The LRP Gene Encoding a Major Vault Protein Associated ...cancerres.aacrjournals.org/content/canres/55/19/4214.full.pdf · When necessary, the slides were destained prior to FISH

(CANCER RESEARCH 55, 4214-4219, October 1, 1995]

Advances in Brief

The LRP Gene Encoding a Major Vault Protein Associated with Drug ResistanceMaps Proximal to MRP on Chromosome 16: Evidence That ChromosomeBreakage Plays a Key Role in MRP or LRP Gene Amplification1

Marilyn L. Slovak,2 Jennifer Pelkey Ho, Susan P. C. Cole, Roger G. Deeley, Lee Greenberger,

Elisabeth G. E. de Vries, Henricus J. Broxterman, George L. Scheffer, and Rik J. ScheperDepartment of Cytoge netics, City Of Hope National Medical Center, Duarte, California 91010 [M. L S., J. P. H.Â¡,Cancer Research Laborlories, Queen's University, Kingston,

Ontario K7L 3N6, Canada IS. P. C. C., R. G. D.I: Wyeth-Ayerst Research, Pearl River, New York 10965 [L. G.]: University Hospital Groningen, 9700 RB Groningen, theNetherlands Â¡E.G. E. d. V.Â¡;and Free University Hospital, 1081 HB Amsterdam, the Netherlands Â¡H.J. B., G. L. S., R. J. S.]

Abstract

A cDNA encoding the novel drug resistance gene, IMI' (originally

termed lung resistance-related protein), was isolated from HT1080/DR4, a220-fold doxorubicin-resistant human fibrosarcoma cell line which dis

plays a multidrug resistance phenotype and overexpresses the multidrugresistance protein (MRP) but does not overexpress P-glycoprotein encoded by the MDR1 gene. Using the full-length 2.8-kb cDNA probe, thegene for LRP was regionally localized to the 16pl3.1-16pll.2 chromo

somal segment in human metaphases. Dual color fluorescence in situhybridization studies refined the localization of LRP to Iftp11.2. a locationapproximately 27 cM proximal to MRP (16pl3.1). Two color hybridization studies indicated that HT10SO/DR4 fibrosarcoma cells contain amplification of both the MRP and LRP genes in a striking striped pattern inthe homogeneously staining region, hsr(7)(pl2pl5). In contrast, only amplified MRP gene sequences were contained within the homogeneouslystaining region, hsr(18q). Amplification of LRP was not identified in anyof seven other drug-resistant tumor cell lines characterized by 20-300-

fold levels of doxorubicin resistance, including two cell lines known tooverexpress LRP (SW1573/2R120 and GLC4/ADR). Amplified MRP genesequences were identified in H69AR, GLC4/ADR, and HL-60/AR whereas

only MDR1 gene amplification was observed in the S1B120 colon carcinoma cell line. These data indicate that although both the MRP and LRPgenes map to the short arm of chromosome 16, they are rarely coamplifiedand are not normally located within the same amplicon. A key role forchromosome breakage in gene amplification is supported by the presenceof non-random karyotypic anomalies near the MRP and LRP normal

cellular loci.

Introduction

Immunohistochemistry using the pllO-specific LRP3-56 mAb has

detected overexpression of a drug resistance-associated protein referred to as LRP in a number of human multidrug-resistant tumor cell

lines derived from various types of malignancy (e.g., lung and breastcancer, fibrosarcoma, and myeloma; Ref. l). Two clinical investigations suggest that LRP overexpression in acute myeloid leukemia andadvanced ovarian carcinoma is associated with an unfavorable prog-

Received 6/29/95; accepted 8/17/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a grant from the Medical Research Council of

Canada (S. P. C. C., R. G. D.). M. L. S. is a member of the City of Hope Cancer ResearchCenter, which is supported by Public Health Service Grant CA-33572. S. P. C. C. is aCareer Scientist of the Ontario Cancer Foundation and R. G. D. is the Stauffer ResearchProfessor (Queen's University, Kingston, Ontario, Canada).

2 To whom requests for reprints should be addressed, at City of Hope National Medical

Center, Department of Cytogenetics, Northwest Building, Room 2255, 1500 East DuarteRoad, Duarte, CA 91010-3000.

