2005Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis

Effect of EBV LMP1targeted DNAzymes on cell proliferation and apoptosis

Zhong-Xin Lu,1Mao Ye,1Guang-Rong Yan,1Qun Li,1Min Tang,1Leo M Lee,2

Lun-Quan Sun,3and Ya Cao1

1Cancer Research Institute,Xiangya School of Medicine,Central South University,Changsha,China;

2Laboratory of Molecular Technology SAIC-Frederick,National Cancer Institute,Frederick,Maryland, USA;and3St Vincent’s Clinical School,Faculty of Medicine,The University of New South Wales,Sydney, Australia.

The latent membrane protein(LMP1)encoded by Epstein–Barr virus(EBV)has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis.RNA-cleaving DNA enzymes are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner.In this study,we explore the potential of using DNAzymes as a therapeutic approach to EBV-associated carcinomas by targeting the LMP1gene.In all,13different phosphorothioate-modified‘‘10–23’’deoxyribozymes (DNAzymes)were designed and synthesized against the LMP1mRNA and transfected into B95-8cells,which constitutively express the LMP1.Fluorescence microscopy was used to examine the cellular uptake and distribution in B95-8cells.As demonstrated in Western blots,three out of13deoxyribozymes significantly downregulated the expression of LMP1in B95-8cells.These DNAzymes were shown to markedly inhibit B95-8cell growth compared with a disabled DNAzyme and untreated controls,as determined by an alamarBlue Assay.It was further demonstrated that these DNAzymes arrested the B95-8cells in G0/G1using flow cytometry.Interestingly,the active DNAzymes could also downregulate the expression of Bcl-2gene in treated cells,suggesting a close association between the LMP1and Bcl-2genes and their involvement in apoptosis.This was further confirmed with the result that the DNAzymes could induce the release of cytochrome c from mitochondria,which is the hallmark of the apoptosis.The present results suggest that the LMP1may present a potential target for DNAzymes towards the EBV-associated carcinoma through cell proliferation and apoptosis pathways.

Cancer Gene Therapy(2005)12,647–654.doi:10.1038/sj.cgt.7700833

Published online1April2005

Keywords:latent membrane protein;DNAzymes;EBV-associated carcinoma;targeted suppression

E pstein–Barr virus(EBV)is a gamma herpesvirus

infecting more than90%of adults worldwide and is the first human virus identified to be associated with human cancers.1EBV has been implicated in the pathogenesis of several human malignancies including lymphoproliferative malignancies such as Burkitt’s and Hodgkin’s lymphomas as well as epithelial tumors, including nasopharyngeal carcinoma(NPC)and gastric carcinoma.2More recently,there have been scattered reports linking EBV with typical epithelial cancers of other primarysites including breast,lung,colon and prostate carcinomas.3These studies suggested that EBV, rather than being associated with restricted groups of uncommon malignancies,might in fact playan oncogenic role in a varietyof human epithelial cancers and possibly also in hyperplasias and certain dysplasias preceding carcinomas.

Latent membrane protein1(LMP1)is considered as a major oncogenic protein encoded byEBV because it could transform rodent fibroblasts and was tumorigenic in the nude mice.4LMP1is an integral membrane protein composed of a short cytoplasmic N-terminus of24amino acids,six transmembrane domains of162amino acids and a cytoplasmic C-terminus of200amino acids.The six hydrophobic transmembrane domains facilitate spontaneous self-oligomerization and create a complex that constitutivelyactivates cell-signaling pathway s via a cytoplasmic C-terminus that mimics tumor necrosis factor(TNF)receptor superfamilymembers.While the C-terminal activating region1(CTAR1)of LMP1is located proximal to the membrane and binds to the TNF receptor-associated factors(TRAFs),the CTAR2 is located toward the far C-terminus of LMP1and binds the TNF receptor-associated death domain protein (TRADD)and receptor-interacting protein(RIP). Consequently,LMP1can trigger several signaling

Received September23,2004.

Address correspondence and reprint requests to:Dr Lun-Quan Sun, Faculty of Medicine,University of New South Wales,Sydney, Australia.E-mail:lun-quan.sun@https://www.360docs.net/doc/ec6249080.html,.au or Professor Ya Cao, Cancer Research Institute,Xiangya School of Medicine,Central South University,Changsha,Hunan,410078,PR China.

