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Research Article

Expression of the RAI gene is conducive to apoptosis: Studiesof induction and interference

Magdalena J. Laskaa, Dorthe Strandbyga°rda, Anette Kjeldgaarda, Mette Mainsa,Thomas J. Corydona, Ashfaque A. Memonb, Boe S. Sørensenb, Ulla Vogelc,Uffe B. Jensena,d, Bjørn A. Nexøa,⁎aInstitute of Human Genetics, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, DenmarkbDepartment of Clinical Biochemistry, Aarhus University Hospital, Aarhus, DenmarkcNational Institute of Occupational Health, Copenhagen, DenmarkdDepartment of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author.E-mail address: nexo@humgen.au.dk (B.A

0014-4827/$ – see front matter © 2007 Elsevidoi:10.1016/j.yexcr.2007.05.006

A B S T R A C T

Article Chronology:Received 24 October 2006Revised version received30 April 2007Accepted 2 May 2007Available online 22 May 2007

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shownthat the region encompassing RAI is important for the development of cancer in youngand middle-aged persons. It has been speculated that the RAI product induces apoptosisby blocking NF-κB or inhibits apoptosis by blocking p53. Either way the gene couldinfluence the survival of precancerous lesions. Here we report that the expression of RAImRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergoapoptosis by various means, such as treatment with etoposide, calcium ions, orinterleukin-2 and/or serum deprivation. Treatment with etoposide increased the contentof RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAIexpression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatmentwith the NF-κB-inhibiting substance sulfasalazine relieved this dependence. In thetransformed cell line HEK-293 the association between RAI induction and apoptosisseemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficientfor apoptosis induction in non-transformed cells. Our results could be explained by aNF-κB mediated mechanism.

© 2007 Elsevier Inc. All rights reserved.

Keywords:ApoptosisLymphocytesGene RAINF-κBEtoposide VP-16

Introduction

The gene RAI, an acronym for RelA-associated inhibitor, is alsoknown as iASPP and PPP1R13L. Recent investigations haveshown that the region of chromosome 19, encompassing thisgene is important for the development of cancer in young andmiddle-aged persons [1]. Persons that are homozygous for aspecific haplotype in this region: ERCC1 Asn118AsnA, ASE-1

. Nexø).

er Inc. All rights reserved

G-21AG, RAI IVS1 A4364GA , are at 5 to 12-fold increased risk ofdeveloping cancer of the breast, lung and skin before age 55[2–4]. Importantly, persons that are heterozygous for thehaplotype have approximately the same risk as the basepopulation. This suggests that the gene belongs to the generalgroup of tumour-suppressor genes, i.e. has a tumour-pre-ventive function that somehow is defective in the haplotypehomozygotes.

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These findings are easy to reconcile with reports that ex-pression of RAI induces apoptosis by blocking one subunit ofthe transcription factor NF-κB [5,6]. The direct interaction bet-ween RAI and p65 (RelA) most likely contributes to theinhibition of NF-κB activity through interfering with its NF-κB DNA binding activity [5]. Thus, it may act as a novel NF-κB-binding protein, causing a repression of transcriptionalactivity, and preventing precancerous cells to survive. Therecessive risk-associated allele would then be defective in thisfunction. On the other hand they seem at odds with otherreports that RAI may be oncogenic, acting by blocking theapoptosis-promoting effect of p53 [7,8], and hence could beselected for in tumour development [8]. In this case, we wouldexpect the risk-associated allele to be dominant. Furthermore,it is a worrisome detail thatmost experiments on the action ofRAI have been performed in tumour-derived cell lines (HEPG-2,MCF-7 and A549) [7], whose intracellular regulation, inparticular the signal transmissions pathways, easily could bederanged.

The structure of the final RAI message and the primaryprotein product has been a subject of discussion. The RAImRNA encodes a protein with high structural homology to theC-terminal 200-amino-acid segment of 53BP2, containing fourankyrin repeats and an SH3 domain that are known to beinvolved in specific protein–protein interactions [5]. Originally,the RAI product was described as a 351-amino-acid peptidechain [5]. However, it was later realized that the publishedsequence contained two frame-shift errors. As a consequencethe protein seems to be much longer, 828 amino acids,consisting of a large N-terminal extension relative to thegene originally described (Diagram 1). In accordance with thelatter results investigations ofmRNA sizeswithNorthern blotshave revealed a 3.2 kb mRNA, and not a 2.6 kb mRNAcorresponding to the 351 amino acid short form. However,we tested early on that an RT–PCR assay for the short mRNA,designed so that it reacts with its 5′-UTR, and positioned in anintron of the long mRNA consistently produced positiveresults, which might indicate that a form of RNA containingthis region exists.

To resolve some of these issues we have studied the asso-ciation of RAI expression with apoptosis in short termcultures of normal cells as well as in non-tumorigenic andtumorigenic cell lines. We here report that the levels of RAImRNA and RAI protein are increased in cells induced toundergo apoptosis, and that inhibition of RAI expression inthese cells decrease apoptosis. Thus, modulation of RAIexpression seems to be an integral part of the apoptoticresponse.

Diagram 1 – Previously and presently inferred mRNAs forRAI.

Materials and methods

Cell isolation and primary cell cultures

Blood samples from five healthy donors were obtained fromSkejby Hospital, Denmark. Primary peripheral mononuclearcells (PBMCs) of the first three donorswere isolated frombuffy-coat using Ficoll-Paque density centrifugation (AmershamPharmacia, Uppsala, Sweden). From last two donors, primaryperipheral blood lymphocytes were isolated using BDVacutainer® CPT™ cell preparation tubeswith sodiumheparin(Cell PreparationTubes, BectonDickinsonVacutainer Systems,Franklin Lakes, NJ, and USA) according to the manufacturers.Human PBMCs were stimulated with 5 μg/ml phyto-hemagglutinin (PHA) and 30 U/ml interleukin-2 (IL-2) andfurther cultured in AIM-V medium (Gibco Invitrogen, GrandIsland) supplemented with 3% fetal bovine serum, 30 U/mlinterleukin-2 (IL-2).

Cell lines

WI-38 (human lung fibroblasts) was obtained from AmericanType Culture Collection (Rockville, MD). The cells were cul-tivated in advance modified Eagle's medium (A-MEM; Gibco,Life Technology, Rockville, MD, USA) supplemented with 10%fetal bovine serum (Gibco, Life Technology), 100 U/ml of peni-cillin and streptomycin and 2 mM L-glutamine.

HEK-293 (adenovirus-transformed embryo kidney cells) wasobtained fromAmerican Type Culture Collection. HEK-293 cellswere cultivated in high glucose (4.5 g/l) Dulbecco's modifiedEagle's medium (DMEM; Gibco, Life Technology) supplementedwith 10% fetal bovine serum (Gibco, Life Technology), 100 U/mlof penicillin and streptomycin and 2 mM L-glutamine.

Apoptosis induction

For induction of apoptosis following chemotherapeutic drugsand methods were tested:

Etoposide, VP-16 treatmentHumanPBMCs,WI-38cells,andHEK-293cellswereculturedunderstandard conditions. For apoptosis inductions cells were washedonce in PBS and culture medium supplemented with VP-16, eto-poside (Sygehusapotekerne, Denmark) was added to the cells atthe indicated concentrations and for the indicated time periods.Control cells were left untreated in complete culture medium.

Sulfasalazine treatmentHuman PBMCs and WI-38 cells were cultured under standardconditions. For apoptosis induction cells were washed once inPBS and culture medium supplemented with sulfasalazine(Biochemica, Fluka, Switzerland) was added to the cells at theindicated concentrations and for the indicated time periods.Sulfasalazine was freshly dissolved in 1 M ammonia at 50 mM.Control cells were left untreated in complete culture medium.

Interleukin-2 and FCS withdrawalHuman PBMCs were cultured under standard conditions incomplete medium. For apoptosis induction cells were washed

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once in PBS and resuspended in AIM-V (Gibco Invitrogen),culture medium deprived of either FCS, IL-2, or both compo-nents. Control cells were grown in complete culture medium.Cells were cultured under indicated conditions and for indi-cated time periods.

Immunofluorescence-microscopic detection of RAI protein

The mouse monoclonal antibody, LX49.3, reacting with RAIprotein was a generous gift from Dr. Xin Lu, London, UK. Forthe detection of RAI protein WI-38 cells were grown on cover-slips, washed with PBS, fixed for 10 min with 3% paraformal-dehyde in isotonic PBS, washed 2 times in PBS, permeabilizedfor 30 min with standard buffer [0.1% bovine serum albumin(BSA) and 0.3% Triton in PBS]. They were then treated with theprimary antibody in standard buffer at a dilution of 1:500,washed in PBS 3 times for 3 min, treated with Alexa 546-con-jugated goat anti-mouse second antibody (1:500) in standardbuffer for 1 h at room temperature. Finally, the slides weremounted with glycogel with 2.5% antifade (T48, DAKO, Glos-trup, Denmark). The pictures were taken on a Leica ConfocalMicroscope (Heidelberg, Germany) and analyzed using LeicaConfocal Software LCS Lite 2611537.

Determination of apoptosis by FACS

For quantitative determination of apoptosis, cells were treatedas indicated and acquired on a FACS Aria (Becton Dickinson,USA). Samples were analyzed using FACSDiva Software (BDBiosciences). Phosphatidylserine exposure in short term cul-tured lymphocytes, or cultured fibroblasts, as a marker of celldeath, was measured by the binding of Annexin V–fluoresceinisothiocyanate (FITC), by using Annexin V–FLUOS (RocheDiagnostics) according to the instructions of themanufacturer.For differentiation of apoptosis and necrosis, cells were alsostained with 100 ng/ml propidium iodide (PI) to detectmembrane integrity and were immediately analyzed. Fluores-cence of Annexin V–FITC and PI were acquired in log mode onchannels 1 and 2, respectively. Forward-scatter versus side-scatter was used to exclude cell debris. A minimum of 10,000events were collected on each sample and acquired.

Sorting of apoptotic cells

The cells were sorted using low sheet pressure (20 psi) andstandard purity mask (Diva software). One million cells weresorted, immediately centrifuged, and processed for furtheranalysis. The sorting step was performed in less than 15 minon ice. In all sorting experiments, the viable population ofuntreated cells were also sorted and used as a referencepopulation.

Apoptosis assay by microfluorometry

The effect of RAI silencing on apoptosis was assessed usingAlexa Fluor® Annexin V (Molecular Probes, Invitrogen, USA) asper themanufacturer's protocol. The cells were plated in 6-wellplates (3.5×105). 24 h later, the cells were transfectedwith FITC-conjugatedRAI siRNAsornon-silencing siRNA(QiagenSciences,Inc., Germantown, MD), using Lipofectamine 2000™ Reagent

(Invitrogen, USA) as described by the manufacturer. 36 h posttransfection, cells were washed once with phosphate-bufferedsaline (PBS) and new complete growth medium or mediumsupplemented with 40 μM VP-16, etoposide (Sygehusapote-kerne, Denmark) was added. Cells were incubated for another48 h. At this time, the cells were ready for further apoptosisdetection experiment. Briefly,mediawas aspirated and the cellswere washed with PBS. The cells were then incubated withannexin binding buffer (1×), Alexa Fluor® Annexin V (1×) for30minat roomtemperature indark. The cellswerewashedwithannexin binding buffer (1×) followed by PBS. Next, cells werevisualized and counted with a Leica DM IRBE microscope.

RNA extraction, RT–PCR, and real-time PCR

RNA was isolated using NucleoSpin® RNA II (Macherey-Nagal,Germany) according to the instruction of the manufacturer.cDNAwas synthesized using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the instruction of the manu-facturer. Real-time PCR analysis was performed using a Light-Cycler 3.0 (Roche Diagnostics). About 2 μl of cDNA (from a total20-μl reaction) was used in a 20-μl real-time PCR. The reactionscontained 13.6 μl water, 4 μl LightCycler (Fast Start DNAMasterSYBR Green I; Roche Diagnostics), 0.2 μl forward primer, and0.2 μl reverse primer. PCR amplifications were performed, afteran initial denaturation at 95 °C for 10 min, for the abovespecified number of cycles according to the following regime:denaturation at 94 °C for 30 s, annealing at the above specifieddegrees for 30 s and extension at 72 °C for 30 s. The primersequences and annealing temperatures were as follows:

β-actin (annealing temperature 59 °C)Actin-s 5′ CTG TGC TGT CCC TGT ATG CC 3′,Actin-as 5′ GTG GTG AAG CTG TAG CC 3′,RAI Assay 1 (annealing temperature 64 °C)Forward primer 5′ CAG GCG GTG AAG GAG 3′Reverse primer 5′ AAA TCC ACG ATA GAG 3′,RAI Assay 2 (annealing temperature 60 °C)Forward primer 5′ ACT CGA CTC AGC CAA TAA TGC 3′Reverse primer 5′ GTG GTG AAG CTG TAG CC 3′

The crossing point (CP) for each transcript was measuredand defined at constant fluorescence level in the LightCyclersoftware 3.3 (Roche Diagnostics). The value for the test genewas normalized to the β-actin value and relative quantifica-tion was determined using mathematical model presented byPE Applied Biosystems (Perkin Elmer, Foster City, CA).

Construction of siRNA

The siRNAs sequences targeting RAI (GenBank accession noAF07803) corresponded to the coding regions of the gene. Thetarget sequences of siRNA duplex 1 and siRNA duplex 2 wereas followed:

RAIsiRNA01 TAC AAT AAG GTA GGC ATG TAARAIsiRNA02 CAC TGG TTT AAT GTT TAT AAA

A negative control siRNA was purchased from QiagenSciences, Inc. (Germantown, MD), with the target sequence,

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AAT TCT CCG AAC GTG TCA CGT, which has no matcheseither in the viral or human genome. Synthetic siRNAs wereobtained from Qiagen Sciences, Inc. (Germantown, MD). Toobtain a 20 μM solution, the lyophilized siRNAswere dissolvedin 1 ml siRNA suspension buffer, then heated to 90 °C for1 min, incubated at 37 °C for 60 min, and stored at −20 °C.

siRNA transfection

Human PBMCs, and WI-38 cells were transfected with smallinterference siRNAs using Lipofectamine 2000 reagent (Invi-trogen, USA), following the protocol set by the manufacturer.48 h post transfection cells were stimulated for apoptosis asdescribed previously. Transfected untreated cells were used asa control.

Western blot analysis

Following the specific treatment, as described above, humanPBMCs or WI-38 cells were harvested and washed with ice-cold PBS (10 mM, pH 7.4). Ice-cold RIPA buffer (150 mMNaCl, 50 mm Tris–HCl pH 7.4, 1 mM EDTA, 1% Np-40) withfreshly added PMSF (1 mM) and protease inhibitors (RocheDiagnostics) was added. The suspension (cells with lysisbuffer) was transferred into a centrifuge tube and wasplaced on ice for 15 min with occasional inversion to ensurea complete lysis of the cells. The cell suspension wascleared by centrifugation at 14,000×g for 15 min at 4 °C, andthe supernatant (total cell lysate) was either used immedi-ately or stored at −70 °C. The protein concentration wasdetermined using Coomassie Protein Assay Kit (Pierce) asper the manufacturer's protocol.

For immunoblot analysis 20 μg protein was subjected toSDS–PAGE, using 10% Tris–HCl gel (Bio-Rad). The protein wastransferred to a nitrocellulose membrane and blocked withTBS–Tween (0.1%) plus 5% skim milk. The membrane wasprobed with an appropriate primary antibody followed by asecondary horseradish peroxidase (HRP)-conjugated antibody.Membranes were visualized using ECL and exposure to ECLHyperfilm (both Amersham Biosciences, UK). The followingantibodies were used: anti-iASPP/RAI (Invitrogen, USA), goatanti-rabbit immunoglobulins/HRP (DAKOCytomation, Den-mark A/S), and anti-tubulin (Abcam, UK).

Results

In order to study the role of the RAI gene in apoptosis wecultured normal human primary lymphocytes, and the non-transformed fibroblast strain WI-38 under various conditionsleading to apoptosis. To quantify the level of RAI mRNA weused two real-time RT–PCR assays. ASSAY1: an assay for the 5′part of the 3.2 kb form of RAI, which should not detect theputative short 2.6 kb form of RAI mRNA. ASSAY2: an ampli-fication of a region, which should be specific for the putative2.6 kb of RAI mRNA and located in its 5′-UTR, while located inan intron and removed in the 3.2 kb mRNA. Thus, the 3.2 kbmRNA should not be detected by the ASSAY2. The sizeanalyses and sequencing of the PCR amplified regionsconfirmed that the expected fragments were amplified.

To confirm the specificity of the assayswe size fractionatedmRNA on agarose gels, eluted mRNA of different size bandsand performed RT–PCRs on the eluted RNA (results notshown). Importantly, ASSAY2 did not detect a shorter 2.6 kbmRNA, but rather a roughly 6 kb mRNA, presumably corre-sponding to the previously described 6.5 kb Northern band [5].This suggests that this band is actually a pre-message stillcontaining intron 7 of the gene. There was no evidence of a2.6 kb message. ASSAY1 detected both 6.5 kb and 3.2 kb RNAs(results not shown).

In the following we use two cell strains and a permanentcell line. We consider the short term cultures of lymphocytes(MJL3) non-malignant, as they have a finite life span andnormal karyotypes, and also consider WI-38 non-malignant,since it has a finite life span, is essentially diploid, has a non-transformed morphology and does not form tumors in nudemice [9,10]. In contrast, we consider HEK-293 malignant as itwas transformed with the early region of adenovirus 5 [11].

To quantify apoptosis we used Annexin V–FLUOS stainingof cells followed by FACS analysis. This staining detects theinversion of the cell membrane leading to the exposure ofphosphatidylserine groups on the external cell surface, acharacteristic part of apoptosis. Fig. 1 shows the induction ofapoptosis in MJL3 quantified in this manner after exposure tothe etoposide drug VP-16. Clearly the fraction of cells, whichbinds high levels of Annexin V and thus registers to the rightin the plots (above 400), increased after the treatment, inconcordance with the fact that VP-16 induces apoptosis [12]. Itis also obvious that these cells had a certain level ofspontaneous apoptosis.

Table 1 shows the fraction of apoptotic MJL3 cells atdifferent time-points after beginning of treatment of thecells with the optimal concentration (20 μM) of etoposide,VP-16, as well as the induction of RAI mRNA levels in the cellsat these times. Clearly the fraction of cells, which bound highlevels of Annexin V increased after the treatment, inconcordance with the fact that VP-16 induces apoptosis [12].It is also obvious that these cells had a certain level of spon-taneous apoptosis. The table also shows that the RAI mRNAsbecame elevated at about the same time as apoptosis wasinduced, with a tendency that the 6.5 kb RNA, measured byASSAY2, responded earlier than the combined mRNAs,measured by ASSAY1 . To further substantiate the associationof the RAI mRNA induction with apoptosis in these cells, wealso FACS-sorted the MJL3 cells into non-apoptotic and apop-totic cells 24 h after beginning of the treatment with VP-16. Fig.2 shows the mRNA levels in untreated cells and in the sortedcells. The level of RAImRNAwas highest in the apoptotic cells.The level was also somewhat elevated in the treated cells,which had not (yet) become apoptotic.

To see if the association of RAI mRNA induction and apop-tosis was a specific effect of the VP-16 treatment, we inducedapoptosis in MJL3 with various other drugs andmanipulationsof the culture medium. Dose response curves for VP-16, sulfa-salazine and calcium ions were developed, as were time-curves. Similarly, time curves were developed for cultivationin the absence of serum, interleukin-2 or both. Table 2 lists thelevels of apoptosis and RAI mRNA in the cultures at the op-timal concentrations and time. All modes of inducing apop-tosis also induced RAI mRNA.

Fig. 1 – Apoptosis induction in primary human lymphocytes (MJL3 cells). Flow Cytometry analysis of Annexin V–FLUOS/Propidium iodide-stained MJL3 cells in complete medium (control), or medium supplemented with increasing doses ofetoposide VP-16. The results are from one experiment out of three independent experiments.

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Similar to Fig. 2 we sorted cells induced by sulfasalazine orcalcium ions, into apoptotic and non-apoptotic cells. Asexpected the mRNA levels were increased and highest in theapoptotic cells (results not shown).

To see if the association of apoptosis and RAI mRNAinduction was a peculiarity of lymphocytes, we treated othercell cultures with VP-16 and quantified apoptosis and RAImRNA levels (Table 3). The WI-38 cells had to be tested after48 h to see a strong effect, presumably a reflection of the slowgrowth of these cells. All the cells induced RAI mRNA duringthe VP-16 treatment. However, only the non-transformedlymphocytes and WI-38 went into apoptosis, whereas thetransformed HEK-293 cells died without registering in ourassay, i.e. without signs of membrane inversion. Presumablythese cells did not go to apoptosis under those conditions, but

Table 1 – Time curve of induction of apoptosis (with 20μMetoposide VP-16) andmRNA RAI level asmeasured by twoRT–PCR assays in MJL3 lymphocytes

Time Average percentapoptotic cells

RAI mRNAASSAY1a

RAI mRNAASSAY2a

0 10 1 14 19 3 68 25 6 724 55 31 10

a Average of three experiments.

rather died by necrosis, which could be explained by the hightoxicity of the treatment.

To show that induction of RAI also happened at the proteinlevel we stained WI-38 cells with a monoclonal antibodydirected towards the C-terminal part of the RAI protein andstudied its presence in normal cells and after etoposide-treatment using immunofluorescencemicroscopy (Fig. 3). Theresults showed a marked increase in the amount of RAIprotein during the treatment. Moreover, the protein clearlyaccumulated in the nucleus. In contrast, when the resultswere repeated with HEK-293 cells, RAI protein levels in-creased, but the protein remained in the cytoplasm. Westernblots of RAI protein also confirmed that the protein levelincreased during apoptosis (Fig. 4).

In the following set of experiments we chose to see, ifinduction of RAI mRNA was instrumental for apoptosis innon-malignant cells. For this purpose we used siRNAs specificfor the RAI mRNAs. One siRNA, RAIduplex1, was homologousto the 3′-UTR of the RNA and presumably reacted with both3.2 kb mRNA proper and the 6.5 kb pre-message. One siRNA,RAIduplex2, reacted to sequences from intron 7 representingthe 6.5 kb pre-message, but not present in the 3.2 kb mRNAproper. We also used a non-silencing siRNA, i.e. an analogousRNA, whose sequence did not correspond to any humansequence. The siRNAs were transfected into cultures of MJL3lymphocytes. 48 h later the cultures were analyzed for RAImRNA. Figs. 5A and B shows that the RAI mRNAs weresuppressed by RAIduplex1 as measured by RT–PCR assays.

Table 2 – Induction of apoptosis and RAI mRNA level inMJL3 cells after treatment with various regimens for 24 h

Treatment Sample Percentapoptotic

cells

Foldincrease inRAI mRNAASSAY1a

Foldincreasein RAImRNA

ASSAY2a

VP-16 (20 μM) Untreated 10Treated 55 31 6

– Serum Untreated 1.8Treated 8 1.3 0.8

– IL-2 Untreated 1.8Treated 10.5 2 2

– Serum – IL-2 Untreated 1.8Treated 15 5.1 4.0

Sulfasalazine(2.5 mM)

Untreated 5Treated 60 25 3

Calciumchloride(10 mM)

Untreated 1.5Treated 15.5 5.6 1.5

a Average of three experiments.

Fig. 2 – mRNA levels in MJL3 cells sorted for apoptosisafter induction with 20 μM VP-16. (A) mRNA levels asmeasured by ASSAY1. (B) mRNA level as measured byASSAY2. Statistical comparisons show that the apoptoticcells have more RAI mRNA than the non-apoptotic cells: (A)p=0.0008 (t-test; two-sided); (B) p=0.002 (t-test, two-sided).

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RAIduplex2 also suppressed the 6.5 kb RNA and to a lesserextent the 3.2 kb RNA. We also tested if the siRNAs reducedthe level of RAI protein in MJL3 cells. The Western blotshowed a moderate decrease of the RAI protein by eithersiRNA (Fig. 6).

To see if the siRNAs influenced apoptosis MJL3 cultureswere transfectedwith either siRNA orwith non-silencing RNA.24 h later the cells were treatedwith VP-16. 30 h later again thecells were analyzed for apoptosis. Fig. 7 shows that inductionof apoptosis was reduced to approximately half by treatmentwith siRNA RAIduplex1 relative to treatment with non-silencing RNA. Fig. 8 shows in more detail the statistics ofapoptosis in these cultures. This is direct evidence that RAIinduction plays an active role in the induction of apoptosis byVP-16. We also showed that apoptosis caused by deprivationof serum and IL-2 could be reduced by the siRNAs (results notshown). Finally, we made similar experiments, where apop-tosis was induced in WI-38 by VP-16. Again inhibition of RAImRNA reduced the level of apoptosis (Fig. 9). RAI also played arole in induction of apoptosis in these cells.

In the above experiments transfection with siRNA wasincomplete and typically reached 30–70% transfected cells.This limited the effects obtainable. In order to test the effect ofthe siRNAs further we transfected WI-38 cultures on cover-slips with non-silencing RNA or RAIduplex2 that werefluorescent (green). 36 h later we treated some of the cultureswith 20 μM etoposide VP-16, and 48 h later we stained the cellswith Annexin V (red) and analyzed in fluoromicrography. Thevisual impression of the cultures transfected with siRNAs wasthat cells were either red or green but usually not both, i.e.transfection reduced apoptosis. In cultures transfected withnon-silencing RNA, the cells were red and green indiscrimi-nately (results not shown).

