Flow cytometric analysis of MDM2-mediated growth arrest

Methods Mol Biol. 2003;234:257-67. doi: 10.1385/1-59259-408-5:257.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • Antimetabolites
  • Growth Inhibitors
  • Nuclear Proteins
  • Proto-Oncogene Proteins
  • DNA
  • MDM2 protein, human
  • Mdm2 protein, mouse
  • Proto-Oncogene Proteins c-mdm2
  • Bromodeoxyuridine