DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).

RESEARCH ARTICLE Open Access DNA me hyla ion egula es exp ession o VEGF-R2 (KDR) and VEGF-R3 (FLT4) Hilma Quen meie 1* , Sonja Ebe h 1,2 , Julia Romani 1 , He be A Weich 3 , Ma ga e e Zabo ski 1 and Hans G D exle 1 Abs ac Backg ound: Vascula Endo helial G ow h Fac o s (VEGFs) and hei ecep o s (VEGF-Rs) a e impo an egula o s o angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs a e no only exp essed on endo helial cells bu also on a ious sub ypes o solid umo s and leukemias con ibu ing o he g ow h o he malignan cells. This s udy was pe o med o examine whe he VEGF-R2 (KDR) and VEGF-R3 (FLT4) a e egula ed by DNA me hyla ion. Me hods: Real- ime (RT) PCR analysis was pe o med o quan i y KDR and FLT4 exp ession in some nine y leukemia/lymphoma cell lines, human umbilical ein endo helial cells (HUVECs) and de mal mic o ascula endo helial cells (HDMECs). Wes e n blo analyses and low cy ome ic analyses con i med esul s a he p o ein le el. A e bisul i e con e sion o DNA we de e mined he me hyla ion s a us o KDR and FLT4 by DNA sequencing and by me hyla ion speci ic PCR (MSP). Wes e n blo analyses we e pe o med o examine he e ec o VEGF-C on p42/44 MAPK ac i a ion. Resul s: Exp ession o KDR and FLT4 was obse ed in cell lines om a ious leukemic en i ies, bu no in lymphoma cell lines: 16% (10/62) o he leukemia cell lines exp essed KDR, 42% (27/65) we e FLT4 posi i e. None o hi y cell lines ep esen ing six lymphoma sub ypes showed mo e han ma ginal exp ession o KDR o FLT4. Wes e n blo analyses con i med KDR and FLT4 p o ein exp ession in HDMECs, HUVECs and in cell lines wi h high VEGF-R mRNA le els. Ma u e VEGF-C induced p42/44 MAPK ac i a ion in he KDR - /FLT4 + cell line OCI-AML1 e i ying he model cha ac e o his cell line o VEGF-C signal ansduc ion s udies. Bisul i e sequencing and MSP e ealed ha GpG islands in he p omo e egions o KDR and FLT4 we e unme hyla ed in HUVECs, HDMECs and KDR + and FLT4 + cell lines, whe eas me hyla ed cell lines did no exp ess hese genes. In hype me hyla ed cell lines, KDR and FLT4 we e e-inducible by ea men wi h he DNA deme hyla ing agen 5-Aza-2’deoxycy idine, con i ming epigene ic egula ion o bo h genes. Conclusions: Ou da a show ha VEGF-Rs KDR and FLT4 a e silenced by DNA me hyla ion. Howe e , i he p omo e s a e unme hyla ed, o he ac o s (e.g. ansac i a ion ac o s) de e mine he ex en o KDR and FLT4 exp ession. Backg ound Vascula endo helial g ow h ac o s (VEGFs) and hei co esponding ecep o s (VEGF-Rs) a e impo an egu- la o s o angiogenesis and lymphangiogensis. VEGF-A binds VEGF-R1 (FLT1) and VEGF-R2 (KDR). Bo h y o- sine kinase ecep o s a e exp essed on blood essel endo helial cells. VEGF-C and VEGF-D bind o VEGF- R3 (FLT4) and he ully p ocessed, ma u e o ms also o KDR. FLT4 is p ima ily exp essed on cells o he lympha ic endo helium [1]. VEGFs and VEGF-Rs a e impo an o essel o ma ion in heal hy indi iduals, bu also o umo angiogenesis [2]. Mo eo e , he VEGF-Rs a e no only exp essed on endo helia, bu also on di e en ypes o solid umo cells and on leukemic cells [3-11]. The in e ac ion o ecep o s wi h hei ligands media es su i al and can lead o p oli e a ion o he malignan cells [2,12]. E en wen y yea s a e hei disco e y, li le is known abou he egula ion o he h ee VEGF-Rs. On he an- sc ip ional le el, NF-Band heNF-B a ge P ox1 ha e been desc ibed as ac i a o s o FLT4 in lympha ic endo helial cells [13]. Epigene ic mechanisms con ibu e * Co espondence: [email p o ec ed] 1 Depa men o Human and Animal Cell Cul u es, Ge man Collec ion o Mic oo ganisms and Cell Cul u es, B aunschweig, Ge many Full lis o au ho in o ma ion is a ailable a he end o he a icle Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 © 2011 Quen meie e al; licensee BioMed Cen al L d. This a icle is published unde license o BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. o he egula ion o FLT1 and KDR bu his is no in es- iga ed in g ea de ail [14,15]. We se ou o es whe he DNA me hyla ion is also esponsible o he silencing o FLT4.Wede e mined he me hyla ion s a us o KDR and FLT4 in human umbilical ein endo helial cells (HUVECs), de mal mic o ascula endo helial cells (HDMECs) and in a la ge panel o leukemia and lymphoma cell lines. Con- i ming ha exp ession o KDR and FLT4 is epigene i- cally egula ed, we obse ed an in e se co ela ion be ween p omo e me hyla ion and ecep o exp ession. Fu he mo e, he deme hyla ing agen 5-Aza-2’deoxycy- idine (5-Aza-dC) induced exp ession o KDR and FLT4 in me hyla ed, bu no in unme hyla ed cell lines. Me hods Cell lines and p ima y cell cul u es The cell lines in his s udy we e aken om he s ock o he cell bank (DSMZ–Ge man Collec ion o Mic oo - ganisms and Cell Cul u es; h p://www.dsmz.de). De ailed e e ences and cul i a ion p o ocols ha e been desc ibedp e iously[16].P ima yHDMECswe epu - chased om Clone ics/Lonza (Ve ie s, Belgium). P i- ma y HUVECS (pooled) we e pu chased om P omoCell (Heidelbe g, Ge many). HDMECs and HUVECs we e cul u ed in endo helial cell g ow h med- ium MV (P omo Cell). CpG island sea ch CpG island sea ch was done wi h Me hyl P ime Exp ess 1.0 so wa e and EMBOSS CpG plo (h p://www.ebi.ac. uk/Tools/emboss/cpgplo /index.h ml). The c i e ia o an island we e: GC con en > 50%; CpG obse ed e sus CpG expec ed a io > 0.6, leng h > 100 bp. Me hyla ion-speci ic polyme ase chain eac ion (MSP) Bisul i e con e sion o DNA was pe o med as desc ibed by he supplie (Epi Tec Bisul i e Ki , Qiagen, Hilden, Ge many). Fo de ec ing FLT4 and KDR p omo e me hyla ion, we pe o med nes ed PCR wi h i s ound p ime s (FLT4 BSP wd 5’-AAA TAT TTG GGG GAG TTT TAA A-3’,FLT4 BSP e 5’-CCC AAT CTC AAA AAT AAA CAA A-3’;KDR BSP wd 5’-AAG TTG TTG TTT TGG GAT GTT T-3’,KDR BSP e 5’-AAA TAA ACT CCT TAC CCA CAA A-3’)ampli yingcon e ed DNA independen ly o he me hyla ion s a us (bisul i e- speci ic PCR o BSP; annealing emp.: 54.9°C o FLT4 BSP, 54.7°C o KDR BSP, 