regulation of p-glycoprotein gene expression in hepatocyte cultures

Nucleic Acids Research, Vol. 20, No. 11 2841-2846

Regulation of P-glycoprotein gene expression inhepatocyte cultures and liver cell lines by a frans-actingtranscriptional repressor

Timothy W.Gant, Jeffrey A.Silverman and Snorri S.ThorgeirssonLaboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA

Received January 22, 1992; Revised and Accepted May 4, 1992

ABSTRACT

Previously we have demonstrated that expression ofthe multidrug resistance (mdr) genes in rat liver andprimary rat hepatocyte cultures is induced by exposureto 2-acetylamlnofluorene and 3-methylcholanthrene.The mdr expression induced by both of thesecompounds occurs primarily via increased genetranscription. To determine the nature of possibleregulatory proteins involved in mdr gene regulation weinhibited protein synthesis using cycloheximide oremetine in primary rat hepatocyte cultures, mouse(HePa 1), human (Hep G2) and rat (H4-II-E) cell lines.Each cell type responded by strongly increasing itssteady state mdr1 mRNA levels. In hepatocytesIncreased mdr expression was observed after greaterthan 50% inhibition of protein synthesis, and was firstdetected after 2h of protein synthesis inhibition withmaximal induction occurring by 24h. Nuclear run-onanalysis showed that the increased steady state mRNAlevel was due to increased gene transcription withoutalteration of the transcription start site. Combined thesedata indicate that one regulatory mechanism by whichmdr gene expression is controlled is via a trans-actingtranscriptional repressor.

INTRODUCTION

Cells selected in vitro for drug resistance often overexpress aprotein of 170 kDa, P-glycoprotein (1). This protein mediatesmultidrug resistance (mdr) by acting as a trans membrane pump(2—4) to transfer xenobiotics across the cell membrane at theexpense of ATP (2, 4). Overexpression of P-glycoprotein isusually achieved in these cells by an amplification of the mdrlgenes (1, 5 -7) . However, amplification of the mdr gene familyhas not been observed in most human tumor samples that showelevated levels of P-glycoprotein (8—10). Only one clinical studyhas reported a mdr gene amplification in a single human tumor(11). This suggests that in vivo mdr gene regulation may bedifferent from that in cells selected for resistance in vitro. Also,early passages of drug selected cells show an elevated mdr geneexpression in the absence of mdr gene amplification (12).Expression of the mdr gene is induced in vivo following partialhepatectomy (13 — 15), during chemical carcinogenesis (15), andafter exposure to several xenobiotics (16, 17), for example

2-acetylaminofluorene (AAF) and 3-methylcholanthrene (MC).For AAF and MC the increased steady state mdr mRNA levelis due to increased gene transcription (17), which results in anincreased P-glycoprotein level. Hepatocytes isolated from ratspreviously exposed to AAF demonstrate increased resistance toseveral cytotoxic drugs including actinomycin D (18).

MC and AAF mediated induction of the mdr gene family obeysa Iog1()/response relationship consistent with that expected of areceptor mediated response (17). This suggests that mdr inductionmediated by AAF and MC occurs via a receptor like proteinwhich binds AAF and MC but not TCDD. The 4S protein hasthese characteristics (19, 20), but as yet no demonstrated rolein mdr gene regulation.

Transient transfection analysis of the 5' regulatory regions ofthe mouse mdr genes has revealed the presence of positive andnegative cis acting regulatory regions situated 5' of thetranscription initiation site in the mouse mdrlb (21,22) and mdr lagenes (23). In the human gene sequences both 5' and 3' of thetranscription initiation site appear to be important for themodulation of mdrl gene transcription (24). The human promoterregion has several recognized 5' regulatory regions in commonwith the mouse mdrla promoter except a TATA box which isfound in the mouse mdr la and mdr lb promoters but not in theequivalent human gene (21, 25, 26). TATA boxes are commonlyfound in many regulated gene promoter regions (27).

Inhibition of translation has been previously utilized to identifyproteins involved in transcriptional (28-33) and post-transcriptional (34—38) gene regulation. This approach has beensuccessful because protein synthesis affects expression of thosegenes under the control of relatively labile proteins (37) ratherthan causing a global alteration of gene expression. In this studywe used protein synthesis inhibitors to demonstrate the presenceof a labile transcription repressor of the mdrl genes in rat, mouseand human liver cells.

