Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells

Patricia V Schoenlein, D. W. Shen, John Thomas Barrett, I. Pastan, M. M. Gottesman

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Abstract

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-Ch(R)-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-Ch(R)-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Original languageEnglish (US)
Pages (from-to)507-520
Number of pages14
JournalMolecular Biology of the Cell
Volume3
Issue number5
DOIs
StatePublished - Jan 1 1992

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KB Cells
Circular DNA
Colchicine
Human Chromosomes
Dimerization
Multiple Drug Resistance
MDR Genes
Carcinoma
Gene Amplification
Pulsed Field Gel Electrophoresis
Genes
Pharmaceutical Preparations
Cell Line
P-Glycoprotein
Metaphase
Electrophoresis
Digestion
Chromosomes
Gels
Cell Membrane

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-Ch(R)-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-Ch(R)-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.",
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