E-mail:

ycao98@https://www.360docs.net/doc/ec6249080.html, Cancer Gene Therapy(2005)12,647–654

r2005Nature Publishing Group All rights reserved0929-1903/05$30.00 https://www.360docs.net/doc/ec6249080.html,/cgt

pathways that lead to the activation of several transcrip-tion factors,including NF-k B,STATs and AP-1.5–7Activation of NF-k B or AP-1byL MP1has been linked to the upregulation of some cellular proteins and inhibition of apoptosis.Depending on the cell types,expression of L MP1has been shown to playdifferent roles in response to biological and physiological stimulus.For example,the LMP1expression was essential in the immortalization of the EBV-infected B cells,and in human epithelial cells LMP1was suggested to be involved in tumor progression and invasions.More significantly,L MP1is expressed in the majorityof EBV-associated human malignancies,which supports the notion that the L MP1maybe one of the keyfactors contributing to EBV-mediated tumorigenesis.8Therefore,genetic manip-ulation of the L MP1expression mayprovide a novel strategyfor the treatment of EBV-associated human cancers.

Currently,there are several commonly used technologies for gene suppression,which include antisense oligonucleo-tides,ribozymes,DNAzymes and RNA interference (RNAi).The relative merits of antisense,RNAi,ribozymes and DNAzymes for sequence-specific knockdown of target RNA in different situations were recentlycompared by Scherer and Rossi.9DNAzymes are synthetic,single-stranded DNA catalysts that can be engineered to bind to their complementarysequence in a target messenger RNA (mRNA)through Watson–Crick base pairing.10A general model for the DNAzyme has been proposed,and is known as the ‘‘10–23’’model.DNAzymes following the ‘‘10–23’’model,also referred to simplyas ‘‘10–23DNAzymes’’,have a catalytic domain of 15deoxyribonu-cleotides,flanked bytwo substrate-recognition domains of seven to nine deoxyribonucleotides each.In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions.11These agents have been used in a number of in vitro and in vivo applications to inhibit the expression of their target gene 12and their dependent genes.13Their capacityto block development of a diverse range of pathologies in animal models suggests a potential use of DNAzymes as therapeutic tools.12–17

In this report,we designed and constructed the specific DNAzymes that were shown to be effective in suppressing expression of the target protein LMP1and affecting some LMP1-associated biological pathways.These results demonstrated the feasibilityof targeting the L

MP1mRNA as a potential therapeutic strategyfor EBV-associated carcinomas.

Materials and methods

Cell culture

The B95-8cell line (ATCC CRL-1612)was grown in RPMI1640medium supplemented with 10%fetal bovine serum (FBS,GIBCO).All media were supplemented with 10mg/ml streptomycin and 10IU/ml penicillin and all cultures were incubated in a humidified atmosphere of 5%CO 2at 371C.

Design and construction of DNAzymes

EBV has been shown to possess some mutations in LMP1and these sequence changes maybe associated with the viral oncogenic potential.In order to identifysome conserved regions in the LMP1gene for DNAzyme targeting,we aligned the LMP1sequences from B95-8cells with a panel of eight EBV isolates,including representatives of four defined groups of European LMP1variants (groups A–D)and Chinese NPC-derived LMP1.18All DNAzymes contained the 10–23catalytic motif (ggctagctacaacga)and 9bp hybridizing arms specific for RU cleavage sites (R ?G or A)in the conserved regions of the LMP1transcript.DNAzymes were selected in the studyif their hy bridization stability with their targets (D G 1)is o à25kcal/mol.19Based on the analyses of sequences,thermodynamics and site distribution within the LMP1gene,13DNAzymes were synthesized with two phosphorothioate linkages on both arms (Table 1).The control oligonucleotide of DZ10was designed byinverting cataly tic core sequence.Where indicated,DZ10DNAzyme was labeled with a fluorescein isothiocyanate (FITC)at the 50-end for cellular uptake study.

DNAzyme transfection of B95-8cells

Cells were split the daybefore transfection.On the dayof transfection,cells were harvested and washed once with cold PBS.2?106cells per well were seeded in a six-well plate in 1ml of fresh RPMI 1640medium containing 10%FBS.The DNAzymes/tetra(4-methylpyridyl)porphyrine (TMP)mixtures were made at a charge ratio of 1with 2m M DNAzyme oligonucleotides as described.20The mixture was then added to the cells and incubated at 371C for 4hours,followed bythe addition of complete medium to the wells and further incubation for 20hours.

Fluorescence microscopic analysis

At 20hours after transfection ,cells were rinsed twice with PBS,once with 2ml of .2M glycine (pH 2.8)buffer,and then once with PBS.Cell nuclei were stained with 5ng/ml Hoescht 33258or 50ng/ml PI in PBS for 5minutes prior to fixing with 4%paraformaldehyde in PBS for 30minutes at ambient temperature.21Cells were rinsed with PBS and then mounted in 75%glycerol.Microphoto-graphs were acquired under UV (for Hoescht staining)and B2(for FITC-labeled DNAzymes)filters using a Nikon Optiphot-2microscope (Tokyo,Japan)equipped with a Nikon Microflex UFX-DX photomicrographic attachment.

Western blot analysis

B95-8cells grown in six-well plates were transfected as described above and incubated for 20hours at 371C in medium supplemented with 10%FBS.Cells were then washed with ice-cold PBS and lysed in the lysis buffer (10mM Tris-HCl,pH 8.0,1mM EDTA,2%SDS,5mM DTT,10mM PMSF,Proteinase inhibitors mix)for 30minutes on ice.The lysate was centrifuged for 5

minutes

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at 14,000rpm in a microfuge.After protein quantification bythe BCA AssayReagent (Pierce Chemical,Inc.),50m g of the total proteins from the DNAzyme-,control ODN-or mock-transfected cells were mixed with sample buffer and boiled for 5minutes.The samples were then resolved on 10%polyacrylamide SDS gel and transferred onto a nitrocellulose membrane byelectroblotting.The mem-brane was incubated in blocking buffer (TBS containing 5%skim milk and .1%Tween 20)for 2hours,followed byincubation with primaryantibodydiluted in the same buffer (1:1000).The membrane was washed in TBS containing .1%Tween 20and further incubated with a secondaryantibodythat could be detected using a peroxidase-conjugated anti-IgG at 1:10,000.Protein expressions were determined using a supersignal chemi-luminescence system (ECL,Pierce),followed by exposure to autoradiographic film.The studyemploy ed antibodies against LMP1(cs1-4,DAKO),a -tubulin (sc-5286,Santa Cruz BiotechnologyInc.),Bcl-2(C-2,sc-7382,Santa Cruz BiotechnologyInc.)and Cy toC (K257-100,BioVision),I k -B a (sc-203,Santa Cruz BiotechnologyInc.)and NF-k B p65(sc-109,Santa Cruz BiotechnologyInc.).

Cell proliferation assays

B95-8cells grown in 96-well micro-titer plates were transfected at .5,1and 2m M of DNAzymes or control ODN as described above.Cells were then incubated at 371C and 25m l of alamarBlue Assayreagent (Serotec L td)was added to each well at different time points.The plates were allowed for a further incubation of 3hours and analyzed spectrophotometrically (absorbance at 570and 600nm,Bio-Tek Inc.).

Cell cycle analysis

The transfected B95-8cells were incubated for different times at 371C in medium supplemented with 10%FBS.Untreated and treated cells were then washed with

ice-cold PBS and suspended in 75%ethanol at à201C overnight.Fixed cells were centrifuged and washed with PBS twice.For cell cycle analysis,cells were stained with 50m g/ml of PI and .1%of RNase A in 400m l PBS in a light-proof tube at 251C for 30minutes.Stained cells were assayed on FACSort (Becton Dickinson)and the cell cycle parameters were determined using the CellQuest software program (Becton Dickinson).

Cytochrome c release assay

The cells were transfected as described above.The proteins from cytosolic fraction and mitochondrial fraction were extracted using cytochrome c apoptosis assaykit (BioVision),according to the manufacturer’s instruction.Western blots were performed using a CytoC-specific antibody(K257-100,BioVision).

Results and discussion

Cellular uptake and distribution of LMP1DNAzymes A cell line B95-8was used to examine DNAzyme efficacy in downregulation of the LMP1gene expression and impact on cellular functions.To facilitate deliveryof DNAzyme oligonucleotides into cells,a cationic porphy-rin,TMP,was used as a transfection reagent for intracellular delivery.20In order to increase DNAzyme stabilityin cells,two phosphorothioate linkages were incorporated into each of the arms in DNAzymes,which has been shown to be an efficient wayto protect DNAzyme oligonucleotides from enzymatic degradation.22When the transfected cells were subjected to fluorescent microscopic analysis,efficient delivery was observed using the TMP-complexed phosphorothioate-Dz.Importantly,a significant nuclear deliverywas seen at 24-hour time point (Fig 1).

Table 1Location of DNAzyme target sites in the LMP1mRNA and DNAzyme design Name Cleavage site a DNAzyme sequences

Modification b Dz10a a (1)u 50cgtgttccaGGCTAGCTACAACGAggtcagggt3050FITC/2+2PS Dz10a (1)u 50cgtgttccaGGCTAGCTACAACGAggtcagggt30

2+2PS Dz1a (167)u 50gcaaaggaaGGCTAGCTACAACGAagaggacaa302+2PS Dz2a (211)u 50ttctgaagaGGCTAGCTACAACGAaaagatgat302+2PS Dz3g (233)u 50ccaagtggaGGCTAGCTACAACGAagagaaggt302+2PS Dz4a (476)u 50tgttgtagaGGCTAGCTACAACGAagagagcaa302+2PS Dz5g (492)u 50tagagtccaGGCTAGCTACAACGAcagttttgt302+2PS Dz6g (505)u 50ggagatcaaGGCTAGCTACAACGAcaatagagt302+2PS Dz7a (593)u 50agggagtcaGGCTAGCTACAACGAcgtggtggt302+2PS Dz8a (785)u 50tgtgggccaGGCTAGCTACAACGAtgtcatcag302+2PS Dz9a (893)u 50ccattgtcaGGCTAGCTACAACGAcagtgttgt302+2PS Dz11a (950)u 50gggcctccaGGCTAGCTACAACGAcatttccag302+2PS Dz12g (1109)u 50tcatctccaGGCTAGCTACAACGAcggaaccag302+2PS Dz13

g

(1138)u

50ttagctgaaGGCTAGCTACAACGAtgggccgtg302+2PS Control ODN

50cgtgttccaGGTATGTACAACGAggtcagggt30

2+2PS

a Cleavage sites are defined as AU or GU along the LMP1mRNA.Numbers in brackets are the nucleotide positions of A or G.b

2+2PS,2phosphorothioate linkages at each end of DNAzyme

oligonucleotides.

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As for all the oligonucleotide-based agents such as antisense,ribozyme or siRNA,the DNAzyme delivery has been the major hurdle in assayof its efficacy .Many types of delivery agents have been widely used for this purpose,including liposomes,nanoparticles or electro-poration.23In this study,TMP was shown to be a simple and efficient transfection reagent due to its high complex-ing efficiency,low cellular toxicity and preferential nuclear uptake.

Inhibition of LMP1protein expression in B95-8cells using DNAzymes

DNAzymes are enzymatic molecules composed entirely of DNA that can be made to bind and cleave any RNA molecule both in vitro and in vivo .However,like other agents that function byhy bridization with single-stranded RNA,the DNAzyme must compete with the target’s own stable intramolecular base pairing,which forms its characteristic secondarystructures.Fortunately ,the 10–23DNAzyme cleavage sites are plentiful in most biological substrates and thus provide a host of opportunities to achieve maximum cleavage efficiency.In addition to target site accessibility,the thermody-namics of enzyme–substrate interactions also have a strong influence on the efficiencyof DNAzy me-mediated hydrolysis of a long RNA.24

Taking into consideration these factors,we designed 13different 10–23DNAzymes targeted against AU or GU sites along the full-length LMP1mRNA (Table 1).These DNAzymes were transfected into B95-8cells and assayed

for their effect on the LMP1expression at the protein level using Western blotting.As shown in Figure 2,DNAzymes DZ1,DZ7and DZ10(2m M)strongly inhibited LMP1protein expression in B95-8cells,compared with a control ODN,TMP transfection reagent and untreated controls.Based on the initial screening results,one of the efficient DNAzymes,DZ10,was used for further studyand confirmed that DZ10could inhibit LMP1expression in B95-8cells in a dose-dependent manner (Fig 3).