35 cycles), and second ound p ime s o M- and U-PCR speci ically ecognizing he me hyla ed o unme hyla ed e sions o he p omo e (FLT4 M wd5’-GTC GGT TAT TTC GGG TGT TTC -3’,FLT4 M e 5’-AAT ATC GAC GAA CAA TAT CGA CG-3’,FLT4 U wd 5’-GGG TTG GTT ATT TTG GGT GTT TT-3’,FLT4 U e 5’-ACA CAA TAT CAA CAA ACA ATA TCA ACA-3’,KDR M wd5’-CGT TTT CGC GTT TTA GAG TTT C-3’,KDR M e 5’- GCG CAA ATA ATA CCC GAC G-3’,KDR U wd5’- TTT TGT TTT TGT GTT TTA GAG TTT T-3’,KDR U e 5’-ACA CAC AAA TAA TAC CCA ACA-3’). PCR p oduc s o he ini ial BSP we e dilu ed 1:100 o 1:4.000 o subsequen M- and U-PCR. Annealing em- pe a u e was 61.2°C o FLT4 M- and U-PCR, 58°C o KDR M- and U-PCR wi h 30 cycles each. Epi ec PCR Con ol DNA (Qiagen) was used as con ol o me hy- la ed and unme hyla ed empla es. Bisul i e sequencing To con i m he me hyla ion s a us o he FLT4 and KDR p omo e s, genomic DNA was bisul i e con e ed acco ding o he manu ac u e ’s ins uc ions (Qiagen). Subsequen ly, ampli ica ion o he p omo e egions (FLT4: 337 bp; KDR:612bp)waspe o medusingBSP p ime s, speci ically binding bisul i e con e ed DNA ( o p ime sequence and PCR condi ions see MSP sec- ion). Resul ing PCR p oduc s we e pu i ied, cloned in o he pGEM-TEasy ec o (P omega,Madison,WI,USA) and sequenced. Sequences we e e alua ed using BiQ Analyze (h p://biq-analyze .bioin .mpi-sb.mpg.de) and had o con o m o a leas 90% bisul i e con e sion a e. In addi ion, iden ical clones we e excluded om he analysis. Gene exp ession analyses Quan i a i e PCR was pe o med on a 7500 Applied Biosys ems (Da ms ad , Ge many) eal- ime PCR sys em using he manu ac u e ’s p o ocol. RNA was p epa ed using he RNeasy Mini ki (Qiagen). This ki includes a DNase diges ion s ep o a oid alse posi i es esul ing om con amina ing genomic DNA. Fo mRNA quan i i- ca ion, e e se ansc ip ion was pe o med using he Supe Sc ip II e e se ansc ip ase ki (In i ogen, Ka ls uhe, Ge many). TaqMan p obes (Applied Biosys- ems) we e used o quan i y human FLT4 (Hs 01047677 m1) and KDR (Hs 00911700 m1) exp ession le els wi h TATA box binding p o ein (TBP) as endogenous con ol. Fo in e e on gamma inducible p o ein 10 (IP-10)and umo nec osis ac o alpha (TNFa), SYTO-82 (Molecu- la P obes, Leiden, Ne he lands) was used as luo escen dye, ImmoMix (Biline, Luckenwalde, Ge many) as PCR mas e mix, and ibosomal p o ein S9 (RPS9)asendo- genous con ol. The ollowing p ime s we e used: TNFa exon 2 wd 5’-CCC CAG GGA CCT CTC TCT AA-3’, TNFaexon 3 e 5’-TGG GCT ACA GGC TTG TCA CT-3’;IP-10 exon 1 wd 5’-GCC ATT CTG ATT TGC TGC CTT A -3’,IP-10 exon 2 e 5’-TGA TGC AGG TAC AGC GTA CAG-3’;RPS9 exon 2 wd 5’-GGG AAG CGG AGC CAA CAT G-3’,RPS9 exon 3 e 5’- GTT TGT TCC GGA GCC CAT ACT-3’.Rela i e Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 2 o 10 exp ession le els we e calcula ed using he ΔΔC - me hod. [ 3 H]-Thymidine up ake Assays o [ 3 H]- hymidine inco po a ion we e execu ed as ollows: 1.25 × 10 4 cells (in 100 μl) we e seeded in iplica e in 96-well la -bo om mic o i e cell cul u e pla es. Inhibi o s we e added as 2x concen a ed solu- ion in a 100 μl olume. Fo he las 3 h o he incuba- ion pe iod, 1 μCi [ 3 H]- hymidine (Ha mann Analy ic, B aunschweig, Ge many) was added o each well. Wes e n blo analysis, an ibodies, eagen s Samples we e p epa ed as desc ibed p e iously [17]. An i FLT4, ERK and pERK an ibodies we e pu chased om San a C uz (Heidelbe g, Ge many). An i IB, pIB, KDR, p38 MAPK and pp38 MAPK an ise a we e ob ained om Cell Signalling (New England Biolabs, F ank u , Ge - many). The an i GAPDH monoclonal an ibody (mAb) was pu chased om Abcam (Camb idge, UK). Speci ic bands on ni ocellulose memb anes we e isualized wi h he bio in/s ep a idin-ho se adish pe oxidase sys em (Ame sham, F eibu g, Ge many) in combina ion wi h he “Renaissance Wes e n Blo Chemoluminescence Reagen ”p o ocol (Pe kin Elme , Wal ham, MA, USA). Syn he ic mac ophage ac i a ing ac o o 2 kDa molecu- la mass (MALP-2) was a gi om P. Mühl ad o H. Weich. The p epa a ion was ee o endo oxin. Analysis o FLT4 and KDR p o ein exp ession by low cy ome y Fo de ec ion o CD31 (Bec on Dickinson Biosciences, Heidelbe g, Ge many), FLT4 (R&D Sys ems, Wiesbaden, Ge many), KDR (Relia ech, Wol enbü el, Ge many) and podoplanin (Relia ech) on he cell su ace, cells we e washed and incuba ed wi h he mouse mAb o wi h he iso ope-ma ched con ol mouse immunoglobulin (BD Biosciences) o 30 min a 4°C. Subsequen ly, cells we e ea ed wi h FITC conjuga ed an i-mouse seconda y Ab (Biozol, Eching, Ge many) and p opidium iodide. Labeled cells we e analyzed on a FACSCalibu (BD Bios- ciences) using CellQues P o so wa e. T ea men wi h DNA deme hyla ing agen 5-Aza-2’- deoxycy idine (5-Aza-dC) 5-Aza-dC (Sigma Ald ich, Tau ki chen, Ge many) dis- sol ed in DMSO was used o e i y he e ec o me hy- la ion on exp ession o FLT4 and KDR.Cellswe e seeded a a cell densi y o 5 × 10 5 cells/ml, 5-Aza-dC was added a a inal concen a ion o 5 μM. Con ol cells we e ea ed wi h 0.05% DMSO. A e 2 d, hal o he medium was eplenished wi h medium wi h/wi hou 5-Aza-dC (5 μM). A e 3 d, cells we e ha es ed o p e- pa e RNA and p o ein. Resul s and Discussion Exp ession o KDR and FLT4 in leukemia and lymphoma cell lines The VEGF-Rs KDR (VEGF-R2) and FLT4 (VEGF-R3) a e no only exp essed on blood endo helial and lymphen- do helial cells, bu also on solid umo s and leukemias. Leukemia-de i ed VEGFs may induce he g ow h o leu- kemic cells in an au oc ine o pa ac ine ashion [7,10,18,19]. The p omo e s o VEGF-Rs and hei ligands con ain CpG islands, egula o y egions ha a e ypically me hyla ed in epigene ically silenced genes [14]. Recen epo s show ha exp ession o FLT1 and KDR a e con olled by p omo e me hyla ion [14,15]. Howe e , only a limi ed numbe o leukemia and lym- phoma cell lines ha e been es ed o VEGF-R exp es- sion and p omo e me hyla ion hi he o. To ind model sys ems o VEGF-R egula ion, we es ed some nine y leukemia and lymphoma cell lines o KDR and FLT4 mRNA exp ession. Bo h genes we e egula ly exp essed in leukemia bu no in lymphoma cell lines: 10/62 (16%) cell lines om a ious leukemic en i ies exp essed KDR, 27/65 (42%) exp essed FLT4 (Table 1). In con as , 0/30 lymphoma cell lines exp essed KDR, and only 1/30 (3%) exp essed FLT4 (Table 1). Cell lines wi h high VEGF-R ansc ip le els exp essed also he co esponding p o eins: cell lines CMK, HEL and MEG-01 exp essed KDR, whe eas cell linesHEL,MHH-CALL2,OCI-AML1andSUP-B15 we e FLT4 posi i e (Figu e 1, Table 2). OCI-AML1: a model sys em o VEGF-C induced cell signaling Cy okine-dependen cell lines ha e o en and success ully been used as model sys ems o signal ansduc ion s u- dies. In con as o p ima y cells, no con amina ing cell ac ion e ec s “ alse”signals in cell lines, and in con as o cy okine-independen ly g owing cell lines, cy okine s a a ion silences he ele an enzymes in cy okine- dependen cell lines. We chose cell line OCI-AML1 as his was he only cy okine dependen , FLT4 posi i e cell line es ed (Table 2). The cy okine esponse p o ile o his cell line has been published p e iously [20]. Cell line OCI-AML1 did no show a p oli e a i e esponse on VEGF-C (da a no shown). Howe e , sho - e m (5 min) s imula ion wi h VEGF-C induced phospho yla ion o ERK1/2 (Figu e 2). P eincuba ion wi h he FLT4 inhibi o MAZ51 inhibi ed his e ec , con i ming he speci ici y o he VEGF-C induced EKR1/2 ac i a ion (Figu e 2). ERK1/2 phospho yla ion was es ed because he p42/44 MAPK pa hway is a known FLT4 a ge [21,22]. The esul s o cell signaling expe imen s shown in Figu e 2 con i m ha cell line OCI-AML1 is a model sys em o FLT4 signaling, expecially as KDR, he second ecep o o VEGF-C is no exp essed in his cell line (Table 2). Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 3 o 10 KDR: p omo e me hyla ion and gene exp ession To es whe he KDR is epigene ically egula ed, we pe - o med bisul i e sequencing o KDR nega i e and posi- i e cell lines and o p ima y endo helial cells. The KDR nega i e cell line DOHH-2 had a highly me hyla ed KDR p omo e , he KDR posi i e cell line HEL was nea ly unme hyla ed (Figu e 3). La gely unme hyla ed we e also HDMECs and HUVECs, bo h exp essing KDR (Figu e 3). To assess he KDR me hyla ion s a us o a la ge numbe o cell lines, we pe o med me hyla ion- speci ic PCR (MSP), a echnique less cos ly and labo - ious han bisul i e sequencing. The majo i y o KDR nega i e cell lines we e me hyla ed, KDR posi i e HUVECs we e unme hyla ed (Figu e 4). Howe e , e en HDMECs we e U-and M-PCR posi i e al hough hey exp essed high KDR le els and al hough only a small mino i y o clones we e me hyla ed acco ding o sequencing analysis (Figu es 3 and 4). Appa en ly, a low p opo ion o me hyla ed CpGs was su icien o yield signals in he M-PCR. The same was ue o U-PCR: he KDR nega i e cell line DOHH-2 - highly me hyla ed acco ding o he esul s o bisul i e sequencing–showed signals in M- and in U-PCR (Figu e 3, Table 2). In spi e o