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Amplification of 4q21-q22 and the MXR gene in independently derived mitoxantrone-resistant cell lines
1Medicine Branch, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, MD 20892, USA. turidk@Box-t.nih.gov
Abstract:
Molecular cytogenetic studies were conducted on three multidrug-resistant cancer sublines which are highly resistant to the chemotherapeutic agent mitoxantrone, an anthracenedione. The three independently selected sublines were derived by exposure to mitoxantrone or Adriamycin and do not overexpress MDR1 or MRP. Two sublines, MCF-7 AdVp3000 and MCF-7 MX, showed an amplification peak at 4q21-q22, as demonstrated by comparative genomic hybridization (CGH), while the third, S1-M1-80, did not. FISH using a whole chromosome 4 paint demonstrated multiple rearrangements involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX, while S1-M1-80 contained only a simple reciprocal translocation. The parental cell lines had no chromosome 4 rearrangements and no copy number gain or amplification of chromosome 4. Spectral karyotyping (SKY) analysis revealed a balanced translocation, t(4;17)(q21-q22;p13) in S1-M1-80 and multiple clonal translocations involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX. A novel cDNA, designated MXR, which encodes an ABC half-transporter and is highly overexpressed in the three sublines, was localized to chromosome 4 by somatic cell hybrid analysis. Southern blot analysis demonstrated amplification of the MXR gene in MCF-7 AdVp3000 and MCF-7 MX, but not in S1-M1-80. FISH studies with a BAC probe for MXR localized the gene to 4q21-22 in the normal chromosome 4 and revealed in both MCF-7 AdVp3000 and MCF-7 MX amplification of MXR at one translocation juncture, shown by SKY to be t(4;5)(4qter-->4cen-->4q21-22::5q13-->5qter++ +) in MCF-7 AdVp3000 and t(6;4;6;3)(6pter-->6q15::4q21-q22::hsr::6q?::3q?27-->+ ++3qter) in MCF MX; neither of the breakpoints in the partner chromosomes showed amplification by CGH. The data are consistent with the hypothesis of a transporter, presumably that encoded by the MXR gene, mediating mitoxantrone resistance. The MXR gene encodes a half-transporter and the absence of cytogenetic evidence of coamplification of other regions suggests that a partner may not be overexpressed, and instead the MXR half-transporter homodimerizes to mediate drug transport. Genes Chromosomes Cancer 27:110-116, 2000. Published 2000 Wiley-Liss, Inc.
Insights
Multidrug-resistant cancer cells develop resistance to mitoxantrone via overexpression of the MXR gene, an ABC half-transporter. This transporter likely homodimerizes to mediate drug resistance, independent of MDR1 or MRP.
Area of Science:
- * Molecular cytogenetics
- * Cancer genetics
- * Drug resistance mechanisms
Background:
- * Multidrug resistance (MDR) is a major challenge in cancer chemotherapy.
- * The anthracycline agent mitoxantrone is used to treat various cancers.
- * Mechanisms of MDR, particularly those not involving MDR1 or MRP, require further investigation.
Purpose of the Study:
- * To investigate the molecular cytogenetic basis of mitoxantrone resistance in three human cancer sublines.
- * To identify novel genes or chromosomal alterations involved in this resistance.
- * To explore the role of a novel transporter, MXR, in mediating mitoxantrone resistance.
Main Methods:
- * Comparative Genomic Hybridization (CGH) to detect copy number changes.
- * Fluorescence In Situ Hybridization (FISH) for chromosomal rearrangements and gene localization.
- * Spectral Karyotyping (SKY) for detailed analysis of chromosomal translocations.
- * Somatic cell hybrid analysis to map gene location.
- * Southern blot analysis to assess gene amplification.
Main Results:
- * Two sublines (MCF-7 AdVp3000, MCF-7 MX) showed amplification at chromosome 4q21-q22, with multiple chromosome 4 rearrangements.
- * A third subline (S1-M1-80) had a simple reciprocal translocation involving chromosome 4.
- * A novel ABC half-transporter gene, MXR, was identified, overexpressed in all three sublines, and localized to chromosome 4.
- * MXR gene amplification was observed in MCF-7 AdVp3000 and MCF-7 MX, localized at translocation junctures.
- * The parental cell lines lacked these chromosomal abnormalities and MXR overexpression.
Conclusions:
- * The MXR gene, encoding an ABC half-transporter, is implicated in mitoxantrone resistance.
- * Amplification and overexpression of MXR, potentially through homodimerization, mediate drug resistance.
- * These findings highlight a novel mechanism of MDR independent of MDR1 and MRP.