Impact of LMP1DNAzymes on B95-8cell growth There have been manyreports showing that the EBV LMP1is one of several latentlyexpressed genes essential for the transformation of B lymphocytes and affects cellular growth and differentiation of epithelial cells in vitro .25,26The broad expression of LMP1in undiffer-entiated NPC suggests this viral oncoprotein as a keyeffector molecule in NPC pathogenesis.8We pre-viouslyshowed that the EBV L MP1might modulate EGFR promoter activityin a NF-k B-dependent manner in NPC.27Thus,downregulation of the LMP1expression could result in a growth inhibition in LMP1-expressing cells.

To examine the effect of downregulation of LMP1on cell growth,active DNAzymes DZ1,DZ7and DZ10were transfected into B95-8cells and the proliferation rate was assessed byalamarBlue assay .As shown in Table 2,the proliferation rate was inhibited in all the Dz-treated cells at 24-hour time point,followed bya recoveryat 48-

and

Figure 1Cellular distribution of fluorescent labeled DNAzyme.B95-8cells were incubated with 2m M 33-mer FITC-labeled phosphorothioate-modified LQ10DNAzyme complexed with TMP at C/R of 1.Cells were transfected for 24hours,treated with glycine (pH 2.8)buffer,stained with 5ng/ml Hoescht 33258or 50m g/ml PI,and fixed with 4%paraformaldehyde.Nuclei stained with Hoescht dye display a bright blue color,and the PI dye exhibits a red hue.(a )Nuclei stained with Hoescht dye.(b ,d )FITC-labeled DNAzyme.(c )Nuclei stained with PI dye.?

150.

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72-hour points,which maysuggest a gradual degradation of the transfected oligonucleotides within the transfected cells.When three DNAzymes were combined and transfected into the cells,an increase in the inhibitory effect was observed,compared with anyof individual DNAzyme treatment.This combined effect may be due to the multiple targeting of the accessible site within the LMP1mRNA structures.

To further investigate the cause of the reduced cell proliferation rate in the DZ-treated B95-8cells,flow cytometry was employed to study the effect of the DNAzymes on cell cycle.The results showed that DZ-7-treated cells were arrested in G0/G1at 24hours post transfection,with a significant reduction of cells in G2/M

phase and a marginal reduction of cells in S phase (Table 3).This result correlated well to the data from the cell proliferation assayat 24hours time point (Table 2),further suggesting an involvement of the LMP1gene in cell proliferation in NPC development.

Inhibition of Bcl-2protein expression and induced apoptosis in B95-8cells with LMP1DNAzymes There have been reports that the tumorigenicityof the EBV-positive cells was associated with an increase in the antiapoptotic protein Bcl-2and resistance to apoptosis.28Abdulkarim et al.29reported that antiviral agent Cidofovir could downregulate the EBV oncoprotein LMP1expression,which was associated with a decrease in antiapoptotic Bcl-2protein and an increase of the proapoptotic Bax protein in Raji (BL)and C15(NPC)cells.However,the mechanism of Codofovir’s effect on the LMP1and bcl-2protein expression was not defined.Here,we used the LMP1-specific DNAzymes to further investigate the association of the viral LMP1and cellular Bcl-2protein in B95-8cells.Western blotting results indicated that the active LMP1DNAzymes could concomitantly downregulate the ex-pression of Bcl-2gene in the treated cells (Fig 4a and b).Analyses of the proteins of cytosolic fraction and mitochondrial fraction,respectively,using cytochrome c apoptosis assaykit (BioVision)confirmed that the active DNAzymes could also induce the release of cytochrome c from mitochondria,which is the hallmark of the apoptosis (Fig 5a and b).

Taken together,these results,for the first time,demonstrated a direct relationship between the LMP1and Bcl-2at a gene-specific level and their involvement in

apoptosis.

