Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL.
Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P-glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug-selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5' to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to "capture" of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)-derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin-selected resistant colon cancer line (S48-3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC-derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500-1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48-3s/ActD), a hybrid MDR1 gene composed of heme oxygenase-2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel-selected MCF-7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC-derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1 (at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC-derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500-1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance.
Knutsen
T
T
Medicine Branch, Division of Clinical Sciences, NCI, NIH, Bethesda, Maryland 20892, USA. turidk@Box-t.nih.gov
Mickley
L A
LA
Ried
T
T
Green
E D
ED
du Manoir
S
S
Schröck
E
E
Macville
M
M
Ning
Y
Y
Robey
R
R
Polymeropoulos
M
M
Torres
R
R
Fojo
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002869:Chromosome Aberrations; D025063:Chromosome Disorders; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics*; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D015321:Gene Rearrangement; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D003609:Dactinomycin; D004317:Doxorubicin