MATERIALS AND METHODS

Hepatocyte IsolationHepatocytes were isolated and cultured as previously described(17). The isolated cells were maintained in culture for 12 — 16hrs prior to treatment with cycloheximide. If protein synthesiswas inhibited before this period had elapsed the cells detachedfrom the plate and died. After this period protein synthesis couldDownloaded from https://academic.oup.com/nar/article-abstract/20/11/2841/2383534

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be inhibited without apparent detrimental effect to the hepatocytesfor at least 48 hrs.

Northern AnalysisExtraction of RNA and Northern analysis was performed aspreviously described (17). Each lane contained 20 fig of totalcytoplasmic RNA. Probes used for the detection of mdr geneexpression were pHDR5A (39), a 85Obp 3' pstl fragment ofpC 1.5 (40) or a 2 kb (p2B13-68) or 353 bp (pRDR-155)fragment of the rat mdrlb cDNA (41). Gene specific mdr probeswere used to determine which specific members of the mdr genefamily were being induced. These probes were probe A, a 122bppiece of the mouse la cDNA and probe C an approximately23Obp piece of the mouse mdr2 cDNA (40). Additionally a human276bp mdr2 gene specific probe, pMDRIIPvun, was used toattempt identification of the MDR2 in the HepG2 cells(M.M.Gottesman, personal communication).

The probes were labelled by random primer labelling using[a-32P] dCTP (42, 43). Blots were prehybridized for 4h in asolution containing 50% formamide, 6xSSC, 5xDenhardts(1X = 10 mg/ml each of BSA fraction V, ficoll andpolyvinylpyrrolidone) and 1 % sodium dodecyl sulphate (SDS)at 42 °C and hybridized in the same buffer containing 106 cpmof probe/ml for two days also at 42°C. After hybridization, blotswere washed to final stringency of 0.1xSSC/0.1% SDS 65°C(pRDR155, p2B13-68, pC1.5, pHDR5A to their respective samespecies), 0.1 xSSC/0.1 % SDS 50°C (rat probed with PHDR5A),0.1xSSC/0.1% SDS 45°C (pMDREPvuII to HepG2) or0.1 xSSC/0.1 % SDS 37°C (pA and pC against rat, mouse andhuman) for 1 - 2 h. Bands were visualized by autoradiographyusing intensifying screens at —80°C.

Analysis of transcription rate by nuclear run on analysisNuclear run-on analysis was performed as previously described(17). Briefly, nuclei were isolated from primary hepatocytecultures by lysis and centrifugation through a sucrose gradient.The nuclear pellet was collected and stored at — 80°C until use.

For analysis, nuclei were thawed on ice, washed, andtranscripts run off using 250 /iCi [a-32P]-GTP per reaction for45 min at 26°C. At the end of the incubation the nuclei weresedimented by pulse centrifugation and the supernatant aspirated.Nuclei were lysed by the addition of 900 /xl of 4 M guanidinethiocyanate, 0.1 M Tris pH 7.5 and 7% /S-mercaptoethanol andthe DNA sheared by several passages through a 25 G needle.The RNA was isolated by centrifugation over 900 /il of CsCl(1.7 g/ml). The incorporated radiolabeled dCTP was assessedby precipitation in 5% TCA/1% sodium pyrophosphate ontoglass fibre filters and scintillation counting. Equal quantities of32P labelled mRNA were hybridized in 1.5 ml of hybridizationbuffer (as used for Northern analysis) to 0.5 jtg of each isolatedand gel purified cDNA fragment, UV crosslinked to nylonmembranes (Magnagraph, MSI). Hybridization was allowed toproceed for four days at 42 °C and the filters then washed at 60°Cin 0.1xSSC/0.1% SDS for lh and exposed to RNAase A(20 ftg/ml) at 37°C for 30 min in 2xSSC beforeautoradiography. The probes utilized for mdr detection wasp2B 13-68 and that for the GAPDH control a full length GAPDHcDNA (44).

Primer extension analysisThe transcription start site was measured by primer extensionanalysis as previously described (45). The primer used had the

sequence 5'-GCGAAGTCCGCCAAGATGTAGAATT-3' whichis homologous to bases —33 to —57 of the rat mdrlb gene (41).Comparison of this primer to the mouse mdr2 cDNA (46)indicates that a minimum of 10 mismatches would be requiredfor binding, and we therefore hypothesize that this primer wouldnot bind to the rat mdr2 gene.

Cell linesThe cell lines used in this study were: Hep G2 a humanhepatocellular carcinoma (ATCC HB 8065), Hepa 1 a mousehepatoma cell (47) and RC3 a single cell clone of the rat H4-II-E hepatoma cell line (ATCC CRL 1548).

Densitometry analysisDensitometric analysis was performed using a computer basedanalysis system as previously described (48). Assessment ofincreased gene expression, in the cells used for transcription rateanalysis, was made from a slot blot (5 ng total RNA per slot)of the same RNA's shown in figure 3 panels B and D.

Assessment of protein synthesis inhibitionProtein synthesis inhibition was determined by measuring the totalincorporation of DL-[4,5-3H]-leucine (1 /xCi/ml into

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Figure 1. Increased mdr gene expression with protein synthesis inhibition inprimary rat hepatocyte cultures. Hepatocytes were cultured as described inMaterials and Methods and protein synthesis inhibited by cycloheximide in 0.1 %DMSO for 24h prior to harvesting of the RNA. Mdr gene expression (upperpanel) was determined by Northern analysis using pHDRSA. Albumin expressionis shown in the lower panel detected using the Psr I fragment of the rat albumingene (55) as a loading control. Inhibition of protein synthesis was determinedby DL-[4,5-3HJ-leucine uptake into TCA precipitable macromolecules.normalized to protein concentration and expressed relative to the control.

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Nucleic Acids Research, Vol. 20, No. 11 2843

trichloracetic acid precipitable macromolecules. Results areexpressed relative to total protein measured by the method ofBradford (49).

RESULTSInhibition of protein synthesis causes increased mdr geneexpressionInduction of mdr gene expression was detected followinginhibition of protein synthesis in primary rat hepatocyte cultures(Fig. 1). Increased mdr gene expression was first detected at acycloheximide concentration of 0.5 /tM which corresponded toa 50% inhibition of protein synthesis and increasedproportionately thereafter with increased protein synthesisinhibition (Fig. 1). Two transcripts, 5.2 and 4.4 kb, are evidenton the blot. This is not due to the use of two different promotersites as has been described in the human (26) and mouse mdr la(25) genes as only one major transcription initiation start site wasdetected by primer extension analysis (Fig. 6). We used the genespecific probes A and C (40) to determine in Hepa 1 and RC3cells that these bands corresponded to the mdrla and lb genesat 5.2 and 4.4 kb respectively (see below).

A time course analysis indicated that mdr gene expression waselevated at 2—4h after protein synthesis inhibition and wasmaximal at 12 to 24h (Fig. 2A and B). Two experiments areshown at different cycloheximide concentrations to demonstratethe early time points (Fig. 2A) and that mdr gene expression wasnot increased purely as a function of time in culture (Fig. 2B,last lane). Hepatocytes maintained in culture under theseconditions do not demonstrate increased mdr gene expression oversix days in culture (17).

Nuclear run-on analysis in hepatocytes (Fig. 3A) and RC3 cells(Fig. 3C) indicated that in both cell types induction of mdr geneexpression occurred primarily by increased transcription. At both12 and 24 hrs mdr gene transcription was increased.Densitometric analysis showed 9-fold (hepatocytes at 12 hrs),27-fold (RC3 cells at 12 hrs), 4-fold (hepatocytes at 24 hrs) and3-fold (RC3 cells at 24 hrs) increases in mdr gene transcriptionover controls after normalization for GAPDH transcription.Corresponding increases of mdr gene expression measured byslot blot analysis and densitometric scanning were 4.5, 9, 12 and7.5 fold over controls, after normalization for GAPDHexpression.

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Figure 2. Time course of increased mdr gene expression in primary hepatocytecultures following protein synthesis inhibition. Hepatocytes were isolated andcultured as described in Materials and Methods. Protein synthesis was inhibitedusing either 10.0 (A) or 2.5 pM (B) cycloheximide in 0.1 % DMSO and the RNAharvested at the indicated time points thereafter. Mdr expression was determinedas described for figure 1. Lower panels show cytochrome P450 1A2 expression(A) detected using the gene specific p72 probe (56) and albumin (B) detectedas described in figure 1, as loading controls.O O Control• • Cycloheximide 2.5 fiMA A Cycloheximide 10.0 pM

Figure 3. Nuclear run on analysis of nuclei from protein synthesis inhibitedhepatocytes. Protein synthesis was inhibited by cycloheximide in 0.1 % DMSOfor the indicated times before the nuclei and RNA were harvested, and a nuclearrun-on assay performed. The mdr probe was p2B13-68. The GAPDH probe wasa full length cDNA clone (44). The slots were washed to 60°C in 0.1 xSSC/0.1 %SDS for lh followed by treatment with RNAse (20 /ig/ml) for 30 min in 2xSSC.Panel A, nuclear run on of rat hepatocyte nuclei for the times and treatmentsindicated; panel B, Northern analysis of mdr gene expression using mRNA isolatedfrom the same cells used for the run-on in panel A. Panels C and D are the sameexperiments but performed in RC3 cells. Results are representative of at least3 independent experiments. The changes in transcription were assessed bydensitometric scanning.



Induction of mdr gene expression by protein synthesisinhibition in human rat and mouse cellsInhibition of protein synthesis had similar effects on mdr geneexpression in all other cell lines examined, as in primaryhepatocyte cultures. Cell lines from three species, Hepa 1(mouse), Hep G2 (human) and RC3 (rat) displayed an increasedmdr gene expression in response to protein synthesis inhibition(Fig. 4). To determine if the two bands observed in panels I,HI and V were due to the increased expression of differentmembers of the mdr gene family, or differences inpolyadenylation as has been previously described for the mousemdrla (25), mdr gene specific probes were utilized. The 85Obp3' Pstl piece of PCI .5 though not specifically described as genespecific detected only one band at 4.4 kb in Hepa 1 cells. Asecond band at 5.2 kb was detected by the 122bp probe A (40)which is specific for mdrla. No signal was detected using probeC (40) the mdr2 specific probe (data not shown). Therefore wedetermine that mdrla and mdrlb were induced in these cells.Had mdr2 been expressed we would have expected to detect itat 4.1 to 4.3 kb, the transcript size that we (unpublished) andothers (40, 50) have previously determined for mdr2. Weobtained a similar result in the RC3 cells. The pRDR155 probewhich spans a conserved region of the rat mdrlb gene includinga nucleotide binding site, detected two transcripts of 4.4 and 5.2

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kb (Fig. 4m). On the same blot probe A detected only the upper5.2 kb transcript (Fig, 411), indicating this to be mdrla. As withthe Hepa 1 cells nothing was detected with probe C. In both ofthese blots probe A detected only one band indicating that wewere not observing the use of two polyadenylation sites as hasbeen described for the mouse mdrla (25). It is not known if therat mdrla gene also has two polyadenylation sites. To detect thepossible expression of MDR2 in the HepG2 cells we used a Pvullfragment of the human MDR2 gene. This probe failed to detectany transcript in the Hep G2 cells. We have previously used thisprobe to detect MDR2, with success, in monkey samples (Gantet al , unpublished). Expression of MDR2 has not been detectedin either drug sensitive or colchicine resistant KB cells, thoughMDR1 is detected in abundance in the KB colchicine resistantcell line (51). Therefore, it appears cycloheximide is inducingexpression of the mdrla and mdrlb, but not that of mdrl underthe conditions used in this study. The primer extension results(see below) were consistent with this conclusion.

Effect of protein synthesis by emetine on mdr gene expression

To determine that the alterations in mdr gene expression weobserved were due to the action of cycloheximide as a proteinsynthesis inhibitor and not as an agonist per se we conductedan experiment with emetine, another protein synthesis inhibitor.Emetine has a different structure from that of cycloheximide andinhibits protein synthesis by inhibition of ribosomal translocation,as opposed to inhibition of transpeptidase, the mechanism ofaction of cycloheximide (52, 53). In hepatocytes (Fig. 5) andRC3 cells (data not shown) emetine induced mdr gene expressionto a similar degree as cycloheximide.

Transcription start site mapped by primer extension analysisPrimer extension analysis mapped the major start site oftranscription to be —156 bp upstream to the ATG (Fig. 6). Thetranscription start site in the mouse mdrlb {mdrl) genes has beenreported to be at -148 bp (21) and -151 bp (22) upstream ofthe ATG. These data suggest that the major induced band at 4.4kb we observed was that of the mdrlb. Additionally, in both our

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Figure 4. Induction of mdr gene expression in cell lines with protein synthesisinhibition. Cells were grown to near confluency before protein synthesis wasinhibited by cycloheximide in 0.1 % DMSO at the concentrations indicated. After24h RNA was harvested and mdr expression determined by Northern analysisusing probes of PCI.5 (850P«/picce) cDNA (panel 0, pRDR155 (panel III),pHDR5A (panel V) and probe A (panels II and IV). For a full description ofthe probes see materials and methods. These blots were additionally probed withprobe C (Hepal and RC3) and pMDRUPvuII (HepG2), no hybridization wasdetected in either case. Cycloheximide <jM) concentrations (A to E) were: forHepa 1 cells 0 , 5, 10, 25, and 50 ^M; for RC3 cells 0, 1 , 5 , 10, and 50 pM;and for Hep G2 cells 0, 2.5, 5, 10 and 50 pM, respectively. The lane designationsare the same for panels II and IV as for I, III and IV.

EAPDW • • •

Figure 5. Induction of mdr gene expression in primary hepatocyte cultures byemetine initiated inhibition of protein synthesis. Protein synthesis inhibition wasfor 24h by emetine at the concentrations indicated in 0.1 % DMSO. Expressionof the mdr gene was determined by Northern analysis as described in Materialsand Methods using pRDR155.


Nucleic Acids Research, Vol. 20, No. 11 2845

study and that of Cohen et al (21), of the mouse mdrlb promoter,gene transcription initiated at one of a pair of guanidines in ahighly conserved region, further indicating that the major geneinduced by protein synthesis inhibition in this system was themdrlb. In this assay we hypothesize that the primer we utilizedwould not bind to the rat mdr2 cDNA based on analogy withthe mouse mdr2 sequence (see materials and methods). Thesequence shown in figure 6 is the sequence of the rat mdr lbwhich identifies the transcription start site as a guanidine. Theprimer extension also confirms the results of the Northerns thatthe steady state mdr mRNA level was being increased bycycloheximide.

DISCUSSION

In this report we offer strong, though indirect evidence that mdrlaand mdrlb gene expression in the rat and mouse and MDR1 inthe human are, at least partially, controlled by a trans-actingtranscriptional repressor. Expression of the mdr2 gene does notappear to similarly regulated.

Three previous studies on the mouse mdrla and mdrlb genepromoters have indicated the presence of 5' negative actingregions using constructs containing the chloramphenicolacetyltransferase (CAT) as a reporter gene (12, 22, 23). Cohenet al. (21) observed a negative regulatory region between bases—230 and —206 by transfection of mouse mdrlb CAT constructsinto COS-1 (monkey) cells. Raymond and Gros (22) detecteda similar element, using the same methodology, between bases-305 and - 9 3 , also in the mouse mdrlb gene promoter usingCMT-93 (mouse rectal carcinoma) cells. In the mouse mdrlagene a negative regulatory region is reported to span the AP-1site (23). In this study we have demonstrated that there is a

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Figure 6. Primer extension analysis of the transcription start site. Primer extensionwas carried on mRNA from cycloheximide treated cells using a 25 mer primerwhich spanned bases - 3 3 to - 5 7 of (he rat mdrlb gene (see materials and methodsfor a full description). Arrow indicates the transcription start site. The sequenceladder shown is the sequence of the rat mdrlb gene in the region of the transcriptionstart site.

negative regulatory element in the mouse and rat mdr la andmdr lb and human MDR1 genes by using protein synthesisinhibition and observing an increased mdr gene expression.

Regulation of the mdr responseBoth cycloheximide and emetine inhibit protein synthesis bydistinctly different mechanisms; cycloheximide by inhibitingtranspeptidase and hence the elongation of the growingpolypeptide chain, and emetine by inhibiting the translocationof the ribosome along the mRNA (53). That both emetine andcycloheximide increased expression of the mdr gene (Fig. 5),and the correlation between the degree of protein synthesisinhibition and the increased steady state mdr mRNA level (Fig. 1)suggests that the effects we observed were not due to thecompounds per se, but rather their ability to inhibit proteinsynthesis.

Several previous studies have examined the effects of proteinsynthesis on other genes. Included among them are theglycoprotein hormone a-subunit (28), c-myc (29, 36, 54), c-fos(29), ets-2 (35), j8 interferon (31), /3 and 7 actin (30, 32),ornithine decarboxylase (29) and tubulin (34). For these genesthe mechanisms by which expression is regulated have beendemonstrated, using protein synthesis inhibition, to be either post-transcriptional (34-36, 38) or transcriptional (28-31). Thus,increased mdr gene expression following protein synthesisinhibition could be due to an elevated gene transcription ordecreased degradation of the mRNA. We differentiated betweenthese mechanisms by use of the nuclear run-on assay. Our results(Fig. 3) demonstrate that the mechanism for increased mdr geneexpression appears to be primarily transcriptional, though wecannot rule out a post transcriptional component. This effect isnot due to an indiscriminate increase of gene expression sincethe levels cytochrome P450IA2 (Fig 2A.) and albumin (Figs.1 and 2B) mRNAs were unaffected during protein synthesisinhibition. Previous investigations have shown that expressionof a (but not /3) tubulin (29) and a actin (30) are also unaffectedby protein synthesis inhibition. Additionally, treatment oflymphocytes with cycloheximide affects the expression of only9 of approximately 400 genes, determined by in vitro translationand two-dimensional gel analysis (37). We therefore propose thatthere is a trans-acting transcriptional repressor involved in theregulation of mouse and rat mdr la and mdr lb and in humanMDR1 gene expression. Upon inhibition of protein synthesis thisprotein rapidly degrades allowing transcription to proceed.

We tested the effect of protein synthesis inhibition on severalcell lines to determine if the effect on mdr gene expression wasspecies specific or required a factor present only in rathepatocytes. These data (Fig. 4) showed that mdr gene expressioncould be induced in mouse and human cells. Therefore the trans-acting transcriptional repressor we describe appears not to be cellor species specific and thus may be an important regulator ofmdr gene expression.

Site of transcription initiation, and identity of the mdr genefamily member being inducedUse of gene specific probes (Fig. 4) indicated that we wereobserving the induction of mdrla and mdrlb, the two genes whichcode for the mdr phenotype. Primer extension demonstrated thatthe start site of transcription was at guanidine nucleotide-156 upstream of the ATG site and that the same start site wasutilized both in non-induced and induced cells (Fig. 6). This startsite is slightly revised from our previously published figure (17,



41) due to our cloning and sequencing of the 5' region of therat mdrlb gene (Silverman, unpublished results). That thetranscription start site we observed in the rat was in a similarposition to that in the mouse mdrlb gene and started from anadjacent base in an identical DNA region (21), suggests the mdrgene induced to the greatest degree by protein synthesis inhibitionin the rat was the mdrlb gene. This data therefore corroboratesthat which we obtained using the gene specific probes. This startsite is the same as that used for initiation of transcription afterinduction by AAF or MC (17).

In conclusion, inhibition of protein synthesis resulted in anelevated expression of the mdrl genes in primary rat hepatocytecultures and mouse, rat and human cell lines. Increasedtranscription appears to be the primary mechanism by which theelevated expression was achieved. Transcription was initiated atthe same base in the induced as in the non-induced cell. Initiationof transcription at this guanidine indicates that the mdr geneinduced by protein synthesis inhibition was the mdrlb. Analysisusing gene specific probes on Northerns showed that the mdrlbwas the major gene induced, but that expression of mdrl a wasalso increased. No expression of mdr2 could be detected in eitherinduced or non-induced cells. From these data we conclude thattranscription of the mdrl genes are partly, or wholly regulatedby a trans-acting transcriptional repressor.

ACKNOWLEDGEMENTS

We thank the following for providing probes; Dr MichaelM.Gottesman (pHDR5A and pMDRnPvuII), Dr John B.Fagan(p72) and Dr Susan B.Horwitz (pC1.5 piece, pV1.3 from whichprobe A is derived and probe C).

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