Figure 2Inhibition of LMP1protein expression in B95-8cells by DNAzymes.B95-8cells grown in six-well plates were transfected with DNAzymes (DZ1-13or Control ODN,2m M),or mock-trans-fected and incubated in medium containing 10%FBS at 371C for 24hours.Total cell lysates were analyzed by Western blots using mouse monoclonal antibodies targeting EBV LMP1(a ),and the LMP1expression level was quantitated by densitometry (b

).

Figure 3Dose–response analysis of anti-LMP1Dz10.B95-8cells grown in six-well plates were transfected with different concentra-tions of DZ10(.5,1,2),or mock-transfected and incubated in medium containing 10%FBS at 371C for 24hours.Total cell lysates were analyzed by Western blots.Expression level for each protein was estimated by densitometry and presented as a ratio to the loading control a -tubulin (lower

panel).

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Modulation of LMP1-induced cellular NF-k B signal pathway in CNE1-LMP1cells by DNAzymes

LMP1-mediated activation of NF-k B is one of the major signal transduction pathways involved in cell growth and apoptosis in the EBV-associated NPC.30In normal cells,NF-k B is sequestered byinhibitorymolecules,I k Bs.When LMP1activates the NF-k B pathway,one of the inhibitorymolecules,I k Ba,is degraded by uniquitination,which leads to release and nuclear transportation of NF-k B.31To elucidate the mechanism wherebythe L

MP1-specific DNAzy mes promote apoptotosis in LMP1-expressing cells,we examined if downregulation of L MP1byDNAzy mes could affect the NF-k B pathway.As shown in Figure 6,the NF-k B level in DNAzyme-treated cells remained unchanged while the I k B a level was significantlyincreased compared with controls.This result suggests that suppression of LMP1expression leads to accumulation of the I k Bs in cells,which could have a global effect on cell functions regulated via the NF-k B pathway,including cell prolif-eration and apoptosis.

In conclusion,we have developed the active DNA-zymes that could suppress the EBV LMP1expression,inhibit the cell proliferation in the LMP-expressing B95-8cells and increase apoptosis bydownregulation

Table 2Inhibition of B95-8cell proliferation by active LMP1DNAzymes

Proliferation rate (%)a

DNAzymes Concentration (m M)

12hours 24hours 36hours 48hours 72hours DZ1

.599.063.987.689.887.7195.255.487.187.391.3289.446.382.475.589.5DZ7

.596.168.586.490.693.3188.256.885.487.588.2285.542.488.085.687.3DZ10

.587.666.782.184.185.7181.051.378.777.081.9272.339.669.879.981.1DZ1+7+10

.595.959.673.885.388.5192.451.073.079.677.3285.928.161.267.981.5Control ODN

.598.895.687.999.489.4193.098.786.994.686.72

96.8

98.6

89.6

86.8

85.5

a

Proliferation rate is calculated as a relative value of OD readings in each well to untreated cells.

Table 3Effect of LMP1DNAzyme on cell cycle in B95-8cells a Phases Cell control TMP control DZ-7treatment

G0/G141.8246.4654.23S 44.1044.0140.26G2/M

14.08

9.53

5.52

a

The numbers represent the percentage of cells in different phases of cell

cycle.

Figure 4Inhibition of Bcl-2protein expression in B95-8cells by DNAzymes.B95-8cells grown in six-well plates were transfected with DNAzymes (DZ1,DZ7or DZ10at 2m M),or mock-transfected and incubated in medium containing 10%FBS at 371C for 24hours.Total cell lysates were analyzed by Western blots (a ).Expression level for each protein was estimated by densitometry and presented as a ratio to the loading control a -tubulin (b

).

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of the bcl-2expression.The biological activityof the DNAzymes was shown to be mediated via the NF-k B signaling pathway.The present results suggest that the

LMP1could be a potential molecular target for nucleic acid-based therapeutics,such as DNAzymes,towards the EBV-associated carcinomas through cell proliferation and apoptosis pathways.

Acknowledgments

This research was supported bythe National Natural Science Foundation of China for Distinguished Young Scholars (No.39525022),State KeyResearch Program,Fundamental Investigation on Human Carcinogenesis of China (No.G1998051201),and the National Natural Science Foundation of China (No.30200009).

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