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.

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 Pglycoprotein, 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 highvoltage 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-ChR-8-5-1 1, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-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.

A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.

“…The amplification unit in the KB-C1 cell line was determ i n e d as a discrete 890-kb region encompassing the PGY1 and PGY3 genes. This is consistent with the results reported by Schoenlein et al (1992). The amplification unit in the Kst-V500 cell line spans >1.5 Mb, and the middle of the unit, near the PGY1 gene, was amplified more t h a n the end of the unit.…”

“…Figure 5 summarizes the estimation of the amplification unit. The amplification unit of the MDR gene region in the KB-C1 cell was determined to be delimited in a 700-to 1300-kb range, consistent with Southern blot results on PFGE analyses of cellular DNA digested with NotI (890 kb; data not shown), as well as with results reported by Schoenlein et al (1992). The amplification ratio of KB-C1 to parental KB is uniform (16.4 times) across the amplified region.…”

“…The amplification unit in the Kst-V500 cell line spans >1.5 Mb, and the middle of the unit, near the PGY1 gene, was amplified more t h a n the end of the unit. DMs are often observed in KB-C4 cells, which were isolated by stepwise selection to further increasing doses of colchicine from KB-C1 cells (Fojo et al 1985;Schoenlein et al 1992). Although DMs have not been observed in the KB-C1 cells, the data reported by Schoenlein et al (1992) Figure 4 Southern blot analysis of the DNA isolated from drug-resistant cell lines.…”

“…DMs are often observed in KB-C4 cells, which were isolated by stepwise selection to further increasing doses of colchicine from KB-C1 cells (Fojo et al 1985;Schoenlein et al 1992). Although DMs have not been observed in the KB-C1 cells, the data reported by Schoenlein et al (1992) Figure 4 Southern blot analysis of the DNA isolated from drug-resistant cell lines. Genomic DNAs were isolated from KB-C1 and Kst-V500 and their parental cell lines, KB and Kst-6, respectively, and subjected to Southern blot analysis as described in Methods.…”

“…4) and the amplification unit in each cell lines were estimated by the relative amplification ratio of the drug resistant cell lines to the parental cell lines according to the calculation described in Methods. The KB-C1 cell line was originally isolated as a colchicine-resistant cell, and the MDR1 region was amplified in an extrachromosomal DNA state (Akiyama et al 1985;Fojo et al 1985;Schoenlein et al 1992 Table 1). …”