Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon.
Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon. In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation. In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA. Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR). By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length. Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification. Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
E W
EW
Eijdems
M
M
De Haas
J M
JM
Coco-Martin
C P
CP
Ottenheim
G J
GJ
Zaman
H G
HG
Dauwerse
M H
MH
Breuning
P R
PR
Twentyman
P
P
Borst
F
F
Baas
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002885:Chromosomes, Human, Pair 16; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D005796:Genes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured / Q000187:drug effects
D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009218:Myosins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm