In estrogen receptor (ER)-positive breast cancer cell lines, very lowexpression of glutathione peroxldase-1 (GPX-1) activity and hgpxl mRNAhasbeenobserved.Suchcell lines havebeenusedasmodelsin studiesofresistanceto redoxcyclinganticancerdrugS.In particular,largeincreasesin GPX-1 activitylevelsby expressionof transfectedGPX-1 cDNA havebeen shown to confer some resistance to such drugs. It has never beendetermined that such low GPX-1 expression Is a common feature of breastcancer. Based on previous limited surveys of breast cancer cell lines, It hasbeen suggested that there may be an inverse correlation between ERstatus and GPX-1 production. Here we report the results from a largersurvey of breast cancer cell lines, including six recently isOlated cell lines.
A near absence of hgpxl mRNA expression was observed in 3 of 13ER-negativecelllines;1of 4 ER-positIvecelllineshadhighproductionofGPX-1.Both observationsweakenthe proposedinversecorrelationbetween ER status and GPX-1 production. We have evidence to suggest thatone cell line, COH-BR-5 (ER-negative), lacked hgpxl gene expressionprior to culture. This Is based on the finding of stable hgpxl gene expresslon during serial culture of ER-negative breast cancer cell lines newlyisolated from malignant effusion and absence ofhgpxl mRNA expressionin COH-BR-5. Expressionof hgpx2mRNA (producingGPXGI, the GItract GPX) was detected in several long and newly establlshed, ER
negative breast cancer cell lines. Cell lines, COH-BR-5 and MDA-MB-175,expressed only hgpx2 mRNA. The hgpx2 mRNA was detected in COHBR-5 and COH-BR-7 at low passage number, suggesting that hgpx2 geneexpression occurs In breast cancer malignant effusion. Thus, studies of therole of GPX in redoxdrug resistancemay accountfor changesIn hgpx2geneexpression.PhospholipidhydroperoxideGPX activitywasnotfoundto be generallyelevatedabovenormal tissuelevelsin newlyestablishedbreast cancer-derivedcell lines.
INTRODUCTION
Several perplexing points have arisen surrounding the expression ofselenium-dependentGPX3in breastcancer-derivedcell lines. Longestablished ER-positive breast tumor cell lines have been found to benearly devoid of selenium-dependent GPX activity due to a near lackof hgpxl mRNA expression(1). The ER-negativelines have beenreported to have much higher levels of GPX activity. These observations have been confirmed for two of the ER-positive lines (MCF-7and T-47D) under conditions of optimum selenium availability, whichstabilizes hgpxl mRNA in expressing cell lines and increases thesteady-statelevels of GPX-l several times over thatobtainedin mediawith 5—10%FBS (2—4).Expressionof the hgpxl mRNA in MCF-7
Received9/1/94;accepted12/15/94.The costsof publicationof this article were defrayed in part by the paymentof page
charges.This articlemustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicate this fact.
1 TIth work was supported by NIH Grant DK46921 (to F-F. C.) and Cancer Core Grant33572from the City of Hope.
2 To whom requests for reprints should be addressed, at Department of MedicalOncology,City of Hope National Medical Center,1500E. Duane Road,Duarte,CA91010.
3 The abbreviations used are: GPX, intracellular tetrameric selenium-dependent glutathione peroxidases, GPX-1 and GPXGI; GPX-1, classical glutathione peroxidase;GPXGI, gastrointestinaltract GPX isoenzyme;ER, estrogenreceptor;FBS, fetal bovinesera; COH-BR, City of Hope, breast cancer-derived; SDS-PAGE, SDS-polyacrylamidegel electrophoresis;PHGPX, monomeric phospholipid hydroperoxide glutathioneperoxidase;kbp, kilobasepalr(s).
ADRR hasbeen implicated in establishment of doxorubicin resistance(5, 6). In this instance, the resistance is partially due to expression of
P-glycoprotein (7, 8). In two published examples involving thelymphoid cell lines K562 and HL-60, doxorubicin resistance occurredwithout changesin hgpxl expression(2). ParentK562 andthe doxorubicin-resistant K562 subline do not have significant GPX-l activity(2, 9). K562 and MCF-7 can be distinguished from cell lines likeLl2lO, not only by a lack of GPX-l activity but by their response toselenium starvation under atmospheric 02. The latter line amplifiesthe catalase gene, resulting in vastly increased catalase-specificactivity, while catalase activity in the former two lines is notaffected under these conditions, nor are catalase activity levels inthese cell lines particularly high (9). This suggests that there islittle need for efficient H202 clearance in the K562 and MCF-7 celllines during routine culture.
The terms “enhanced―and “overexpression―have used to describethe GPX activity status of MCF-7 ADRR (10) relative to MCF-7 andin MCF-7 and T-47D hgpxl cDNA transfectants with high GPX-lproduction (3, 4). The terminology may be misleading if construed toimply that the GPX-l levels in MCF-7 or T-47D are typical of eithernormal breast tissues or breast cancer. While it can be doubted thatMCF-7 and T-47D are representative of normal breast, little is reallyknown about hgpx mRNA expression in breast epithelial cells (3).Normal human tissues generally have GPX activities comparable tothose found in ER-negative breast cancer cell lines (11). Two exceptions are testis and gastrointestinal tract mucosal epithelium (3, 12).PHGPX and GPXGI (a product of hgpx2 gene) are produced in thesetissues, respectively, while GPX-1 is relatively underproduced.Thevery high activity levels of PHGPX found in the testis could providefor efficient free fatty acid hydroperoxide clearance (12) and very lowbut detectable H202 clearance (13). GPXGI is more similar to GPX-lin structure, substraterange, and subcellular localization than PHGPX(2), and its production in the gastrointestinal mucosal epithelium maybe in lieu of GPX-l. Assays of tumor breast samples demonstratehigher GPX activities than normalbreastsamples (11). This may bedue to a greater epithelial cell content in such samples. But, this doessuggest that GPX-l activity is not diminished in primary tumors. It islegitimate to question just what MCF-7 and similar cell lines derivedfrom metastatic cancer represent in terms of GPX expression and therelevance of GPX responses to doxorubicin selection in these celllines as models of drug resistance mechanisms (1). The options arethat these lines really represent GPX expression in advanced metastatic cancer with some relevance to this stage of cancer or that thecell culture process has selected atypical GPX phenotypes from agenerally GPX-producing population.
In this study,we havesurveyeda largersampleof breastcancercelllines than that used to advance the case for an inverse correlationbetween ER expression and GPX-l expression in order to test thestrength of the correlation. The survey included new cell lines isolatedfrom malignant effusion. In using the newer breast cancer cell lines,we attempted to address the issue of whether there is instability ofhgpx gene expression during culture that may bias our perception ofGPx expression in breast cancer. The inverse correlation betweenGPX-l and ER expression is weakened by the results of the survey.
957
Expression of Selenium-dependent Glutathione Peroxidase inHuman Breast Tumor Cell Lines1
R. Steven Esworthy,2 Margaret A. Baker, and Fong-Fong Chu
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duane. California 91010 (R. S. E., F-F. C.], and Zynaxis, Inc., Malvern,Pennsylvania 19355 (M. A. B.]
One ER-positive, GPX-1-positive cell line was found; and GPX-1-negative, ER-negative lines were found. GPX-1 expression does notseem to be particularly unstable during culture of the ER-negativeclass of breast cancer cell lines. Therefore, a lack of GPX-1 expression in one newly isolated ER-negative cell line may indicate thatGPX-1 loss of production does occur in situ. However, GPXGIexpression was detected in 4 of 11 ER-negative cell lines, including 2of the GPX-1 null lines. The hgpx2 gene may be activated in breastcancer so that GPX-1-negative lines may not always lack GPX
activity. PHGPX expression was also examined. The activity is notparticularlyhigh in the newer cell lines.
MATERIALS AND METhODS
Cell Lines. The breast cell lines DU 4475, HS578BST, HS578T, MCF-7,MCF-1OA, MCF-1OF, MDA-MB-134-VI, MDA-MB-157, MBA-MB-175-VII, MDA-MB-231,SK-BR-3,T-47D,and ZR-75—lwereobtainedfromtheAmericanType Culture Collection (Rockville, MD). The cell line MCF-7ADRR was obtained from Dr. K. Cowan (National Cancer Institute; Ref. 10).
Cell lines COH-BR-1 to COH-BR-7 (City of Hope breastcell lines) wereobtained from Dr. J. Doroshow (City of Hope). These cell lines are epithelialcell lines isolated from pleural effusion or ascites fluid of patients with adiagnosis of primary breast cancer using methods described by Engel et a!.(14). COH-BR cell lines and human tissue samples were obtained from discard
materials from medically necessary procedures as approved by the City ofHope Institutional Review Board.
Growth Conditions. Exceptasnotedbelow,the longerestablishedbreastcell lines and COH-BR-l were grown in Richter's modified MEM, zinc option(GIBCO-BRL, Grand Island, NY) with Earle's salts, L-glutamine nonessentialamino acids, and 10% heat-inactivated FBS (Irvine Scientific, SantaAna, CA).For thecell linesHS578BST,MDA-MB-134, andMDA-MB-175, the mediawassupplementedwith 10%NCFC-109(GIBCO-BRL)andepidermalgrowthfactor (10 ng/ml) in early studies (Ref. 1; American Type Culture Collection).These media did not support rapid growth ofthe MDA-MB-175 cellline or anygrowth in DU 4475andwerereplacedwith themediausedfor cultureof theMCF-1OAcell line, further supplementedwith conditionedmediafrom theHS578BSTandFHS741ntlines in laterexperiments.The GPX andPHGPXactivity values reported for MDA-MB-175 are prior to growth stimulation and
for DU 4475 after growth stimulation. The MCF-1OA and -1OF lines weregrown in DMEM-F12 (GIBCO-BRL) with insulin (5 ,.@g/ml), transferrin (5
The lots of FBS (10% fmal concentration; v/v) used in this study supportedGPX and PHGPXactivity at 10—40%the levels achievedby supplementingthe media with 100 flM selenium as sodium selenite (2). The activity valuesreported for GPX and PHGPX of all cell lines are from cells grown in 100 nM
seleniumfor three to four passages.COH-BR-5 and MDA-MB-175 wereinducedfor 1—2monthswithoutpassagedueto slowgrowth.Sodiumseleniteandmostof theserumreplacementfactorsusedin thepreparationof themediawerepurchasedfrom the SigmaChemicalCo. (St. Louis, MO).
The COH-BR cell lines, exclusiveof COH-BR-1, were maintainedinEagle'sMEM with 5% FBS until study,when the mediawassupplementedwith 100 nM selenium for induction of GPX and PHGPX. All lines weremaintained at 37°Cin a humidified atmosphere of air and 5% CO2.
Selenoprotein Labellng ProtocoL [75Se]Sodium selenite (3—7mCi/mg)was obtainedfrom Amersham(Arlington Heights, IL). The [75Se]sodiumselenite was used as the sole selenium supplement when it was used to labelcells(2). Cellswerelabeledby serialpassagein [75Sejsodiumselenitefor 3 to4 passages (COH-BR-5 and MDA-MB-175 excepted) with the cells splitapproximately1:4.
Preparation of CytosolSamples.Humantissueswerehomogenizedwitha Polytron homogenizer in buffer A as described by Chu et a!. (3) with 0.2 Msucrose. Attached cell lines were harvested by scraping in PBS (no calcium,pH 7.4; GIBCO-BRL). This provedto be too harshfor the harvestof theMCF-1OA and COH-BR-6 cell lines. These two cell lines were extensivelywashed as monolayers with PBS, rinsed as a monolayer with buffer A, andscrapedoff the flask surfacein this buffer andthensonicated.The othercelllines were rinsed once in buffer A and processed as described by Chu et a!. (3).
Crudehomogenateswerecentrifugedto obtaina postmitochondrialsupernatant (15,000 x g for a minimum of 30 mm).
Protein and Enzyme Assays. All methods used were as described by Chuet a!. (3).
SDS-PAGE. Assessment of the selenoproteinpatternof each cell line,except COH-BR-5, was based on the results of two independently labeledsamples and SDS-PAGE runs (3).
Northern Analysis of RNA. See Chu et a!. (2) for procedures. The hgpxl(GPX-1), hgpx2(GPXGI), andhgpx4(PHGPX) cDNA probeshavebeendescribed (3, 17).
Characterization of the COH-BR Cell Lines. The COH-BR lines, exceptCOH-BR-2whichdied-offpriortocharacterization,wereexaminedbyDr. S.Wilczynski (Department of Pathology, City of Hope) for evidence consistent
with breast epithelial origin by morphology, ER, progesterone receptor status,
andothercell typemarkers(epithelialmembraneantigen,grosscysticdiseasefluid protein,humanmilk fat globularprotein,andkeratin)usingimmunohistochemistry(18—20).This was performedin 1994.Cell lines MCF-7 andSK-BR-3 were examined as controls and to verify the ER status of SK-BR-3as reported in the literature (21). Cytogenetic analysis of the lines was per
formedexceptfor COH-BR-2andCOH-BR-5.COH-BR-2expired,andtheCOH-BR-5cell line hasa doublingtimegreaterthan3 weeksandhasnotpresentedmitotic cellsfor study.Analysisofthe otherCOH-BRcell lineswasperformed using Colcemid (50 @.tWml)for 45 mm prior to cell harvesting.Individual chromosomes were identified by GTG-banding, according to theclassificationin the Guidelinesfor CancerCytogenetics,Supplementto AnInternational System for Human Cytogenetic Nomenclature (22). Except forCOH-BR-1whichwascharacterizedin 1991,thelineswereexaminedin 1994.
The analysis of the COH-BR cells lines for hgpx gene expression wasperformed: (a) COH-BR-1, passageunknown for all analyses; line was established in 1988; first study date for activity and selenoproteins in 1990; mRNAanalysis done in late 1992; all parameters have been checked many times since;GPX statushasnotchanged;(b) COH-BR-2,activityanalysisperformedatpassages7—9;the line died after the secondassay;(c) COH-BR-4,passageunknown for all analyses; line was seeded 6/4/92; the line was first assayed on
9/8/92;mRNA wasexaminedon 11/5/92;this line hasbeenexaminedmanytimes since and has not altered GPX status; (d) COH-BR-5, mRNA expression
wasanalyzedat passage8; at passage13,mRNA, activity, andselenoproteinwere analyzed;at passage15, activity was examined;(e) COH-BR-6, atpassages 10 and 25, activity, selenoprotein, and mRNA were analyzed; nochange in GPX status hasbeen detected; and (f) COH-BR-7, at passages3 and25, activity, selenoprotein, and mRNA were examined; no change in GPX
statushasbeenobserved.
RESULTS
Properties of the COH-BR Cell Lines The COH-BR cell lineswere isolated from malignant effusions of patients with a diagnosis ofprimary breast cancer. The COH-BR cell lines are ER negative orweakly ER positive (COH-BR-7). The cell lines have a morphologyconsistent with a breast epithelial cell origin. All lines were scored aspositive for keratin and human milk fat globule expression. COH
BR-S expressedepithelial membraneantigenand was vimentin negative. Only COH-BR-7 was clearly progesterone receptor positive.
Cytogenetic analysis of the COH-BR-1, -4, -6, and -7 cell linesdemonstrates that the lines are of human origin. COH-BR-5 has notbeen analyzed becausethe doubling time is about 3 weeks. This hasprevented detection of mitotic figures after Colcemid arrest. COHBR-i consistsof two cytogenetically distinct subpopulations, one witha near diploid chromosome number (56 chromosomes, modal number)and one with a near haploid number (27—28chromosomes). COHBR-4 hasa chromosomenumberin the rangeof 52—57,COH-BR-6has a range of 81—87,and COH-BR-7 has a chromosome numberrange of 61—70.The hgpxl gene has been localized to 3p2l (23). Thislocus is in a region that is frequently deleted in breast cancers. Wecharacterized thesecell lines for 3p abnormalities. All of the COH-BRlines had chromosome 3 abnormalities in all mitoses analyzed.However, only two of the cell lines had abnormalities near 3p2l. In
COH-BR-6, this actually involved 3p21-der(1 i)t(3;1 l)(p2l;pl5). InCOH-BR-7, del(3)(p13p22) occurred in many mitoses. Further detailsof the histological and cytogenetic analyses can be obtained bycontacting the authors.4
GPX and PHGPX Activity Levels in Breast Cell Lines. Table 1reports the GPX and PHGPX activity levels of breast cell linescategorizedby origin (newly establishedcancercell lines, COH-BR;older cancer lines; and “normal―or near diploid lines) and by ERstatus. GPX specific activity levels (H202, substrate) as low as 2—4%of the levels found in diploid, “normal―breast cell lines (Table 1) andin normal and tumor breast tissues(Table 2) were found in threecategories of breast cancer cell lines: ER-negative new cell lines(COH-BR); ER-negative long-established lines; and ER-positivelong-established cell lines. With one exception (MDA-MB-175, seebelow), the remaining breast cancer cell lines have GPX activitylevels in the range of the diploid cell lines and tissues. This includesone ER-positive cell line, MDA-MB-134. In this survey of 15 celllines (COH-BR-2 and MCF-7 ADRR excluded), 4 are observed toviolate the inverse correlation of ER and GPX-1 expression. As notedabove, COH-BR-6 has a translocation with a 3p2l breakpoint (site ofthe hgpxl gene), and COH-BR-7 has deletions of 3pl3 and 3p22.Both cell lines have high GPX activity. GPX-1 production has notbeen significantly affected by the chromosome 3 abnormalities, or itis compensated by expression from the other homologue. Underexpression of hgpxl mRNA is not routinely associated with frankdeletion or gross rearrangement of the gene. Genomic DNA wasisolated from cell lines COH-BR-1, MCF-7, MDA-MB-231, SKBR-3, T-47D, and ZR-75—1.The DNA was digested with BglII,fractionated on 0.9% agarose, Southern transferred, and probed withthe hgpxl cDNA probe. All of these cell lines exhibited an identicalpattern of three hybridizing bands with apparent sizes of 6, 4.4, and3.5 kbp. Based on the results presented by McBride et al. (24) and
Diamond et a!. (25), the 6- and 3.5-kbp BglII bands contain hgpxlpseudogenes on chromosomes X and 21, respectively, while the4.4-kbp band contains the active hgpxl gene. Application of PCR fordetection of exon 2 of hgpxl appearedto confirm its presence in celllines MCF-7, T-47D, and ZR-75—1(results not shown). The results forMCF-7, in particular, support the result of Moscow et a!. (26) thatMCF-7 transcribes an intact hgpxl gene, whereas the mature mRNAis not detected.
Cytosolic PHGPX activity (phosphatidyl choline hydroperoxide,substrate) is detected in all of the long-established breast cancer celllines with no correlation or inverse correlation detected with ERexpression. The activity levels range from 150 to 600% of normal andcancerbreasttissuelevels andfrom 70 to 300% of diploid cell levels(Tables 1 and 2). PHGPX activity levels in the COH-BR cell lineswhich were directly assayedranged from 20 to 70% of the lowestPHGPX activity level measured in long-established cell lines (Table1). We have no direct evidence that the PHGPX activities reported forCOH-BR-1 and COH-BR-4 derive from the selenium-dependentPHGPX enzyme. The PHGPX activity in COH-BR-1 appears to beselenium-independent (data not shown). It is possible that the activityattributed to PHGPX in these lines is from microsomal glutathioneS-transferase(EC 2.5.1.18; Ref. 27) or nonspecific background in thecell lysate. However, the cell lines that were probed for mRNAexpressionwith a hgpx4 cDNA probe demonstratedexpressionof a0.9-kbmRNA consistentwith thesizeestimateof 0.895kbp observedin a full-length cDNA (28). This included COH-BR-1, COH-BR-4,COH-BR-5, and COH-BR-7. PHGPX activity levels were notstrongly correlated with the level of mRNA detected (data not shown).
aGPXandPHGPXassaysperformedoncytosolsofcellsgrowninsodiumselenitesupplementedmedia (100 nM) for several passages,except for COH-BR-5 and MDAMB-175, which were grown for 1 to 2 monthswith supplementationwithout passage.H202(60 @sM)isthesubstratefortheGPXassay,andphosphatidylcholinehydroperoxide(25 @M)is the substrate for the PHGPX assay.
b ER status for breast cell lines only based in part on Ref. 21. SK-BR-3 and COH
BR-i-i weredeterminedusingimmunohistochemistryby the Departmentof Pathology,City of Hope.
C n = 2 or more for all samples; SD.
d Expressed hgpx2 mRNA alone (MDA-MB-175 and COH-BR-5)or coexpressed with
hgpxl mRNA (COH-BR-7 andDU 4475). HS578BST was not examinedfor hgpxmRNAexpression.If not listed in this footnote, the line was positive for hgpxl mRNA andnegativefor hgpx2mRNA.
e ND, not determined; +, positive detection; —, not detected, +/-, weak positive.
1For line MDA-MB-i75, activity levelsarefrom cellsbeforeexposureto choleratoxinandconditionedmediato stimulategrowth rates.DU 4475would not grow without thesupplements;therefore, the activities are reported subsequentto cholera toxin andconditionedmediaexposure.
Normal breast(4)b@ ±9ic 5.9±7.6(2.2±2)dTumor breast (4) 503 ±127 3.4 ±1.6Kidney (4) 191±74 8.1±4Liver(4) 352±89 5.0±0.7
aSpecificactivity,nmol/min/mgpostmitochondrialsupematantprotein.b Number of independent samples. Normal breast and tumor breast were matched sets
of biopsy material from the uninvolved breastand tumor site of the involved breastofcancerpatients,respectively.
CSD.d Mean and SD with one sample excluded for a very high PHGPX value.
GPX-specific activity is correlatedwith M@22,000 selenoproteinsubunit 75Selabeling intensity, and PHGPX-specific activity is correlated with Mr 18,000 selenopolypeptide 75Selabeling intensity onautoradiographs of SDS-PAGE fractionated cytosol for the breast celllines (Fig. 1, left-hand panel). These cell lines were cultured undersimilar conditions and have similar growth rates. For the remaininglines, detection of GPX and PHGPX activity is qualitatively associated with the detection of these two selenopolypeptides (Figs. 1 and2). Among the COH-BR cell lines, only COH-BR-6 appears to havePHGPX protein levels (Fig. 2) that match thoseof the longer established cell lines, using the relative intensities of the Mr 18,000 and Mr22,000 selenopolypeptides as an index (see COH-BR-1 in Figs. 1 and2 for a reference). In COH-BR-5, it was not possible to observe 75Seincorporation into either GPX or PHGPX. The Mr 57,000 selenium4 R. S. Esworthy and F-F. Chu, unpublished observations.
Fig. 1. Cytosolic selenoprotein subunit detection in long-established breast cell lines selectedfrom Table 1. Cells were grown in 75Se-containingmedia (100 ni@i)for 3 to 4 passages (1:4 split).Analysis was performed by fractionating 100 @g/lane (left panel) or 50 @gprotein/lane (middle andright panels) on SDS-polyacrylamide gels (12%acrylamide). Autoradiography was perfonned for 7days (left panel), 14 days (middle panel), and 12days (right panel) to visualize the selenoproteins.MDA.MB-175(—), cells grown without choleratoxin (middle panel). MDA-MB-175(+), cellsgrown with cholera toxin (right panel). GPX, sdenopolypeptide subunit of selenium-dependent, tetrameric glutathione peroxidase (M, 22,000).PHGPX, Mr 18,000 selenoprotein.
@--@@
because hgpxl mRNA has never been detected in this cell line. Thisinterpretationis supportedby the fmding that down-regulationofhgpx2 mRNA in MDA-MB-175 resulted in a loss of GPX activity inthe cell line (see next section). The COH-BR-5 cell line also expressesonly the hgpx2 mRNA. The COH-BR-7 and DU 4475 cell linescoexpress hgpxl and hgpx2 mRNA. The results for COH-BR-5 andCOH-BR-7 are based on RNA isolated from low-passage cultures.This may indicate that hgpx2 gene expression was occurring in thecancer cells in the malignant effusion from which these lines wereisolated.
Stability of hgpxl and hgpx2 Gene Expression. We did notobserve any loss or gain of GPX-1 expression (data not shown) in thecourse of following GPX activity expression in several of the longestablished cancer-derived cell lines for up to 1 year of continuousculture in the presence of selenium. Inclusion of breast epitheial cellgrowth factors used to support the growth of the MCF-10 cell linesand conditioned media as used in the case of the cell lines MDA-MB175 and DU4475 to the media of MCF-7, T-47D, SK-BR-3, andZR-75—1failed to induce GPX-1 production (data not shown). Comparison of GPX-1 activity and hgpxl mRNA during the early passageof the ER-negative, COH-BR cell lines showed that GPX-1 production was stable, provided it was detected in the earliest passage. Theactivity data listed for COH-BR cell lines (COH-BR-2 and COH
BR-5 excepted) in Table 1 is pooled from the earlier passages andlater passages (see “Materialsand Methods―)since no real differenceswere detected. Because expression of the hgpxl gene is stable inculture, the results may indicate that lack of hgpxl gene expression in
the COH-BR-5 cell line was a feature of a substantial subpopulationof cells in the malignant effusion.
The expression of the hgpx2 gene in culture may not be stable inbreast cancer cell lines. MDA-MB-175 grew at a very slow rate underthe conditions that resulted in high hgpx2 mRNA expression (Fig. 4),GPx subunit production (Fig. 1), and moderate levels of GPX H202activity (Table 1). Successful growth stimulation in media describedin “Materialsand Methods―resulted in reductions in GPX-specificactivity levels, GPX subunit levels, and hgpx2 mRNA levels (Figs. 1and 4). GPX-speciflc activity levels corresponding to the reducedlevels of hgpx2 mRNA were 7 ±5 nmol/min/mg. Comparison ofhgpx2 mRNA expression in COH-BR-5 at passage 8 (Fig. 5) and
Fig. 2. Selenoprotein subunit labeling pattern from the COH-BR cell line cytosols.Analysis consist of metabolically labeling cells by growth in culture media with 5% FBSsupplemented with 100 n@ [75Selsodium selenite for 3 to 4 cell passages, with theexception of COH-BR-5, which was labeled for 7 weeks without passage. Analysis wasperformed by fractionating 25 @gprotein on SDS-polyacrylamide gels (12% acrylamide).Autoradiography was performed for 15 days on the dried gels to visualize the selenoproteins. GPX, selenopolypeptide subunit of selenium-dependent, tetrameric glutathioneperoxidase (M, 22,000); PHGPX, M@18,000 selenoprotein. The samples correspond tocell mRNA expression shown in Fig. 3.
960
f4),@
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@ci: @alu@cor@1@N•- l@ll
Li@i@i =lfll()LL@<O@Hct@ oc,nooc@
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binding protein shows binding of 75Se, indicating that the radioisotopewas taken up by the cells. Failure to detect significant GPX activitymay indicate a very low rate of synthesis linked to the very slowgrowth rate of this cell line. This may make it nearly impossible toequilibrate the GPX and PHGPX protein with the selenium pool aftersupplementing the media with sodium selenite.
Expression of hgpxl and hgpx2 mRNA in Breast Cell Lines.Production of GPX can be related in a qualitative way to detection ofhgpxl mRNA in both the diploid MCF-1OA and MCF-1OF cell linesand the cancer cell lines, with the exception of the MDA-MB-175line. In the case of MDA-MB-175, hgpx2 mRNA expression seems tobe completely responsible for the production of GPX (Figs. 3 and 4)
I I I I I I I I IIa: cta:ci:o::@co a@a@a@tn a@ nctcü@
Fig, 3. Northern analysis ofRNA from the morerecently isolated COH-BR breast tumor cell lines . [email protected] hgpx2 and hgpxl mRNA expression at later . . . ‘@@passage. For coH-BR-5, this was passage 13; for
@OH-BR-6and BR-7, this was passage 25. COHBR-i and COH-BR-4 had been in culture for 4 and1.5 years, respectively. Twenty gsg of total RNAwere fractionated on 1.2% agarose-formaldehydegels, and transferredmembraneswere used forprobing with hgpx2 cDNA and hgpxl cDNA. Theethidium bromide staining of the gel demonstratedthatthe loadingwasequalamongsamplesfor thissetandinthecaseofFig5. . “@hgpx1@ • “hgpx2
passage 13 (Fig. 3), using COH-BR-7 as a standard, suggests that asignificant decline in mRNA levels has occurred.
DISCUSSION
Various studies have been made of the impact of GPX expressionin breast cancer and the potential impact of changes in GPX expression on the response of these cancers to treatment with redox cyclingantitumor drugs (1, 4—6).Since it is not clear when or how GPX-1production was lost, we questioned the validity of using some GPX1-underproducing cell lines in such studies. Established cell linesisolated from normal breast or from solid tumors have high hgpxlmRNA expression and no hgpx2 expression (MCF-10 and HS578T),which may reflect the findings in tumor breast biopsy samples presented in Table 2. Long-established cell lines from metastatic cancercan lack hgpxl gene expression. Newer breast cell lines were examined with the intention of observing whether hgpxl gene expressionwas unstable during early culture or whether low GPX-1 activity wasa feature of metastatic breast cancer. The sample set did not containER-positive members, which provide a significant portion of theGPX-1-underproducing, long-established cell lines. However, withinthe new lines, we did observe stable hgpxl gene expression for thoselines in which expression was observed early in culture. Additionally,one ER-positive cell line, MBA-MB-134, was found with high hgpxlmRNA expression. The results of this study support the possibilitythat loss of hgpxl gene expression occurs in ER-negative metastaticbreast cells in situ. This is based on confirmation that hgpxl geneexpression is stable during early culture of ER-negative cell lines (1)and the finding of one ER-negative cell line (COH-BR-5) withouthgpxl expression soon after isolation. It remains to be seen whetherloss of hgpxl expression is a frequent event and whether it occurs inearlier stages of cancer. If so, this could enhance genetic instability inbreast cancer by increasing the levels of clastogenic free radicals.
