Breast Cancer Research and Treatment 69: 2938, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Report

Modulation of expression of ribosomal protein L7a (rpL7a) by ethanol inhuman breast cancer cells

Yunfeng Zhu, Hong Lin, Zheng Li, Mei Wang, and Jia LuoDepartment of Anatomy, West Virginia University School of Medicine, Morgantown, WV, USA

Key words: alcohol, differential display reverse transcription PCR, metastasis, northern blot, stress response,transcriptional regulation

Summary

Epidemiological studies indicate that there is a positive correlation between alcohol consumption and the risk ofbreast cancer. Experimental results demonstrate that ethanol is a tumor promoter and chronic ethanol exposureenhances metastasis and growth of breast cancer. The present study used an in vitro model to investigate themolecular mechanism(s) underlying tumor promoting effects of ethanol. With differential display reverse tran-scription polymerase chain reaction, we demonstrated that human ribosomal large subunit protein L7a (rpL7a) wasan ethanol-responsive factor in T47D breast cancer cells. The results of northern blot hybridization revealed thatthe effect of ethanol on L7a expression was duration- and concentration-dependent. Initial exposure resulted in a2-fold increase in rpL7a level, whereas a longer exposure period produced a down-regulation. Ethanol had littleeffect on the stability of rpL7a mRNA; however, the transcription rate of rpL7a was significantly increased byethanol. Ethanol-induced up-regulation of rpL7a was not a simple stress response, because other stress inducers,such as heat shock, did not affect the expression of rpL7a. Furthermore, breast cancer cells expressed higherlevel of rpL7a than normal mammary epithelial cells. Ribosomal proteins are known to play an important role intranslational regulation, and they have been implicated in the control of cellular transformation, tumor growth,aggressiveness and metastasis. Specially, rpL7a activates the trk oncogene by contributing an amino-terminal-activating sequence to the receptor kinase domain of trk. Thus, ethanol-induced alteration of rpL7a expression maymediate the promoting effects of ethanol on breast cancer development.

Introduction

Breast cancer is a leading cause of morbidity andmortality in women [1]. The cause of most breastcancer remains elusive and considerable research at-tention has focused on identifying the endogenous andenvironmental factors that contribute to its etiology.Alcohol, tobacco and diet are the three major humancancer risk factors [2, 3]. High consumption of alco-holic beverages increases the risk of certain cancers[4]. Ethanol as a risk factor for breast cancer was firstsuggested by Williams and Horm in 1977 [5]. Today,over 50 epidemiologic studies have examined the re-lationship between ethanol consumption and breastcancer. Although some controversy remains, most ofthe studies agree that there is a positive correlation

between ethanol intake and risk of breast cancer [seereview 69].

The positive association between ethanol con-sumption and breast cancer is highlighted by severalrecent studies [e.g., 1015]. Findings are summarizedas follows:

(a) Consuming 560 g of alcohol per day increases therisk of breast cancer by 1570%.

(b) Risk is directly correlated with the duration ofdrinking, that is, the greater the total number ofyears of drinking, the greater the risk.

(c) Alcohol consumption is strongly associated withadvanced and invasive breast tumors.

This suggests that alcohol exposure promotes tumorgrowth and metastasis. Despite the documentation

30 Y Zhu et al.

of the epidemiology of alcohol and breast cancer,little information regarding the mechanism of ethanol-induced tumorogenesis is known. Therefore, isolationof specific ethanol-responsive genes (ERGs) in breastcancer cells is important, because identification ofsuch genes will not only clarify the targets of eth-anol but also provide an insight into the molecularmechanisms of cancer development.

T47D human breast cancer cells were used as anin vitro model. These cells are derived from humanmammary duct carcinoma and express estrogen re-ceptors [16]. We have previously demonstrated thatethanol enhanced migration of T47D cells in culture[17]. The current study uses differential display re-verse transcription polymerase chain reaction (DDRT-PCR) to identify ERGs in breast cancer cells. Herein,we demonstrate that ethanol exposure modulates theexpression of ribosomal protein L7a (rpL7a) in T47Dcells.

Materials and methods

Cell culture

Human mammary epithelial cells (MCF-10A), andbreast cancer cells (T47D, MCF-7 and MDA-MB-231) were obtained from American Type Culture Col-lection (ATCC, Rockville, MD). MCF-10A cells weregrown in a mammary growth medium specified byATCC, supplemented with 100 ng/ml cholera toxin(Calbiochem. La Jolla, CA). T47D cells were grownin RPMI1640 medium supplemented with 2 mM L-glutamine, 4.5 g/l glucose, 10 mM HEPES, 1 mM so-dium pyruvate, 1.5 g/l sodium bicarbonate, 7.2 mg/linsulin, 100g/ml Penicilin/Strepmycin, and 10%fetal bovine serum. MCF-7 and MDA-MB-231 cellswere maintained Eagles minimal essential medium(EMEM) with the same supplement as that for T47Dcells. Cells were incubated at 37C in an atmospherecontaining 5.0% CO2 and saturating humidity. Themedium was changed every 23 days.

Ethanol exposure and heat shock treatment

Because of ethanols volatility, a method utilizingsealed containers [18, 19] was used to maintain eth-anol levels in the culture medium. With this method,ethanol was added directly to the culture medium intissue culture dishes, and the dishes then placed insealed containers with an ethanol-containing waterbath in the bottom. The concentration of ethanol in

the bath was the same as that in the culture medium.Ethanol from the bath evaporates into the air of thesealed container and maintains the ethanol concentra-tion in the culture medium. A small volume of CO2was injected into the container prior to sealing. Theethanol bath was changed daily to maintain the ethanolconcentration. Control cultures were maintained con-currently in separate sealed containers; however, thewater bath in the control groups contained no ethanol.All containers were incubated at 37C. Cells were ex-posed to ethanol at 100400 mg/dl. The concentrationsapplied are physiologically relevant to blood ethanollevels observed in alcoholics. Generally higher ethanolconcentration is required in vitro to replicate similareffects in vivo [20, 21]. In previous studies, we haveconfirmed that this sealed container method accuratelymaintains ethanol concentrations in the culture me-dium [19]. The procedure for heat shock treatmentwas similar to that previously described [22]. Briefly,cells grown in a 175 cm2 flask were heated at 45C for30 min. After heat shock, cells were incubated at 37Cfor the time indicated prior to RNA extraction.

DDRT-PCR

DDRT-PCR was first developed in 1992 to identifydifferentially expressed genes, and it has been suc-cessfully applied to isolate specifically induced genesin cancer [23]. Total RNA was isolated using a TRIREAGENT kit (Molecular Research Center, INC, Cin-cinnati, Ohio). The concentration of RNA was de-termined by the absorbance at 260 nm, and the puritywas determined by the 260/280 ratio with a BioPho-tometer (Eppendorf, Westbury, NY). DDRT-PCR wascarried out using a commercially available kit, DeltatmDifferential Display Kit (Cat. # K1810-1, Clontech,Palo Ato, CA). The protocol consists of two stages:cDNA synthesis and differential display PCR. First-strand cDNA was synthesized using each of the RNAsof interest as a template and oligo (dT) as a primer.One of the advantages of this protocol is that it re-quires only a single cDNA synthesis reaction for eachRNA sample, in contrast to 912 syntheses requiredfor each RNA sample in other differential display pro-tocols. The manufacturers instructions were followed.In brief, 2g of total RNA was reverse transcribedwith oligo-dT at 42C for 1 h. cDNA was ampli-fied by PCR reaction. Amplification was performedin the presence of specific primers and [-33P]dATP.The PCR products were separated on 6.0% dena-turing polyacrylamide/urea gel for 24 h. Gels were

Ethanol alters the expression of ribosomal protein 31dried and exposed to Kodak X-Omat film overnightat 70C. Differentially displayed cDNAs were re-covered from the gel and re-amplified with the sameprimers.

DNA cloning and sequence analysis

Differentially displayed PCR products were ligatedinto a pCR4-TOPO, TA vector (Invitrogen, Carls-bad, CA). Competent bacterial cells were transformedwith the ligated construct. Plasmid DNA subcolonescontaining inserts were purified using QIAprep SpinMiniprep Kit (Qiagen, Stanford Valencia, CA). Se-quence analysis on the inserts was performed by Re-search Genetics, INC (Huntsville, AL). Sequencesthus derived were compared for homology to the se-quences available in the current GenBank databasethrough the National Center for Biotechology Inform-ation using the BLAST program [24].

Northern blot hybridization

The cDNA probes were derived from differential dis-play PCR as described above. The probes were labeledwith [32P]dCTP by PCR reaction. The reaction mix-ture contained the following components: [32P]dCTP(3000 Ci/mM, ICN), templates (plasmid subclone,20 ng/l), 50 dNTP (10 mM of dATP, dGTP, anddTTP, and 100M of dCTP), Taq DNA polymerase(5 U/l), and primers (same as for DDRT-PCR). Amp-lification was performed at 95C for 3 min for denatur-ing, followed by 30 cycles of additional reaction (94Cfor 30 s, 55C for 30 s, and 72C for 2 min). Labeledprobes were then purified by G-50 column (Promega,Madison, WI).

Ten micrograms of total RNA per lane were separ-ated by electrophoresis on 1.0% agarose/formaldehydegel. The RNA was transferred to Nytran filters with aBio-RAD Vacuum Blotter Model 785 and ultraviolet-cross-linked. The hybridization was performed withthe following procedure. Pre-hybridization occurredby incubating filters with a hybridization buffer(ULTRAhybTM Buffer, Austin, TX) at 42C for30 min. After pre-hybridization, the filters were hy-bridized with cDNA probes at a final concentration of1 106 cpm/ml in the same buffer (42C, overnight).The filters were washed three times (5 min each) at42C in 2X SSC containing 0.1% SDS. This was fol-lowed by three additional washes (15 min each) at42C in 0.1X SSC containing 0.1% SDS. The signalswere processed with a PhosphorImager.

mRNA stability

The stability of the mRNA was determined by amethod described by Dani et al. [25]. Briefly, totalRNA was extracted from T47D cells before and atvarious times (30 min8 h) after treatment with actino-mycin D (5g/ml). Actinomycin D at this concentra-tion blocks essentially all transcriptional activity. RNAsamples were then subject to northern blot analysis asdescribed above. The signals were processed with aPhosphorImager

Nuclear run-off assayNuclei were isolated by sucrose gradient centrifu-gation [26] and stored at 70C. For analysis oftranscription rate of specific gene, 250l of nuc-lei suspension (4 107 nuclei/100l) was incubatedwith 100Ci of [-32P] UTP and 60l of 5X run-off buffer (25 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2,750 mM KCl, 1.25 mM of ATP, CTP, and GTP) for30 min at 30C. After reaction, the labeled RNA wasisolated using Nick columns (Pharmacia Biotech, Pis-cataway, NJ). The RNA transcripts were partiallyhydrolyzed with 200 mM NaOH at 0C for 10 min,then neutralized with HEPES acid. Nytran filters(Schleicher & Schuell, Keene, NH) were coated with1g of denatured cDNAs for rpL7a or cyclophilin byslot-blotting. Then the filters were UV cross-linkedat 254 nm (Spectro Linkertm, Spectronics Corpora-tion, West Tbury, NY), and pre-hybridized with ahybridization buffer (50% formamide, 250 mM so-dium phosphate, pH 7.2, 1 mM EDTA, 250 mM NaCl,100 mg/ml ssDNA, 40g/ml tRNA and 7.0% SDS) at55C for 4 h. Hybridization in the same solution con-taining 2 106 cpm of the 32P-RNA was performed at55C for 48 h. Filters were washed four times (20 mineach) in 2X SSC, followed by a 15 min wash in 2XSSC containing 5g/ml RNAse A, 100g/ml RNAseT1 at 65C. Finally, the filters were rinsed in 0.3XSSC containing 1.0% SDS for 1 h. The hybridizationsignals were visualized using a PhosphorImager.

Statistical analysis

Differences among treatment groups were tested us-ing a one-way analysis of variance (ANOVA). Dif-ferences in which p< 0.05 were considered statistic-ally significant. In cases where significant differenceswere detected, specific post-hoc comparisons amongtreatment groups were performed using a Student-Newman-Keuls test.

32 Y Zhu et al.

Results

Identification of ethanol-responsive gene

With DDRT-PCR, eleven cDNA PCR products thatwere differentially expressed in T47D cells betweencontrols and ethanol-treated cells were identified. Thedifferentially expressed cDNA fragments were thenexcised from the polyacramide gel and re-amplifiedwith the same primers as used in DDRT-PCR. Amongthe eleven cDNA products recovered, three of themwere verified by northern blots as differentially dis-played fragments. These three candidate ethanol-responsive genes (ERGs) were purified and ligatedinto a PCR4-TOPO, TA cloning vector. The differ-entially expressed candidate ERG cDNA inserts weresequenced and compared for homology to entries inthe existing nucleic acid database available throughthe National Center for Biotechology Information.

Figure 1. Differential display of mRNA in control (Ct) and ethanol(Et)-treated breast cancer cells. T47D cells were exposed to ethanol(400 mg/dl) for 2 weeks. Total RNA was isolated and a sample of2g of total RNA was used for DDRT-PCR. Lanes 1 and 3 wereloaded with the cDNA product diluted at 1:10. Lanes 2 and 4 wereloaded with cDNA product diluted with 1:40. Arrow points to thedifferentially displayed product, rpL7a.

Figure 2. Northern blot analysis of rpL7a expression in control (Ct)and ethanol (Et)-treated breast cancer cells. T47D cells were ex-posed to ethanol (400 mg/dl) for various durations (6 h, 24 h, 4 daysand 2 weeks), and total RNA was isolated. Top panel: Ten micro-grams of total RNA was subject to northern blot analysis using aprobe prepared from the product of DDRT-PCR. Bottom panel: Thesame blot was stripped off cDNA probe for rpL7a, and re-hybridizedwith a probe for the housekeeping gene cyclophilin.

Figure 3. Time sequence for ethanol-mediated rpL7a expression.T47D cells were exposed to ethanol (400 mg/dl) for various dura-tions (6 h2 weeks). rpL7a expression was examined with northernblot hybridization (see Figure 2). The relative amount of rpL7a levelwas determined with a PhosphorImager and expressed as percentageof control. The expression of rpL7a was normalized with the levelof cyclophilin. denotes a statistically significant difference fromcontrol. The result was the mean S.E.M. of four replicates.

Two cDNA fragments of candidate ERGs displayedno homology to any identified genes. One PCRproduct (512 bp) exhibited 100% homology to hu-man ribosomal large subunit protein rpL7a (accession#: NM000972). As determined by DDRT-PCR (Fig-ure 1), ethanol exposure for 2 weeks suppressed theexpression of this gene.

Expression of rpL7a mRNA

The expression of rpL7a mRNA was examined bynorthern blot hybridization using a specific cDNAprobe for rpL7a. The probe hybridized a mRNA bandaround 1 kb. Time sequence analysis of rpL7a expres-sion showed that the effect of ethanol on rpL7a was

Ethanol alters the expression of ribosomal protein 33

Figure 4. Concentration-dependent effect of ethanol on rpL7a expression. A: T47D cells were exposed to ethanol (0400 mg/dl) for either one(Left panel) or 4 days (Right panel), and the total RNA was isolated. Top panel: the expression of rpL7a was examined with Northern blothybridization using a probe prepared from the product of DDRT-PCR. Bottom panel: the same blot was stripped off cDNA probe for rpL7a,and re-hybridized with a probe for the housekeeping gene cyclophilin. B: The relative amount of rpL7a was determined with a PhosphorImager.The expression of rpL7a was normalized with the level of cyclophilin. Left panel: the expression of rpL7a in the cells that were exposed toethanol for 1 day. Right panel: the expression of rpL7a in cells that were treated with ethanol for 4 days. The result was the mean S.E.M. offour replicates. denotes a statistically significant difference from control.

specific and duration-dependent (Figure 2). Ethanolspecifically altered the expression of rpL7a mRNA,but had little effect on cyclophilin expression. Theexpression of rpL7a and cyclophilin was quantifiedwith a PhosphorImager and the relative amount ofrpL7a was normalized with cyclophilin (Figure 3).Ethanol produced an initial increase in rpL7a ex-pression with the amount of rpL7a transcript doub-ling after 24 h of ethanol exposure. Then, the effectof ethanol was diminished thereafter. Although theethanol-mediated increase of rpL7a expression wasreduced, the up-regulation was still evident after 4days of exposure. In contrast to initial increases,long-term exposure to ethanol (2 weeks) significantlyreduced the transcript of rpL7a by more than 60%(Figure 3). Although ethanol did induce up-regulationof rpL7a in other breast cancer cell lines (MCF-7 andMDA-MB231) to various extents (data not shown),T47D cells displayed the most dramatic and consistentresponse.

To examine the effect of ethanol at pharmacologic-ally relevant concentrations, T47D cells were exposedto a range of ethanol concentrations. As shown inFigure 4, ethanol-induced up-regulation of rpL7a wasconcentration-dependent. Expression of rpL7a wassignificantly increased following 1-day of exposureat ethanol concentrations as low as 100 mg/dl, andprogressively higher ethanol concentrations producedenhanced up-regulation. Although the effect of ethanoldiminished, a statistically significant up-regulationwas still observed following 4-days of exposure toethanol at concentrations of 200 and 400 mg/dl.

Figure 5. Effect of heat shock on the expression of rpL7a. T47Dcells received heat shock treatment as described in Materials andmethods. Total RNA was isolated at various time points post heatshock. rpL7a expression was examined with northern blot hybrid-ization. The relative amount of rpL7a level was determined with aPhosphorImager and expressed as percentage of control. The ex-pression of rpL7a was normalized with the level of cyclophilin. Theamount of rpL7a transcript was not signigicantly altered by heatshock treatment at any of the times studied (2, 6, 12, 24 h). Theresult was the mean S.E.M. of four replicates.

Ethanol is known to evoke cellular stress response.Ethanol exposure, and other stress inducers, such asheat shock, induces the expression of stress-relatedproteins (e.g., heat shock proteins and Alu)[22, 2729]. To determine whether rpL7a is a stress-responsiveprotein, we applied heat shock to T47D cells and ex-amined the expression of rpL7a following heat shock.The expression of heat shock protein 27 and 70(HSP27 and HSP70) was up-regulated following thistreatment (data not shown). As shown in Figure 5, heat

34 Y Zhu et al.

Figure 6. Expression of rpL7a in mammary epithelial cells andbreast cancer cells. Total RNA was extracted from various cell linesand equal amount of RNA was subject to northern blot hybridiza-tion. The expression of rpL7a was compared among normal mam-mary epithelial cells (MCF-10A) and various breast cancer cells(T47D, MCF-7 and MDA-MB-231). (1) T47D; (2) MDA-MB-231;(3) MCF-7; and (4) MCF-10A.

Figure 7. Stability of rpL7a mRNA. T47D cells were exposed toethanol (0, or 400 mg/dl) for 24 h. Total RNA was extracted fromT47D cells before and at various times (30 min8 h) after treatmentwith actinomycin D (5g/ml). RNA samples were then subject tonorthern blot analysis as described in the Materials and methods.The signals were processed with a PhosphorImager and expressedas percentage of the amount prior to application of actinomycin D.The result was the mean S.E.M. of four replicates.

shock treatment had little effect on the expression ofrpL7a mRNA in T47D cells.

To determine whether rpL7a is over-expressed inbreast cancer cells, mRNA level of rpL7a was com-pared among normal mammary epithelial cells andvarious breast cancer cells using northern blot hybrid-ization. MCF-10A is a normal mammary epithelialcell line. T47D and MCF-7 are estrogen receptor-positive human breast cancer cells, and MDA-MB-231is an estrogen receptor-negative breast cancer cell line.As shown in Figure 6, the mRNA of rpL7a in all threebreast cancer cell lines (T47D, MCF-7 and MDA-MB-231) was significantly higher than normal mammaryepithelial cells (MCF-10A) and estrogen receptor-positive breast cancer cell lines displayed extremelyhigh level of rpL7a.

mRNA stability and transcription of rpL7a

The ethanol-induced up-regulation of mRNA of rpL7acould result from an alteration in the rate of gene tran-scription or the stability of mRNA, or both. To distin-guish between these possibilities, analysis of mRNAstability and nuclear run-off assay were conducted incontrol and ethanol-exposed cells. T47D cells wereexposed to ethanol at 200 and 400 mg/dl for 24 h. Ana-lysis of mRNA stability and nuclear run-off assay wereperformed as described in the Materials and methods.As shown in Figure 7, ethanol had little effect on thedegradation rate of rpL7a transcript. In contrast, eth-anol at 400 mg/dl almost doubled the transcription rateof rpL7a gene, which was determined by the ability ofnuclei to produce mRNA of rpL7a (Figure 8). As ex-pected, the transcription rate of cyclophilin gene wasnot changed following ethanol exposure.

Discussion

DDRT-PCR revealed that ethanol exposure alters theexpression of the ribosomal large subunit proteinrpL7a. Subsequent study with northern blot hybrid-ization indicated that ethanol modulates expressionof rpL7a in a duration- and concentration-dependentmanner. Ethanol induces an initial up-regulation ofrpL7a expression followed by a suppression of thisgene. The acute effect of ethanol may better mimic thein vivo situation. On the other hand, down-regulationof rpL7a at late time point may reflect a desensitizationor compensatory response to the chronic exposure.Ethanol at pharmacologically relevant concentration(e.g., 100 mg/dl) significantly up-regulates rpL7a ex-pression, and the higher the ethanol concentrationapplied, the stronger up-regulation observed. Ethanol-mediated increase in rpL7a expression results fromenhanced transcriptional activity. In addition, ethanol-induced up-regulation of rpL7a is not a simple stressresponse, because other stress inducers, such as heatshock, did not affect the expression of rpL7a.

Ribosomes are catalyzers of protein synthesis. Eu-karyotic ribosomes are composed of two subunits, 40Sand 60S, according to their sedimentation coefficients.During protein synthesis, mRNA and tRNA are boundto the small subunit, while the peptide bond form-ation is catalyzed by the large subunit. The smallsubunit consists of an 18S ribosomal RNA (rRNA)molecule and about 33 proteins. The large subunit iscomposed of 5S, 5.8S, and 28S rRNA, together with

Ethanol alters the expression of ribosomal protein 35

Figure 8. Transcription rate analysis of rpL7a from control (Ct) and ethanol-treated (Et) T47D cells. A: Cells were exposed to ethanol (0,200, and 400 mg/dl) for 24 h. Nuclei were isolated and subject to nuclear run-off assay as described in Materials and methods. A constantcDNA concentration (1g) was used in each lane. B: The relative transcription rate was determined with a PhosphorImager and expressed aspercentage of control. The result was the mean S.E.M. of four independent experiments. p< 0.05 versus control values.

approximately 49 proteins. Although a large numberof ribosomal proteins exist, many of them do not ap-pear to be essential for ribosome function. Ribosomalproteins participate in various extra-ribosomal activit-ies [3032]. The specific function for many ribosomalproteins remains unknown. Cumulative evidence sug-gests that ribosomal proteins are involved in the ma-lignant transformation, growth of cancer cells, tumoraggressiveness and metastasis [3339]. For example,over-expression of several ribosomal proteins has beenreported in colon [36, 40, 41], liver [39], brain [42],breast [43] and prostate [44] carcinomas.

The ribosomal protein rpL7a is DNA-damage in-ducible. Ben-Ishai et al. [45] have shown that ul-traviolet irradiation and other DNA-damaging agentsspecifically induce transient increases of rpL7a tran-scripts. UV irradiation and DNA damage act as both atumor initiator and as a tumor promoter [4649]. Thissuggests rpL7a may be involved in carcinogenesis anddevelopment of tumors. The involvement of rpL7ain tumorogenesis is supported by the finding thatrpL7a activates the trk (tyrosine receptor kinase) on-cogene by contributing the amino-terminal-activatingsequence to the receptor kinase domain of trk [50].Furthermore, over-expression of rpL7a has been ob-served in prostate, colon and brain tumors [36, 42, 44].Our results suggest that rpL7a is over-expressed inbreast cancer cells. Elevated expression of rpL7a in tu-mor cells suggests that it plays a role in the malignanttransformation and the growth of cancer cells. rpL7ais believed to be involved in cell growth and differenti-ation [51]. For example, thyrotropin (TSH) is a growthfactor for thyroid follicular cells, and TSH stimula-

tion of thyroid follicular cell leads to cellular growth,replication and differentiation. Treatment of thyroidcells with TSH results in a significant up-regulationof rpL7a [51].

It has been suggested that control of cell growthmay be regulated, at least partially, by rpL7as interac-tion with nuclear hormone receptors. rpL7a specific-ally interacts with human thyroid hormone receptor(THR) and retinoic acid receptor (RAR) which in turninhibits transactivation of the two nuclear hormone re-ceptors [52]. Retinoic acid (RA) and thyroid hormone(TH) play an important role in regulation of cell pro-liferation, differentiation and death. RA and TH areknown to inhibit the proliferation of various cancercells including breast cancer cells [53, 54]. Lack ofsensitivity of advanced lung and breast cancer to RAis a suggested mechanism for abnormal growth of can-cer cells [53]. rpL7a is a co-repressor for RAR andTHR by inhibiting RAR and THR-mediated transcrip-tion of target genes [52]. Up-regulation of rpL7a byethanol may diminish the sensitivity of breast cancercells to RA and THR and thereby result in disruptionof growth control.

The precise mechanism(s) by which ethanol mod-ulates the expression of rpL7a is not clear. Ethanolunder certain circumstances induces DNA damage[5557]. It is likely that ethanol activation of rpL7ais triggered by DNA damage. Alternatively, ethanolexposure alters the levels of circulating estrogen andup-regulate the expression of estrogen receptors (ER)in breast cancer cells [5860]. It is noted that the5-flanking sequence of rpL7a gene contains severalimperfect estrogen responsive elements (ERE). Recent

36 Y Zhu et al.

study suggests that ER may interact with these im-perfect or half-sites of ERE and subsequently activatethe regulated genes [61]. Therefore, ethanol may in-directly regulate the transcription of rpL7a gene byaltering ER levels.

Although the exact mechanisms by which chronicalcohol consumption stimulates carcinogenesis and tu-mor development are not known, experimental studiesin animals support the concept that ethanol alone isnot a carcinogen, but a co-carcinogen and/or a tumorpromoter [62, 63]. Ethanol exposure is shown to en-hance the metastasis and aggressiveness of mammarytumor in rat [64]. Recent studies using cultured hu-man breast cancer cells confirm that ethanol promotesproliferation, migration and invasion of tumor cells invitro [17, 60, 65]. The current study identifies thatribosomal protein rpL7a is an ethanol-regulated genein breast cancer cells. rpL7a has been suggested tobe involved in tumorogenesis and growth of cancercells. Therefore, modulation of rpL7a expression maybe a mechanism underlying the promoting effect ofethanol on cancer development. Further exploration ofrpL7a function in malignant transformation and can-cer progression should provide important insights intonot only the target of ethanol toxicity but also themechanisms of carcinogenesis.

Acknowledgements

We thank Drs. Frank Reilly and Michael Miller fortheir critical reading of this manuscript. This researchwas supported by grants from the National Institutesof Health (AA12968 and CA 90385).

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Address for offprints and correspondence: Jia Luo, Ph.D., Depart-ment of Anatomy, West Virginia University School of Medicine,Robert C. Byrd Health Science Center, Morgantown, WV 26506-9128, USA; Tel.: 304-293-0608; Fax: 304-293-8159; E-mail:jluo@hsc.wvu.edu