Flow cytometric analysis of MDM2-mediated growth arrest

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 234; p. 257
• Main Authors: Frum, Rebecca, Deb, Swati Palit
• Format: Journal Article
• Language: English
• Published: United States 2003
• Subjects: Animals; Antimetabolites - metabolism; Bromodeoxyuridine - metabolism; Cell Cycle - physiology; Cell Separation - methods; DNA - analysis; Flow Cytometry - methods; Growth Inhibitors - metabolism; Humans; Mice; Nuclear Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-mdm2
• ISSN: 1064-3745
• DOI: 10.1385/1-59259-408-5:257

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.