The MDR1/ABCB1 regional amplification in large inverted repeats with asymmetric sequences and microhomologies at the junction sites.
A multidrug-resistant lung cancer cell line PTX250, established by treatment with the anti-cancer drug paclitaxel, has been demonstrated to have an increased copy number in the 7q21.12 region including the MDR1/ABCB1 gene. The amplicon is 2.7 megabases in size, and the copy number increase is 11-fold compared with the parental cell line. Here, we examined the amplicon structure and determined nucleotide sequences at both junctions of the amplicon. Fluorescence in situ hybridization analysis using an MDR1 probe demonstrated a cluster of fluorescent signals at the chromosomal end, suggesting an intra-chromosomal amplification. DNA fragments of both junctions were cloned and sequenced. The distal junction was a head-to-head fusion with a 4-base pair (bp) overlap separated by an asymmetric sequence of 1,265 bp, and the proximal junction was a tail-to-tail fusion with a 2-bp overlap intervened by an asymmetric sequence of 2,203 bp. These results suggest that the amplicon has a large palindromic structure with an asymmetric sequence and has been amplified through the breakage-fusion-bridge cycle. Specific sequences, which might be related to the occurrence of double-strand-breakages, were found at or near the junctions of the amplicon -- an inverted repeat in the distal junction and a highly AT-rich region near the proximal junction.
Kitada
Kunio
K
Kamakura Research Laboratories, Chugai Pharmaceutical Co Ltd, 200-Kajiwara, Kamakura, Kanagawa, Japan. kitadakno@chugai-pharm.co.jp
Yamasaki
Tomoaki
T
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D045744:Cell Line, Tumor; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D017931:DNA Primers; D018432:Drug Resistance, Multiple; D005784:Gene Amplification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D017239:Paclitaxel / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers; D017239:Paclitaxel