Next we counted the fraction of green cells that were alsored, i.e. the transfected cells that had gone into apoptosis. Thenon-transfected control cells had no green color and werecounted in toto. The results are shown in Fig. 10. Clearly, the

levels of apoptosis after etoposide were diminished if the cellshad been transfected with siRNA, and there were suggestionsthat the spontaneous level of apoptosis was also reduced bythe transfection. In contrast, non-silencing RNA had no effect.We conclude that the RAI siRNAs reduce apoptosis after VP-16.

In contrast, in experiments where sulfasalazine was usedas inducer in WI-38, apoptosis appeared unaffected by thesiRNAs (Fig. 11A). To be sure the siRNAs were working, wequantified the mRNAs in the cells with both ASSAY1 andASSAY2 (Fig. 11B). The siRNA were clearly active. Presumably,apoptosis can proceed after sulfasalazine treatment in spite oflowered levels of RAI mRNA.

Discussion

Themost important result of this report is that induction of RAImRNA and apoptosis are associated in non-transformed cells.Thus, all the apoptosis-inducing treatments tested inducedRAI mRNA. This was true for short-term cultures of normallymphocytes, and the non-transformed cell strain WI-38. Thesorting experiments confirmed that it was primarily theapoptotic cells, and much less the surviving ones that hyper-expressed RAI. Immunofluorescence analysis confirmed that

Table 3 – Induction of apoptosis and RAI mRNA level with optimal concentration of etoposide VP-16 in the different cells(20 μM VP-16 for all cell stains except WI-38: 40 μM)

Cell type Culture Time ofsampling (h)

Percentapoptotic cells

Fold increasein RAI mRNAmeasured with

ASSAY1a

Fold increasein RAI mRNAmeasured with

ASSAY2a

MJL3 Untreated 24 10Treated 24 55 31 6

WI-38 Untreated 48 10Treated 48 35 6 4.8

293 Untreated 24 1Treated 24 1.4 5.5

a Average of three experiments.

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expression of RAI protein increased during the treatment withetoposide and showed that the protein translocated to thenucleus. The latter may be a possible second regulation of RAIaction. At least the translocation was absent in the 293 cells,which also did not go into apoptosis.

The evidence indicates that our RAI siRNAs were effectiveand reduced both mRNA and protein levels. Moreover,inhibition of RAI induction by siRNA reduced the level ofapoptosis in non-malignant cells. Thus, the burst of RAIexpression not only occurred simultaneous with apoptosis,but also seemed necessary for apoptosis to proceed. In otherwords, RAI induction seemed causatively related to apoptosis.We preferred this proof of causality to studies of over-expression. In the latter situation the high concentration ofthe factor in question might lead to spurious interaction with

Fig. 3 – Immunofluorescence pictures of WI-38 cells cultivated nVP-16. (A, C) Normal WI-38 cells; (B and D) WI-38 cells induced foantibody to RAI protein; C and D: staining with monoclonal antib

other intracellular substances. Moreover, over-expressionexperiments would fail, if the effect of RAI was contingentupon other events in the cell.

A limitation of the experiments involving siRNA transfec-tion was the moderate (twofold) size of the suppression ofapoptosis by the siRNAs. However, transfections with fluor-escent siRNA have suggested that only 30% to 70% of the cellstake up detectable amounts of siRNA. Thus, the simpleinterpretation would be that suppression is virtually completein the transfected cells and virtually all transfected cells avoidapoptosis.

We tried to make a three-color analysis on MJL3 cells,working both with fluorescent siRNA and fluorescent AnnexinV in addition to the propidium iodide. Because of the increaseddemand on discrimination, the results became quite depen-

ormally and after induction of apoptosis with etoposider apoptosis. A and B: staining procedure without monoclonalody to RAI protein.

Fig. 4 – Western blot of RAI protein in MJL3 cells inducedby sulfasalazine. LaneA: Untreated cells. Lane B: Cells treatedfor 24 h with 2.5 mM sulfasalazine.

Fig. 6 – Suppression of RAI protein inMJL3 cells by siRNAs asvisualized in aWestern blot. (A) Primary human lymphocytesMJL3; (B) MJL3 cells transfected with non-silencing RNA;(C) MJL3 cells transfected with RAI siRNA Duplex 1; (D) MJL3cells transfected with RAI siRNA duplex 2.

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dent on gating in the FACS. However, the results supported theconclusions above (results not shown). Experiments with cellsstably transfected with a siRNA vector will probably benecessary to take this issue further.

Fig. 5 – Suppression of RAI mRNA level in MJL3 cells bysiRNAs. Primary human lymphocytes MJL3 were transfectedwith small interference RNA duplex1 or duplex2.Non-silencing siRNA was used as a control. The RAI mRNAlevel was quantified by real-time RT–PCR ASSAY1 (A), orASSAY2 (B) and normalized to mRNA β-actin. The data arepresent as the RAI mRNA expression level (mean±SEM)relative to control cells of three independent experiments.Statistical tests show that ASSAY1 is reduced by bothRAIduplex1 (p=0.04, t-test, two-sided) and RAIduplex2(p=0.04, t-test, two-sided) relative to non-silencing RNA).Statistical tests show that ASSAY2 is marginally reduced bytransfection with RAIduplex1 (p=0.07, t-test, two-sided), butnot significantly reduced by transfection with RAIduplex2(p=0.28; t-test, two-sided).

