Flow cytometric analysis of MDM2-mediated growth arrest.
Although MDM2, the product of mouse double minute-2 (mdm2) gene, or its human homologue possesses the potential to confer tumorigenic properties, it induces G1/S arrest in nontransformed cells. Flow cytometry provides a way to determine the effects of MDM2 on the cell cycle by expressing the protein ectopically, immunostaining cells expressing MDM2 and analyzing their DNA content. The DNA histograms of MDM2-transfected and untransfected cells can then be used to visualize the effect of ectopically expressed MDM2 on the cell cycle. Fluorescence-activated cell sorter (FACS) analysis following bromodeoxyuridine (BrdU) incorporation can be used to determine whether MDM2-expressing cells are synthesizing DNA. Incorporation of BrdU during DNA synthesis or repair can be detected in partially denatured DNA with a BrdU-specific fluorescent antibody. Subsequent staining of transfected MDM2 with a different fluorochrome provides information about whether transfected cells make significant progression through S phase. Further analysis of the growth-regulatory properties of MDM2 will elucidate both its normal function and the ways in which its deregulation leads to tumorigenesis.
Frum
Rebecca
R
Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, USA.
Deb
Swati Palit
SP
D000818:Animals; D000963:Antimetabolites / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006131:Growth Inhibitors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2
D000963:Antimetabolites; D006131:Growth Inhibitors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D004247:DNA; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D001973:Bromodeoxyuridine