Preferential expression of a mutant allele of the amplified MDR1 (ABCB1) gene in drug‐resistant variants of a human sarcoma

Abstract

Activation of the MDR1 (ABCB1) gene is a common event conferring multidrug resistance (MDR) in human cancers. We investigated MDR1 activation in MDR variants of a human sarcoma line, some of which express a mutant MDR1, which facilitated the study of allelic gene expression. Structural alterations of MDR1, gene copy numbers, and allelic expression were analyzed by cytogenetic karyotyping, oligonucleotide hybridization, Southern blotting, polymerase chain reaction, and DNA heteroduplex assays. Both chromosome 7 alterations and several cytogenetic changes involving the 7q21 locus are associated with the development of MDR in these sarcoma cells. Multistep-selected cells and their revertants contain three- to six-fold MDR1 gene amplification compared with that of the drug-sensitive parental cell line MES-SA and single-step doxorubicin-selected mutants. MDR1 gene amplification precedes the emergence of a mutant allele in cells that were coselected with doxorubicin and a cyclosporin inhibitor of P-glycoprotein (P-gp). Allele-specific oligonucleotide hybridization showed that the endogenous mutant allele was present as a single copy, with multiple copies of the normal allele. Reselection of revertant cells with doxorubicin in either the presence or the absence of the P-gp inhibitor resulted in exclusive reexpression of the mutant MDR1 allele, regardless of the presence of multiple wild-type MDR1 alleles. These data provide new insights into how multiple alleles are regulated in the amplicon of drug-resistant cancer cells and indicate that increased expression of an amplified gene can result from selective transcription of a single mutant allele of the gene.

1p31, 7q21 and 18q21 chromosomal aberrations and candidate genes in acquired vinblastine resistance of human cervical carcinoma KB cells

“…It is well recognized that resistant cells with genetic alterations possessing a growth or survival advantage would lead to clonal expansion. Chromosomal 7q21 has also been reported in previous publications of MDR cancers (17)(18)(19). This is particularly helpful for MDR candidate evaluation.…”

The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance

“…Gene amplification is also a potential mechanism for increased ABCB1 transcription, given previously published findings using a variety of drug-resistant cancer cell lines. [20][21][22][23] No ABCB1 gene amplification could be detected in MCF-7 EPI and MCF-7 TAX-2 cells (relative to MCF-7 CC cells) when assessed by Q-PCR experiments using genomic DNA and ABCB1-specific primers (Figure 2). This lack of ABCB1 amplification was further confirmed by FISH studies (Figure 2).…”

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

“…Induction of DNA damage response genes such as p21 were obtained by doxorubicin in breast cancer cell lines (Troester et al, 2004). Both chromosome 7 alterations and several cytogenetic changes involving the 7q21 locus are associated with the development of MDR in sarcoma cells (Chen et al, 2002). Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired doxorubicin resistance including ABCB1, PGY3 (ABCB4) and BAK (Turton et al, 2001).…”