We have quantified two RAI messenger molecules, the3.2 kb mRNA proper and a larger 6 kb molecule, which con-tains at least one large intron, and which could be a pre-

Fig. 7 – Suppression of apoptosis in MJL3 cells aftertransfection with RAI siRNAs. (A) FACS analysis of untreatedcells transfectedwith non-silencing RNA. (B) FACS analysis ofuntreated cells transfected with siRNA RAIduplex1.(C) Comparison of FACS analyses of cells treated with VP-16and either transfected with non-silencing RNA (green) ortransfected with siRNA RAIduplex1 (black line). (Forinterpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

Fig. 8 – Inhibition of etoposide VP-16-induced apoptosis inMJL3 cells by siRNAs. (A) The primary human lymphocytes,MJL3, were left un-transfected, or transfected withRAIduplex1 or non-silencing RNA. After 24 h the cells wereinduced for apoptosis by treatment with increasing doses ofetoposide VP-16. After 24 h exposure cells were stained withAnnexin V–FLUOS/Propidium iodide and analyzed by FlowCytometry. The data present the average number of apoptoticcells (±SEM) in three independent experiments. (B) Primaryhuman lymphocytes MJL3 were left un-transfected, ortransfected with RAIduplex2, or non-silencing RNA. Cellswere induced for apoptosis by treatment with etoposideVP-16 at a concentration of 20 μM. After 24 h exposure cellswere stained with Annexin V–FLUOS/Propidium iodide andanalyzed by Flow Cytometry. The data present the averagenumber of apoptotic cells (±SEM) in three independentexperiments. Statistical comparisons show that theapoptosis levels were lower in cells transfected withRAIduplex1 (p=0.009, t-test, two-sided) or transfected withRAIduplex2 (p=0.02; t-test, two-sided) than in cellstransfected with non-silencing RNA.

Fig. 9 – Inhibition of etoposide VP-16-induced apoptosis inWI-38 cells by siRNA. The human lung fibroblasts cells,WI-38 were left un-transfected, or transfected withRAIduplex2, or non-silencing siRNA. After 36 h the cells wereinduced for apoptosis by treatment with etoposide VP-16 atconcentration 40 μM. After 48 h exposure the cells werestained with Annexin V–FLUOS/Propidium iodide andanalyzed by Flow Cytometry. The data present the averagenumber of apoptotic cells (±SEM) in three independentexperiments. Statistical comparison shows that transfectionwith RAIduplex2 reduced the level of apoptosis relative totransfection with non-silencing RNA (p=0.05, t-test,two-sided).

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messenger. In general we have found the two assays to movein parallel, sometimes with a tendency that the 6.5 kb RNAresponded faster to induction. In another situation, starvationof MJL3 for serum and IL-2, the kinetics of induction alsoseemed different, ASSAY2 having an early peak at 8 h, whichwas not discernible in ASSAY1. Other than that the two assaysseemed almost interchangeable. We also found the twosiRNAs tested to be largely similar in ability to reduce mRNARAI expression level and decrease apoptosis. One of these

siRNAs should not react with the mature 3.2 kb mRNA.However it might easily react with an immature stage, forinstance the observed presumed pre-messenger. It is thereforenot surprising that it was active in reducing RAI mRNA.

Our findings are in accord with the original report that RAIprotein inhibits RelA and thereby inhibits at least some aspectsof NF-κB action, generally considered to be anti-apoptotic [13].The results are also in accord with the finding that RAI-mediated sensitivity to cancer is a recessive feature [13] i.e. RAIis a tumor-suppressor gene. They are not in immediate agree-ment with the report that RAI protein blocks apoptosis byinhibiting the pro-apoptotic tumor suppressor gene p53 [7].However, one should be aware of the complexity of theintracellular signal pathways. Thus, it is possible that RAIperforms both reactions simultaneously. One could be theprimary effect while the second could be a feedback, perhapsnecessary to keep the overall circuitry in balance. It is alsopossible that RAI protein may promote and inhibit apoptosisunder different circumstances. For instance, if the cells have asurplus of active NF-κB, and RAI protein concentration can riseto high levelswithout blocking this factor, or if the cells are ableto survivewithoutNF-κB, anti-apoptotic effectsmay dominate.