We confirm that there is a tendency for ER-negative cell lines toexpress high levels of GPX activity. However, a modest expansion ofthe sample size from that in the study of Townsend et a!. (1) hasweakened the proposed inverse correlation between ER status andGPXlevels;exceptionswerefoundinbothERstatuscategories.It ispossible that the apparent inverse correlation would disappear in alarge sample of cell lines. Underexpression of the hgpxl gene is notuniqueto cell lines establishedfrom breast cancers.We have foundthis in cell lines representing lymphoma (K562; Ref. 2), non-small
cell lung cancer (NCI-H-841 and NCI-H-1299), and colon cancer
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Fig. 4. Northern analysis of long-established breast tumor cell lines selected fromTable 1 for hgpx2 mRNA and hgpxl mRNA expression.Twenty @gof total RNA werefractionated on 1.2% agarose-formaldehyde gels and transferred to membranes for probing with hgpx2 cDNA (A) and hgpxl cDNA (B). A and B, different gel runs of the sameRNA samples. MDA-MB-175(—),cell line grown without cholera toxin; MDA-MB175(+), cell line grown with cholera toxin.
tissue distribution of a new cellular selenium-dependentglutathioneperoxidase,GSHPx-GI.J. Biol. @hem.,268: 2571-2576,1993.
4. Mirault,M-E., Tremblay,A., Beadoin,N., andTremblay,M. Overexpressionofseleno-glutathione peroxidase by gene transfer enhances the resistance of T47Dhumanbreastcellsto clastogenicoxidantsJ. BIOLChem.,266:20752-20760,1991.
5. Sinba, B. K., Katki, A. 0., BatiSt, G., Cowan, K. H., and Myers, C. E. Differentialformation of hydroxyl radicalsby Adriamycin in sensitiveand resistantMCF-7humanbreasttuinorcells: implicationfor themechanismofaction. BiOChCmiStIy,26:3776—3781,1987.
6. Akman, S. A., Forrest, G., Chu, F-F., Esworthy, R. S., and Doroshow, J. H.Antioxidant and xcnobiotic-metabolizingenzymegeneexpressionin doxorubicinresistantMCF-7 breastcancercells.CancerRes.,SO:1397-1402,1990.
7. Cowan, K@H., BatiSt@G., Tulpule, k, Sinha, B., and Myers, C. B. Similar biochemical changesassociatedwith multidrug resistancein humanbreastcancercells andcarcinogen-inducedresistanceto xenobioticsin [email protected]. Sd. USA, 83:9328—9332,1986.
8. FairChild,C. R., Ivy, S. P., Chien-Song,K. S., Wheng-Peng,J., Rosen,N., Israel,M. A, Melera,P.W., Cowan,K. H., andGoldsmith,M. E. IsolationofamplifiedandoverexpressedDNA sequencesfromAdriamycin-resistanthumanbreastcancercells.CancerRes.,47:5141—5148,1987.
9. Liii, F., Thomas,3. P., and GirOtti,A. W. Hyperexpressionof catalasein seleniumdeprivedmurineL1210cells.Arch. Biochem.Biophys.,305: 176-185,1993.
10. Batist,G., Tulpule,A, Sinha,B. K@Aspandiar,0. K., Myers,C. E., andCowan,K. H. Overexpression of a novel glutathione transferase in multidrug-resistant humanbreast cancer cells. J. Biol. Chem., 261: 15544—15549,1986.
11. Howie,A. F., Forrester,L M., Glancey,M. J., Schalger,J. J., Powis,0. Bcckett,G. J., Hayes, J. D., and Wolf@C. R. Glutathione S-transferascand glutathioneperoxidase expression in normal and tumour human tissues. Carcinogenesis (Load.),II: 451—458,1990.
12. Roved, A., Casasco,A., Maiorino, M., Dalan, P., Calligaro, A., and Ursini, F.Phospholipidhydroperoxideglutathioneperoxidaseof rattestis.J.Biol.Chem.,267:6142-6146,1992.
14. Engel, L W., Young, N. A., Tralka T. S., Lippman, M. E., O'Brien, S. J., andJoyce,M. J. Establishmentandcharacterizationof threenew continouscell lines derivedfrom human breast carcinomas. Cancer Res., 38: 3352-3364, 1978.
15. Sonic,R, Maloney, T. M., Wolman, S. R., Peterson,W. D., Jr., Brcnz, R., McGrath,C. M, Russo, J., Pauley, R. J., Jones, R. F, and Brooks, S. C. Isolation andcharacterizationof spontaneouslyimmortalizedhumanbreastepithelialcell line,MCF-10.CancerRes.,50: 6075-6086, 1990.
16. Stampfer,M., Hallowes,R.C.,andHackett,A. J.Growthofnormalhumanmammarycells in culture.In Vitro, 16: 415-425, 1980.
17. Chu, F-F. The humanglutathioneperoxidasegenesgpx2, gpx3, and gpx4 map tochrumosomes 14, 5, and 19, respectively. Cytogenet. Cell Genet., 66: 96-98, 1994.