he high sensi i i y–a ce ain d awback o he PCR-based MSP echnique– he accu acy o KDR M- PCR was 88% suppo ing he no ion ha KDR exp es- sion is egula ed by DNA me hyla ion (Table 2). FLT4: p omo e me hyla ion and gene exp ession Bisul i e sequencing and BSP analysis we e also pe - o med o analyze he me hyla ion s a us o FLT4 in cell lines, HUVECs and HDMECs. Resul s o bisul i e sequencing showed ha FLT4 was la gely me hyla ed in he FLT4 nega i e cell line EM-2 and unme hyla ed in he FLT4 posi i e cell line SUP-B15 as i was in HUVECs and HDMECs (Figu e 5). MSP analysis con- i med ha FLT4 exhibi ed he in e se co ela ion Table 1 VEGF-R mRNA exp ession in leukemia and lymphoma cell lines KDR +++ ++ + (+) - ∑ AML 0 2 0 3 16 21 p e-B ALL 0 0 0 1 10 11 T-ALL 0 0 0 2 9 11 NK 0 0 0 0 5 5 CML 1 0 0 1 12 14 HL 0 0 0 0 5 5 ALCL 0 0 0 0 5 5 BL 0 0 0 0 5 5 DLBCL 0 0 0 0 5 5 FL 0 0 0 0 5 5 MCL 0 0 0 0 5 5 ∑1 2 0 7 82 92 FLT4 +++ ++ + (+) - ∑ AML 0 2 4 3 14 23 p e-B ALL 0 3 1 3 4 11 T-ALL 0 0 2 4 5 11 NK 0 0 0 0 5 5 CML 0 1 1 3 10 15 HL 0 0 0 1 4 5 ALCL 0 0 0 0 5 5 BL 0 0 0 0 5 5 DLBCL 0 0 0 0 5 5 FL 0 0 0 0 5 5 MCL 0 0 0 0 5 5 ∑068146795 KDR and FLT4 mRNA exp ession le els we e de e mined by quan i a i e eal- ime PCR. TBP exp ession was used as endogenous con ol and cell lines CMK (KDR) and HEL (FLT4) we e used o no maliza ion. Rela i e quan i ica ion: +++ ≥5; ++ ≥1; + ≥0.2; (+) ≥0.04;- < 0.04. AML, acu e myeloid leukemia; ALL, acu e lymphoblas ic leukemia; NK, na u al kille leukemia; CML, ch onic myeloid leukemia; HL, Hodgkin lymphoma; ALCL, anaplas ic la ge cell lymphoma; BL, Bu ki lymphoma; DLBCL, di use la ge B cell lymphoma; FL, ollicula lymphoma, MCL, man le cell lymphoma Figu e 1 KDR and FLT4 in HDMECs, HUVECs and leukemia cell lines.KDR and FLT4 mRNA exp ession le els we e de e mined by quan i a i e eal- ime PCR and indica ed unde nea h he cell line name. TBP exp ession was used as endogenous con ol and cell lines CMK (KDR) and HEL (FLT4) we e used o no maliza ion. KDR p o ein exp ession le els–de e mined by Wes e n blo analysis–a e highe in HDMECs and HUVECs han in posi i e cell lines (no e 1:10 lysa e dilu ion in p ima y cells). FLT4 p o ein exp ession is highe in HDMECs han in HUVECs (no e 1:10 lysa e dilu ion in HDMECs) and in posi i e cell lines. P o ein exp ession co esponds o mRNA exp ession pa e n. Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 4 o 10 be ween p omo e me hyla ion and gene exp ession ha is indica i e o epigene ic egula ion (Figu e 4, Table 2). Howe e , he accu acy o FLT4 M-PCR (80%) was lowe han o KDR M-PCR (88%). O no e was also ha TF-1 cells did no exp ess FLT4 al hough he p omo e was unme hyla ed (Table 2). These da a sugges ed ha egu- la o y mechanisms o he han DNA me hyla ion a e also impo an o he egula ion o FLT4. E ec o DNA deme hyla ing agen 5-Aza-dC on exp ession o KDR and FLT4 To es whe he KDR and FLT4 we e silenced by p o- mo e me hyla ion, we ea ed me hyla ed and unme hy- la ed