The transformed HEK-293 cells induced RAI when treatedwith a variety of normally apoptosis-inducing chemicals, butfailed to go into apoptosis, and died in a nondescript way notinvolving membrane inversion. Thus, in these cells apoptosiswas not coupled to RAI induction. We speculate that this is aresult of the original transformation of these cells withAdenovirus 5 early region [11], but it could also be a result ofindependent changes in the cellular logics that could havearisen during the years in culture. Other reports that have

Fig. 10 – Suppression of apoptosis in RNA siRNA transfectedWI-38 cells after treatment with 40 μM VP-16. Two cultureswere made for each column and 150 green cells from eachculture were counted. The control cells had no green andwere counted in toto. Statistical comparisons show that thereis a significant decrease in apoptosis in etoposide treatedcells transfected with RAIduplex1 (p=0.008 t-test; two-sided)or cells transfected with RAIduplex2 (p=0.004; t-test;two-sided) relative to treated cells transfected withnon-silencing RNA. Statistical comparisons also show thatthere is a significant decrease in non-treated cells transfectedwith RAIduplex1 (p=0.02, t-test, two-sided) or transfectedwith RAIduplex2 (p=0.003, t-test, two-sided) versusuntreated cells transfected with non-silencing RNA.

Fig. 11 – Non-inhibition of sulfasalazine-induced apoptosisinWI-38 cells by siRNA. (A) The human lung fibroblasts cells,WI-38, were left un-transfected, or transfected withRAIduplex2, or non-silencing RNA. The cellswere induced forapoptosis by treatment with sulfasalazine at a concentrationof 2.5 mM. After 24 h exposure the cells were stained withAnnexin V–FLUOS/Propidium Iodide and analyzed by FlowCytometry. The data present the average number of apoptoticcells (±SEM) in three independent experiments. (B) Therelative RAI mRNA level as quantified by real-time RT–PCRASSAY1, and normalized to mRNA β-actin. The data arepresented as the mRNA expression level (mean±SEM)relative to control cells in three independent experiments.Statistical comparisons show that transfection withRAIduplex2 increased the level of apoptosis marginally(p=0.05; t-test, two-sided) relative to transfection withnon-silencing RNA, that sulfasalazine increased the level ofRAI mRNA in control cells (p=3×10−5, t-test, two-sided), incells transfected with non-silencing RNA (p=0.001,two-sided) or cells transfected with RAIduplex2 (0=0.02;t-test, two-sided), and that treatment with RAIduplex2reduced the mRNA level (ASSAY1) in sulfasalazine treatedcells relative to treatmentwithnon-silencingRNA in the samecells (p=2×10−7, t-test two-sided). There also was an effect ofRAIduplex2 on mRNA level in untreated cells (non-silencingRNA versus RAIduplex2, p=7×10−6; t-test, two-sided).

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found RAI to be anti-apoptotic have largely been concernedwith transformed cells [7].

In one experimental situation, induction of apoptosis withsulfasalazine, apoptosis proceeded, even when RAI mRNAinduction was inhibited with siRNA. This might seem at oddswith the above conclusions, but this drug by itself blocks theformation of active NF-κB, through the blocking of the I-κBkinase, which directs the release of NF-κB subunits from aninactive cytoplasmic complex with I-κB. Thus, cells treatedwith sulfasalazine are expected to loose NF-κB activityirrespective of the RAI siRNA. The results are consonant withthe hypothesis that high RAI expression induces apoptosis byan NF-κB-dependent mechanism.

We are not implying that induction of RAImRNA is the onlysignal for apoptosis. On the contrary, we believe that othersignals are necessary and come together with RAI induction topromote this cellular outcome. Also, several pathways lead toapoptosis, andnot all of themneed to invokeRAI. However, ourfinding, that variations in the region encompassing the RAIgene on chromosome 19 are associatedwith differences in risk

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for 3major cancer forms, cancer of the breast, lung and skin [2–4], strongly suggests that RAI-related apoptosis is a majordefense-mechanism against these diseases.

“There is now considerable evidence that sustained orconstitutive activation of NF-κB is prevalent in cell lines andtumor tissue specimens and contributes tomalignant progres-sion” [14]. One effect of this activation could be to counteractthe RAI-mediated shutdown of NF-κB by providing moretargets for RAI protein than it can cope with. In this way, up-regulation of NF-κB becomes a survival mechanism in cancercells. Indeed, resistance to apoptosis limits the efficiency ofmany anti-cancer drugs, including certain types of tumors thatdo not respond to the effect of VP-16 [15]. One of the proposedmechanisms by which tumor cells may gain resistance tocytotoxic drugs is activation of NF-κB, a condition reported forsome pancreatic carcinoma cells [16]. Interestingly, severalnew anti-cancer agents aim to block NF-κB activity [14] andthus presumably aim to give RAI a helping hand.

In conclusion, expression of RAI mRNA and protein wasincreased in non-transformed cells induced to undergoapoptosis by various means. Moreover, inhibition of RAIexpression in these cells reduced apoptosis, but treatmentwith the NF-κB-inhibiting substance sulfasalazine relievedthis dependence. In other words, RAI induction seemedconducive to and causatively related to apoptosis. Ourfindings are in accord with the original report that RAI proteininhibits RelA and thereby inhibits at least some aspects ofNF-κB action.

Acknowledgments

Wegratefully acknowledge the help of Keld Kaltoft and SteffenJunker in relation to the cell culture work, and the help ofJeppe Prætorius with the immunofluorescence microscopy.We thank Dr. Xin Lu for the generous gift of the monoclonalantibody to the RAI protein. This work was supported by theLundbeck Foundation, The Danish Medical Research Council,The NovoNordisk Foundation, The Danish Cancer ResearchFund, and Fabrikant Einar Willumsens Mindelegat.

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