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.