3 The abbreviations used are: LRP, lung resistance-related protein; MRP, multidrug

resistance protein; FISH, fluorescence in situ hybridization; hsr, homogeneously stainingregion; GTG, Giemsa-trypsin; dmin, double minute chromosomes.

nostic outcome (2, 3); however, whether LRP actually contributes toclinical resistance is unknown. Recently, a cDNA coding for the LRPgene was isolated from the 220-fold doxorubicin-resistant human

fibrosarcoma cell line HT1080/DR4 (4, 5). Comparative sequenceanalysis indicates that LRP shares significant amino acid homologywith the Mr 104,000 rat major vault protein (4). Vaults are multi-subunit structures involved in nucleo-cytoplasmic transport (6), a

finding which suggests that LRP may be involved in the transport ofcytotoxic agents (4). In this study, we mapped LRP in relation to theATP-binding cassette transmembrane MRP gene previously shown tolocalize to the short arm of chromosome 16 (7-9). To determinewhether non-random cytogenetic alterations were associated with

amplification of either LRP or MRP, we performed sequential cytogenetic and FISH analyses using a panel of seven multidrug-resistant

cell lines.

Materials and Methods

Cell Lines. The six parental and seven drug-resistant cell lines used in thisstudy have been described previously (5, 7, 8, 10-15). HT1080/DR4, H69/AR,HL-60/AR, GLC4/ADR, and SW1573/2R120 were selected in the presence ofdoxorubicin and do not overexpress P-glycoprotein but do overexpress MRP.

The S1B120 cell line was selected in the presence of bisantrene and is~300-fold resistant to doxorubicin (14). The mitoxantrone-resistant S1-M1-3.2 is ~44-fold cross-resistant to doxorubicin, although the mechanism(s) of

drug resistance remains unknown. HL-60 and HL-60/AR were kindly provided

by Dr. K. Bhalla (Medical University of South Carolina, Charleston, SC).Classical Cytogenetics. Cytogenetic analyses were performed according to

the standard method using 50 /ig/ml Colcemid for 45 min before cell harvesting. GTG banding was used to identify the individual chromosomes, and thechromosomes were classified according to the 1991 International System forHuman Cytogenetic Nomenclature (16). When necessary, the slides weredestained prior to FISH analysis.

FISH Analysis. Plasmid DNA isolated from three clones containing LRPcDNA inserts (2.8 kb) was pooled and labeled by nick translation with eitherbiotin-16-dUTP or digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN). Similarly, a 4.0-kb fragment of the MRP cDNA (7) and the pMDRVl

5A MDRI probe (kindly provided by Dr. Igor Roninson) were labeled by nicktranslation. FISH was performed using standard methodology. Labeled probeswere mixed with blocking DNA, ethanol precipitated, and resuspended inHybrisol VII (Oncor, Gaithersburg, MD). Pretreatment, denaturation, hybridization, and detection of the labeled probes were performed per manufacturer's

instructions for in situ detection of single-copy sequences in metaphase chro

mosomes. Signal detection and amplification were performed for biotin-la-beled probes using FITC-avidin and anti-avidin antibody from Oncor. Digoxi-genin-labeled probes were detected with rhodamine-tagged antidigoxigenin

antibody (Oncor), rabbit antisheep, and rhodomine-conjugated antirabbit anti

body. Slides with metaphase spreads were prepared from seven cell lines usingstandard cytogenetic procedures. Sequential GTG/FISH studies were used toidentify the specific chromosomal bands harboring amplified gene sequences.

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UP LOCALIZATION AND GENETIC CHARACTERIZATION

When appropriate, a FITC-labeled 16q24-qter control probe (Oncor, P5432)

was used to identify the long arm of chromosome 16.Slides were examined on a Nikon/Labophot II microscope equipped with

epifluorescence and multiple band pass filters. The Oncor Imaging Systemfacilitated fluorescence photography, and the Adobe Photoshop program wasused to enhance the clarity, brightness, and contrast of some images.

Results

By conventional in situ hybridization, we regionally mapped thenative locus of the LRP gene to the proximal short arm of chromosome 16 (4). To refine these data and define the genetic distancebetween the LRP gene and the MRP gene previously localized to16pl3.1 (7, 8), sequential GTG-banded/FISH studies were performed

using metaphases from normal human lymphocytes. Examination ofnormal lymphocyte metaphase chromosomes indicates that the nativecellular locus for LRP is in close proximity to the centromere ofchromosome 16 (Fig. IA). Fig. 2 exhibits a partial normal lymphocytemetaphase which was first GTG banded to identify the chromosome16s followed by FISH analysis. Hybridization with the FITC-labeledLRP and 16q24-qter probes, the latter used as a reference probe for the

long arm of chromosome 16, localized LRP to band 16pll.2. Dualcolor hybridization experiments with normal human lymphocytesusing the 2.8-kb LRP and the 4.0-kb MRP cDNA probes confirmed

the cytogenetic position of LRP to 16pl 1.2 and MRP to 16pl3.1 (Fig.10). Using the PCR-based linkage map and the sex-averaged framework map of human chromosome 16 (17), our molecular cytogenetic-

based mapping data approximates the genetic distance between thesetwo genes as 27 cM or an estimated 18 megabases.

To identify the karyotypic alterations associated with amplificationof the LRP and MRP gene sequences, we carried out cytogenetic andFISH analyses on seven multidrug-resistant cell lines and their cor

responding parental cell lines (Table 1). In all parental lines tested,LRP and MRP mapped to their native sites (16pll.2 and 16pl3.1,respectively). In the 222-fold doxorubicin-resistant HT1080/DR4 cell

line, double hybridization experiments using LRP and MRP cDNAprobes labeled with biotin or digoxigenin, respectively, exhibitedsignals at the 16p cellular loci and a striped amplification pattern inthe hsr(7) for both LRP and MRP genes, indicating that coamplifica-

tion had occurred (Fig. 1C). A breakpoint in the short arm of chromosome 16 at band 16pll.2 was observed by standard cytogenetics(Fig. 3A), however, a rearrangement at or near 16pl3.1 could not beidentified due to the complexity of the karyotype. Interestingly, amplified MRP gene sequences without coamplification of the LRP locuswere localized to a second site, a derivative chromosome 18, inHT1080/DR4 cells (Fig. 1C).

In a previous study, we showed that the H69AR small cell lungcarcinoma cell line is characterized by a near-triploid karyotype with

multiple numerical and structural abnormalities and cytogenetic evidence of gene amplification in the form of hsrs and dmins (8).Amplification of MRP gene sequences was observed in the hsrs,derivative chromosome 7, and some of the dmins in the 54-folddoxorubicin-resistant H69AR cell line. Interestingly, a derivative

chromosome 16 with a 16pl3.1 breakpoint was observed in H69AR(Fig. 35), a finding not present in the parental H69 cells. LRP mappedonly to the normal cellular locus (16pll.2) in both the parental andH69AR cells.

SW1573/2R120 is a 20-fold doxorubicin-resistant non-small celllung cancer cell line used as antigen to produce the LRP-56 mAb (1).

This cell line and the small cell lung carcinoma cell line GLC4/ADRboth overexpress LRP (1). LRP hybridization signals were observedonly at the normal locus for the LRP gene in these doxorubicin-

resistant cell lines (Table 1 and Fig. l, D and Â£), indicating thatincreased expression of LRP may be by a mechanism other than gene

amplification. In both SW1573 parental and SW1573/2R120 cells, thetwo chromosome 16 homologues exhibited a slight difference in theposition of the MRP locus by FISH, an observation consistent with asmall inversion in the short arm identified by GTG banding asÂ¡nv(16)(pl2pl3.1) (Fig. 3C). Both intrachromosomal (hsr) and extra-

chromosomal (dmins) amplified MRP gene sequences were identifiedin the 123-fold doxorubicin-resistant GLC4/ADR cell line (Fig. IF).

This dual gene amplification pattern was observed in both metaphaseand interphase cells by either a diffuse dmin amplification pattern ora discrete domain pattern consistent with integrated MRP gene sequences within a hsr (Fig. IG). Similarly, different cytological patterns of amplified MRP gene sequences were also reported by Eijdemset al. (18). MRP amplification was not evident in either SW1573parental or the 20-fold SW1573/2R120; however, SW1573/2R120

does overexpress MRP (1) and is characterized by a derivative chromosome 16 with a breakpoint within band 16pl3.1 (Fig. 3D).

Because overexpression of LRP appears to have prognostic significance in the acute myeloid leukemias (2), the HL-60 leukemia cellline and its multidrug-resistant, non-P-glycoprotein, MRP-overex-pressing HL-60/AR derivative were chosen as part of the LRP screen

ing panel. Again, the LRP gene mapped only to 16pll.2 in both theparental and the 100-fold doxorubicin-resistant HL-60 cell lines (Fig.

I//). Moreover, MRP gene sequences mapped to 16pl3.1 in theparental HL-60 cell line and to the two variant forms of thehsr(7)(ql 1.2) in the HL-60/AR cell line (Fig. 1, / and J). An augmen

tation on the short (p) arm of chromosome 16 at band 16pl3.1 wasobserved in HL-60/AR cells (Fig. 3Â£)but was absent in the parentalcells, suggesting that a chromosome breakage event due to doxoru-bicin exposure in HL-60/AR cells may be causally related to the

resistant phenotype.The parental colon carcinoma cell line SI and its two resistant

derivatives, the bisantrene-selected S1-B1-20 cell line (~300-foldcross-resistant to doxorubicin) and the mitoxantrone-selected S1-M1-3.2 cell line (44-fold cross-resistant to doxorubicin), also exhibited

hybridization of LRP and MRP at the normal cellular loci only (Fig.l, K and L). In S1-B1-20 cells, amplified MDRl gene sequences were

localized to an insertion in the long arm of chromosome 8 at band8q22 (14). The karyotypes of the resistant SI sublines were notcharacterized by either abnormalities of chromosome 16 or amplification of either LRP or MRP, despite the presence of dmins in 20% ofthe metaphases analyzed in S1-M1-3.2 cells.

Discussion

In this study, we have mapped the LRP gene to the short arm ofchromosome 16 at band 16pll.2 approximately 27 cM proximal toMRP. LRP is near, and possibly within, the region that contains thechromosome 16 low-abundance repetitive DNA sequences

(CH16LAR) also localized to band 16pll.2 (19). A single hybridization site at 16pll.2 in all six parental and six of the seven drug-

resistant cell lines provides strong evidence that the LRP gene encodesa unique sequence and not part of the low-abundance repeat

CH16LAR sequences found within this chromosome band.In four multidrug-resistant cell lines, overexpression of MRP was

associated with gene amplification and a cytogenetic rearrangement ator near the normal cellular locus. The cytogenetic breakage eventsinvolved unbalanced translocations, inversions, and additions to theshort arm of chromosome 16. Taken together with the fact thatcomplex karyotypes are commonly observed in cell lines that exhibitamplification of drug resistance-related genes, these data suggest that

chromosome breakage plays a central role in the generation of amplified gene sequences.

Coamplification of MRP and LRP gene sequences was identified in

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LRP LOCALIZATION AND GENETIC CHARACTERIZATION

Fig. I. FISH analysis using the 2.8-kb cDNA LRP probe and/or the 4.0-kb cDNA MRP probe. A. LRP maps to I6pl 1.2 as a unique sequence in a normal lymphocyte metaphase.fi, dual color hybridization confirms that Â£/?P(redsignal at 16pl 1.2) maps proximal to MRP (yellow signal at 16pl3.1) on the short arm of chromosome 16. C, dual color hybridizationidentifies a striped pattern of alternating LRP (yellow) and MRP (red) gene sequencesin the hsr(7) of HTI080/DR4 cells (arrow). White arrowhead, derivative chromosome 18 whichcontains amplified MRP gene sequencesonly. A normal chromosome In (yellow arrowhead) exhibits hybridization at the LHP and MRP native sites. D. LRP maps to the normal cellularlocus in the 20-fold doxorubicin-resistant SW1573/2RI20 cell line. E, LRP maps only to the native site Ã¬nGLC4/ADR cells, f, amplified MRP gene sequencesare integrated into ahsr-bearing marker chromosome (arrow, bottom and lop tnetaphase) or localized to dmins (bottom metaphase) in GLC4/ADR cells. G. interphase FISH of GLC4/ADR exhibits twodifferent cytological patterns of MRP amplification: diffuse amplification pattern consistent with dmin MRP localization (larger cell) and discrete domains consistent with integrated(intrachromosomal) MRP sequenceswithin a hsr (smaller cell). H. LRP maps only to the native site in HL-60/AR cells. /. MRP mapped to 16pI3.1 in HL-60/AR cells (arrowhead)and to the hsr(7)(q] 1.2) (arrow) observed in one clone of these resistant cells. GTG-banded hsr(7) with bracket indicating hsr region is found on the left. J, MRP mapped to 16pl3.1in HL-60/AR cells (arrowhead) and to the variant hsr described per International System for Human Cylogenetic Nomenclature as ins(7)(ql 1.2) (arrow) observed in the related cloneof HL-60/AR. GTG-banded ins(7) with bracket indicating hsr region is to the right of the arrow. K. SI-M 1-3.2 exhibits native site hybridization of MRP. L. SI-M 1-3.2 exhibits nativesite hybridization of LRP. There was no hybridization to the dmins observed in 20% of the S1-M1-3.2 cells (data not shown).

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Fig. 2. Sequential GTG-handed/FISH normallymphocyte (partial) metaphase. A, GTG handingidentifies the two normal chromosome 16 homologues (arrows). B, same metaphase is destainedand analyzed by FISH using a FITC-laheled LRPprobe and a 16q24-qter reference probe for thetelomeric region of the long arm of chromosome16. LRP maps to band 1ftp 11.2.

Table 1 Muliulrug-resistanl cellline*Cell

lineOriginHT1080

FibrosarcomaHT1080/DR4H69

SCLC*H69/ARSW1573

Non-SCLCSW1573/2R120GLC4

SCLCGLC4/ADRHL-60

AMLHL-60/ARSI

ColoncarcinomaSI-B1-20S1-M1-3.2Level

of doxorubicinresistance"122215412011231IIIe1306''44"Cylogeneticfeaturesadd(16)(pll.2)hsr(7)(pl2pl5)hsr(18)(q?)dmin2-4

hsrshsr(7)(p22)add(lo)(pl3.1)inv(16)(pl2pl3.l)ÃŒnv(l6)(pl2pl3.1)complex;

nodmin/hsrdmin/hsrder(16)t(5;16)(ql3;pl3.1)hsr(7)(qll.2)add(16)(pl2-13)hsr(8)(q22)dmin

(20%)Gene

amplificationOverexpressionLRP.

MRP l.RP.MRPMRP

MRPLRP,

MRPMRP

LRP.MRPMRP

MRPMORI

MDR1efReference(1.8)(7.

8.10)(1)(11.18)(13)(14)

" Level of drug resistance as reported in Ref. 20 unless otherwise noted, b Level of resistance reported in reference #12. c Level of resistance as reported in reference #14 and

Greenberger. personal communication.'SCLC. small cell lung carcinoma; AML. acute myclogcnous leukemia.' Level of resistance reported in Ref. 12.' Level of resistance as reported in Ref. 14 and L. Greenberger, personal communication.' Unknown.

only one (HT1080/DR4) of seven drug-resistant cell lines with doxo-rubicin-resistant levels ranging from 20- to 300-fold (12, 14, 20). The

lack of LRP amplification in four resistant cell lines with MRPamplification suggests that MRP and LRP do not normally residewithin the same amplicon. Furthermore, overexpression of LRP wasnot associated with gene amplification in GLC4/ADR and SW1573/2R120 cells, suggesting that overexpression of LRP by gene amplification is not a common occurrence. Other mechanisms such astranscriptional activation or mRNA stabilization appear more likely tobe responsible for overexpression of this gene.

HT1080/DR4 fibrosarcoma cells exhibited amplified MRP and LRPgene sequences in a similar striped or ladder pattern within the hsr(7p)but only MRP amplified gene sequences were found in the hsr(18q).The unbalanced translocation, der(16)t(16;?)(pl 1.2;?), observed inthese cells suggests that the 16pll.2 breakpoint may have been thecritical chromosomal breakage step needed to generate acentric am-

plicons prior to gene amplification (21). The presence of repetitive

sequences within chromosomal bands 16pl 1.2 and 16pl3.1, adjacentto the LRP and MRP genes, and the fact that two relatively distantmarkers may be brought closer together by an inverted duplicationevent (22) suggests that the genetic composition near these locicontributes to heterogeneous amplification organization. In support ofthis model, Eijdems et al. (18) described different but related chromosome 16-specific and repetitive sequence compositions ranging in1-2 megabases between the dmins and the hsr in GLC4/ADR. This

gene amplification model implies that the initiation of amplification isregulated by a mechanism defined by highly recombinogenic sequences (19) flanking the genes of interest followed by further alterations which delete the nonesscntial sequences, resulting in the formation of advantageous amplicons (23).

To this end, our previous microdissection studies of HT1080/DR4cells indicated that the complex MRP amplicon in these cells iscomposed of chromosomal material from four distinct chromosomalsegments: 16pl 1.2->16pl3.1, 2pll.2, 7q32-7q34, and 10q22 (24). In

4217




!A^DfÂ§BtEjfUCIIF

Fig. 3. Nonrandom chromosome 16 karyotypic alterations in drug-resistant cell lines.

A, add(16)(pll.2) in HT1080/DR4 cells; B, add(16)(pl3.I) in H69AR cells; C,inv(16)(pl2pl3.1) (arrow) in SW1573 parental and SW1573/2R120 cells. Normal chromosome 16 on left. D, der(16)t(5;16)(ql3;pl3.1) in GLC4/ADR cells; Â£,add(16)(pl2-pl3.1) in HL-60/AR cells; F. normal chromosome 16s from the S1-M1-3.2 cell line.

this cell line, the recombination of the atypical MRP/LRP ampliconmay have resulted in a complex secondary arrangement due to sequence homology of chromosome 16-specific repetitive sequences in

bands 16pll.2 and 16pl3.1 known to undergo breakage and rearrangement, and fragile site [2qll.2 (FRA2A), 7q32.3 (FRA7H),10q22.1(FRA10D)] involvement as observed in rearrangements offour different chromosomes, all of which may or may not be associated with drug resistance (24). This recombinogenic sequence hypothesis suggests that replication intermediates of unlinked loci couldaccumulate under limited growth conditions in association with multiple chromosomal alterations (21). Under stringent growth conditions, the formation of stable amplicons composed of gene sequencesessential or most advantageous for cell preservation takes place as alate event. This supposition is supported by the fact that a second site,a hsr(18q), contains amplified MRP gene sequences without coampli-

fication of LRP by FISH analysis. Chromosome microdissection studies are currently being designed to investigate the exact geneticcomposition of this hsr. Although these data do not provide insight onthe derivation of the amplified sequences, the dynamic nature of thesestructures suggests that late amplification events select for genescrucial to survival. Furthermore, this argument is supported by theobservation that the other four MÃ„f-amplified cell lines in our panel

did not contain amplified LRP sequences.Alternatively, a recombination event, assisted by repetitive se

quences known to exist at 16pl3.1 and 16pll.2, may be necessary forthe phenomenon which may ultimately result in amplification ofspecific gene sequences and cell preservation. This theory is attractivebecause the vault protein complex is composed of four gene productsin lower eukaroyotes (6), and presumably amplification of all fourgenes that make up the vault complex would be necessary to functionin drug resistance. However, amplification of all four gene productsthat comprise the vault complex seems to be an illogical strategy forcell survival. Nonetheless, activation of such a complex vault proteinsystem by transcriptional activation may be a frontline defense mechanism against cytotoxic substances and thus may be clinically relevantas has been suggested for acute myelogenous leukemia and ovariancarcinoma (2, 3). Chromosome microdissection and detailed physicalmapping studies are needed to determine the exact composition of theamplified gene sequences derived from the other chromosomal seg

ments in the hsr(7) of HT1080/DR4 cells and define their role, if any,in acquired drug resistance.

Previous studies have led to the contention that multiple mechanisms are responsible for acquired drug resistance. This has beenshown in various in vitro model systems either by sequential orsimultaneous coexpression of MDR1 and MRP (25), MDR1 and LRP(1, 26), or MRP and altered levels of DNA topoisomerase II with orwithout LRP (8, 27-29). Gene amplification and transcriptional acti

vation are the primary regulatory mechanisms responsible for theoverexpression of the drug resistance-associated genes. Thus, cell

preservation appears to occur by multiple routes depending on celltype, mutagenic exposure, and DNA repair processes. Whether over-expression of LRP by itself is essential for the drug-resistant pheno-

type or requires coamplification/assistance of other drug resistancegenes in HT1080/DR4 or other tumor cells requires further study. Totest these possibilities, the role of LRP in mediating the multidrugresistance phenotype must be supported by gene knockout/antisenseexperiments in which LRP expression is suppressed and resistance isreversed.

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1995;55:4214-4219. Cancer Res Marilyn L. Slovak, Jennifer Pelkey Ho, Susan P. C. Cole, et al.

Gene AmplificationLRPor MRPEvidence That Chromosome Breakage Plays a Key Role in

on Chromosome 16:MRPDrug Resistance Maps Proximal to Gene Encoding a Major Vault Protein Associated withLRPThe

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The LRP Gene Encoding a Major Vault Protein Associated ...cancerres.aacrjournals.org/content/canres/55/19/4214.full.pdf · When necessary, the slides were destained prior to FISH