Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

We use two novel approaches to study thecontrol of cell-cycle progression andcell growth. Instead of synchronizingcells and performing biochemical analyses, as intraditional studies, we study actively growingcultures with time-lapse and quantitative imageanalytical approaches. Our results reveal importantaspects of cell-cycle control not previouslyobserved. Our goal is to extend these approachesand observations to the control of cell-cycleregulateddrug targets and to the analysis ofalterations in tumor cell-growth control.Cyclin D1 and the Cell CycleStudies in quiescent cells following growthstimulation have identified numerous moleculesand their interactions that are required to initiatecell-cycle progression. Unfortunately, loss ofsynchrony within such cultures prevents us fromanalyzing how the cell cycle continues andeventually terminates. Within actively proliferatingcultures, we can define the cell-cycle positionof individual cells, determining the molecularcharacteristics of each in relationship to the cellcycle.Our results reveal a critical, cell-cycleregulatedinteraction between cellular Ras andcyclin D1 proteins that controls the cell’s abilityto continue active proliferation. An importantclue was the fact that cyclin D1 is expressed athigh levels in actively dividing G1- and G2-phasecells, but at extremely low levels in S-phase cells(those actively synthesizing DNA). High levelsof cyclin D1 were strictly dependent uponcellular Ras activity in all cell-cycle phases, butthese levels could be stimulated by Ras activityonly during G2 phase. Thus, growth factorsstimulated by Ras activity induced an increase incyclin D1 levels in G2 phase. These levelsremained high through mitosis andG1 phase to promote the initiationof DNA synthesis; thereafter, cyclinD1 dropped to low levels.These facts form the basis forour model defining cyclin D1 as thecritical switch controlling ongoingcell growth. Cyclin D1 is needed toinitiate DNA synthesis; we haveshown this to be true also in activelycycling cells. Once the cell enters Sphase, however, cyclin D1 isautomatically suppressed to lowlevels. Levels remain low through Sphase, but on entering G2 phase, thecell must make a critical decision. Tocontinue proliferating, it must inducecyclin D1 levels during G2 phase.This requires the activity of growthfactors to stimulate cellular Rasactivity. If cyclin D1 levels areThe Department of Molecular BiologyProliferative Signal Transductionand Cell Cycle Regulationincreased, the cell becomes committed to passthrough not only the upcoming mitosis, but throughthe entire next cell cycle, completing the subsequentmitosis as well. But if conditions are not conducivefor continued cell-cycleprogression, cyclin D1 levelsare not induced during G2phase, and the cell entersquiescence immediatelyfollowing mitosis. CyclinD1 thus performs a switchfunction in the control ofcell-cycle progression. This“switch” automatically shutsoff during S phase, forcingthe cell to turn it on againduring G2 phase if the cell isto continue cycling.Topoisomerase II andDrug ToxicityThe technicalapproach utilized in thecell-cycle studies describedabove has also been appliedto the study of topoisomeraseII alpha, the targetof several anti-cancerdrugs. We have detailedthis molecule’s cell-cycleexpression characteristicsand the cell cycle’s role indetermining the cell’ssensitivity to the anti-topoisomerase II drugetoposide. We are determining the molecularcharacteristics of etoposide’s cell-cycle regulationin normal and tumor cells.THE STACEYLABORATORYINVESTIGATORSYang Guo, Ph.D.Masahiro Hitomi, M.D., D.M.S.Ke Yang, Ph.D.Dennis W. Stacey, Ph.D.Hitomi, M., and D.W. Stacey (1999) Cyclin D1 production in cycling cells depends on Ras in a cellcycle-specificmanner. Curr. Biol. 9:1075-1084.Stacey, D.W., Hitomi, M., and G. Chen (2000) Influence of cell cycle and oncogene activity upontopoisomerase IIalpha expression and drug toxicity. Mol. Cell. Biol. 20:9127-9137.Hitomi, M., and D.W. Stacey (2001) Ras-dependent cell cycle commitment during G2 phase. FEBSLett. 490:123-131.Sa, G., Hitomi, M., Harwalkar, J., Stacey, A.W., Chen, G.C., and D.W. Stacey (2002) Ras is activethroughout the cell cycle, but is able to induce cyclin D1 only during G2 phase. Cell Cycle1:50-58.Guo, Y., Stacey, D.W., and M. Hitomi (2002) Post-transcriptional regulation of cyclin D1 expressionduring G2 phase. Oncogene 21:7545-7556.Stacey, D.W. (2003) Cyclin D1 serves as a cell cycle regulatory switch in actively proliferatingcells. Curr. Opin. Cell Biol. 15:158-63.111