.

Yeung, K.C., Seitz, T., Li, S., Janosch, P., McFerran, B., Kaiser, C., Fee, F., Katsanakis, K.D., Rose, D.W., Mischak, H., Sedivy, J.M. and Kolch, W. (1999). Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 401: 173-177.

Wei, W. and Sedivy, J.M. (1999). Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures. Exp. Cell Res. 253: 519-522.

Yeung, K.C, Janosch, P., McFerran, B., Rose, D.W., Mischak, H., Sedivy, J.M. and Kolch, W. (2000). The mechanism of suppression of the Raf/MEK/ERK pathway by the RKIP inhibitor protein. Mol. Cell. Biol. 20: 3079-3085.

Bazarov, A.V., Adachi, S., Li, S., Mateyak, M.K., Wei, S. and Sedivy, J.M. (2001). A modest reduction in c-Myc expression has minimal effects on cell growth and apoptosis but dramatically reduces susceptibility to Ras and Raf transformation. Cancer Res. 61: 1178-1186.

Adachi, S., Obaya, A.J., Han, Z., Ramos-Desimone, N., Wyche, J.H. and Sedivy, J.M. (2001). c-Myc is neces-sary for DNA damage-induced apoptosis in the G2 phase of the cell cycle. Mol. Cell. Biol. 21: 4929-4937.

Wei, W., Hemmer, R.M. and Sedivy, J.M. (2001). The role of p14ARF in replicative and induced senescence of human fibroblasts. Mol. Cell. Biol. 21: 6748-6757.

Yeung, K.C., Rose, D.W., Dhillon, A.S., Yaros, D., Gustafsson, M., Chatterjee, D., McFerran, B., Wyche, J., Kolch, W. and Sedivy, J.M. (2001). Raf kinase inhibitor protein interacts with NF- B-inducing kinase and TAK1 and inhibits NF- B activation. Mol Cell. Biol. 21: 7207-7217.

Obaya, A.J., Kotenko, I., Cole, M.D. and Sedivy, J.M. (2002). The protooncogene c-Myc acts through the cyclin-dependent kinase inhibitor p27Kip1 to facilitate the activation of cyclin-dependent kinase 4/6 and early G1 phase progression. J. Biol. Chem. 277:31263-31269.

Nikiforov, M.A., Chandriani, S., O'Connell, B., Petrenko, O., Kotenko, I., Beavis, A., Sedivy, J.M. and Cole, M.D. (2002). A functional screen for Myc-responsive genes reveals serinehydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism. Mol. Cell. Biol. 22: 5793-5800.

Schorl, C. and Sedivy, J.M. (2003). Loss of protooncogene c-Myc function impedes G1 phase progression both before and after the restriction point. Mol. Biol. Cell., in press.

O'Connell, B.C., Cheung, A.F., Simkevich, C.P., Tam, W., Ren, X., Mateyak, M.K. and Sedivy, J.M. (2003). A large scale genetic analysis of c-Myc-regulated gene expression patterns. J. Biol. Chem., in press.

Wei, W., Jobling, W.A., Chen, W., Hahn, W.C. and Sedivy, J.M. (2003). Abolition of cyclin-dependent kinase inhibitors p16Ink4a and p21Cip1/Waf1 functions permits Ras-induced anchorage-independent growth in telomerase-immortalized human fibroblasts. Mol. Cell. Biol., in press.

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