18. Deamant,F. D., Pombo,M. T., and Battifora, IL Estrogenreceptorimmunohistochemistryasa predictorof siteof origin in metastaticbreastcancer.Appi lmmunohistochem.,3: 188—192,1993.
20. Ceriani, R. L, Blank@E. W., Peterson, J. A., Battifora, H., and Singh, H. Immunotherapeuticspredlinicalevaluationof anti-humanmilk fat globuleMoAbs Mc5 andBrE-1.Antib.Immunoconj.Radiophaum,3: 181—198,1990.
21. Sommers,C. L, Walker-Jones,D., andHeckfOrd,S. E. Vimentinratherthankcratinexpressionin somehormone-independentbreastcancercell lines and in oncogenetransformedmammaryepithelialcells.CancerRca.,49: 4258-4263, 1989.
22. ISCN: guidelines for cancer cytogenetics, supplement to an international system forhumancytogeneticsnomenclature.Basel:S. Karger,1991.
23. Moscow, J. k, He, R., Onarra, J. R., Knutsen, T, Weng, Y., Zhao, W-P, Whang-Peng,J., Linehan W. M., and Cowan, K. H. Examinationof human tumors for rhoAmutations.Oncogene,9: 189—194,1994.
24. McBride, 0. W., Mitchell, A., Lee, B. J., Mullenbach, G., and Hatfield, D. Gene forselenium-dependentglutathioneperoxidasemapsto humanchrumosomes3, 21 and@CBiofactors, 1: 285-292, 1988.
25. Diamond, A. M., Cruz, R., Bcncsics, C., and Hatfield, D. A pacudogenc for humanglutathione pemxidase. Gene (Amst.), 122: 377-380, 1992.
26. Moscow,J.A., Morrow,C.S.,He,R.,Mullenbach,0. T., andCowan,K. H. Structureandfunctionof the5'-flanking sequenceof thehumancytosolicselenium-dependentglutathione peroxidase gene (hgpxl). J. Biol. Chem., 267: 5949—5958,1992.
27. Mosialou,E., andMorgestern,R. Activity of rat liver microsomalglutathionetransferasetowardproductsof lipid peroxidationandstudiesof theeffectof inhibitorsonglutathione-dependentprotectionagainstlipid peroxidation.Arch. Biocbcm. Biophys.,275: 289—294,1989.
28. Esworthy, R. S., Doan, K., Doroshow, J. H., and Chu, F-F. ao@dag and sequencingof the cDNA encodinga human testis phospholipidhydroperoxideglutathioneperoxidase.Gene(Amst.), 144: 317—318,1994.
Fig. 5. Northern analysisof hgpx mRNA expressionin COH-BR-5 at passage8 andCOH-BR-7 at passage3. See Fig. 4 for methods.
ACKNOWLEDGMENTS
We thank Dr. J. Doroshow for access to the COH-BR cell lines, UndaMatsumoto for the isolation and maintenance of the COH-BR cell lines, Dr. S.Wilczynski for the histological and histochemical study of the COH-BR lines,and Dr. M. Slovak and Jennifer Pelky-Ho for the cytogenetic analysis of theCOH-BRcell lines.
2. Maiorino,M., Chu,F-F.,Ursini, F. Davies,K. 3.A., Doroshow,J. H., andEsworthy,R.S.Phospholipidhydroperoxideglutathioneperoxidaseisthei8-kDa selenoprotein
(HUTU 80).@ It is not clear that estrogen dependency or ER expres
sion has any influence on the tendency toward underexpressionof thehgpxl gene in cancer cell lines.
Alterations in GPX status are generally presentedin terms ofGPx-1 expression.With the observationof hgpx2 mRNA expressionin the breastcancercell lines COH-BR-5 and MDA-MB-175, it seemspossiblethat GPXGI could be producedin someGPX-1 null breastcancer cell lines. While it is too soon to know the significance ofhgpx2 gene expression in terms of tumor progression or drug sensitivity, we have shown that hpgx2 gene expression is not rare inadvancedbreast cancer-derived cell lines. If hgpx2 gene expression isunstable during culture, which is suggested by the results, then weunderestimate the extent of hgpx2 gene expression in advancedbreastcancer by looking at long-established cell lines. If GPX status influencessensitivity to redox cycling anticancer drugs, then alterations tohgpx2 gene expression may be important. The functions of PHGPXare unknown except that the properties of the enzyme suggest thatthey could overlap that of GPX-1 only for free fatty acid hydroperoxide substrates(13). We have found that PHGPX activity levels inthe newer cell lines are similar to those found in tumor breast biopsysamples, rather than the high levels found in long-established celllines. The high levels of PHGPX found in some long-establishedbreast cancer cell lines may not have significance in breast cancer.
1995;55:957-962. Cancer Res R. Steven Esworthy, Margaret A. Baker and Fong-Fong Chu Human Breast Tumor Cell LinesExpression of Selenium-dependent Glutathione Peroxidase in
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