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...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 234; p. 257
Main Authors Frum, Rebecca, Deb, Swati Palit
Format Journal Article
LanguageEnglish
Published United States 2003
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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.
AbstractList 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.
Author Frum, Rebecca
Deb, Swati Palit
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Snippet Although MDM2, the product of mouse double minute-2 (mdm2) gene, or its human homologue possesses the potential to confer tumorigenic properties, it induces...
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StartPage 257
SubjectTerms 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
Title Flow cytometric analysis of MDM2-mediated growth arrest
URI https://www.ncbi.nlm.nih.gov/pubmed/12824538
Volume 234
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