cell lines wi h he DNA deme hyla ing agen 5- Aza-dC. In 4/5 KDR-nega i e cell lines, exp ession o KDR was induced by DNA deme hyla ion (Table 3). FLT4 exp ession was up egula ed in 4/4 nega i e cell lines (Table 3). KDR and FLT4 exp ession in posi i e cell lines we e no a ec ed (Table 3). Al hough hese esul s con i med ha p omo e me hyla ion plays a ole o he egula ion o hese VEGF-Rs,wealsono ed subs an ial di e ences in he le els o 5-Aza-dC- ig- ge ed gene induc ion be ween di e en cell lines (Table 3). Fu he mo e, e en in he mos sensi i e cell lines (HL-60 o KDR induc ion, EM-2 o FLT4 induc ion), deme hyla ion did no induce mRNA exp ession ha would ansla e in o p o ein le els de ec able by Wes- e n blo analysis (da a no shown). These esul s sug- ges ha o he mechanisms han DNA me hyla ion a e also in ol ed in he egula ion o KDR and FLT4. Besides DNA me hyla ion, also his one modi ica ions a e epigene ic mechanisms ha a ec he exp ession o indi idual genes. Jus o men ion wo examples, ace y- la ed his one H3 (a lysine 9 and 14) is a ma ke o gene ac i a ion [23], i-me hyla ion o his one H3 lysine 27 s ands o gene supp ession [24]. Fu he mo e, epige- ne ic modi ica ions can in luence each o he : me hyla ed CpGs in a p omo e egion can be a ge ed by p o eins ha in e ac wi h his one deace ylases. The consequence is an inac i e ch oma in s a us and ansc ip ional ep ession [25,26]. Table 2 P omo e me hyla ion s a us and exp ession le els o KDR and FLT4 KDR FLT4 MSP mRNA p o ein MSP mRNA p o ein 697 M 0.08 neg M/U 0 neg ALL-SIL M 0 neg U 0.3 neg AP-1060 M/U 0 n.d. M/U 0 n.d. BV-173 M 0 neg U 1.4 pos CMK M/U 1 pos M 0.1 neg DOHH-2 M/U 0 neg M/U 0 neg EM-2 M/U 0 neg M 0 neg HANK-1 M 0 neg M 0 neg HEL U 2.6 pos U 1 pos HL-60 M 0 neg M 0.1 neg JURL-MK1 U 0.15 n.d. n.d. n.d. n.d. L-82 M 0 n.d. M 0 n.d. LOUCY n.d. 0.13 neg M/U 0 neg M-07e M/U 0.03 neg U 0.1 neg MEG-01 U 5.8 pos U 0.2 pos MEGAL M 0 neg M/U 0.3 neg MHH-CALL2 M/U 0 neg U 1.1 pos MHH-TALL1 M 0.08 n.d. n.d. n.d. n.d. MOLT-4 M 0 n.d. M/U 0 n.d. MUTZ-3 n.d. n.d. n.d. U 0.6 neg MUTZ-8 n.d. n.d. n.d. M/U 0.3 neg NK-92 M 0 neg M/U 0 neg OCI-AML1 M/U 0 neg M/U 1.3 pos SC-1 M 0 n.d. M 0 n.d. SKNO-1 U 0.08 neg n.d. n.d. n.d. SUP-B15 M 0 neg U 1.6 pos TF-1 U 0.1 pos U 0 neg THP-1 M/U 0 n.d. M/U 0 n.d. KDR and FLT4 me hyla ion was de e mined by MSP. M, posi i e in M-PCR; U, posi i e in U-PCR, M/U posi i e in M- and in U-PCR; n.d., no done. VEGF-R mRNA exp ession le els we e de e mined by quan i a i e eal- ime PCR. Cell lines CMK (KDR) and HEL (FLT4) we e used o no maliza ion (se o 1). P o ein exp ession was done by Wes e n blo analysis. The accu acy o KDR M-PCR (mRNA posi i i y > 0.1) is 88%, he accu acy o FLT4 M-PCR (mRNA posi i i y > 0.1) is 80% Figu e 2 Phospho yla ion le el o ERK1/2 in VEGF-C esponsi e OCI-AML1 cells. The cy okine- esponsi e cell line OCI-AML1 was cy okine-s a ed o 18 h, hen s imula ed o 5 min wi h VEGF-C. P e ea men wi h he FLT4 inhibi o MAZ51 (20 μM, 1 h) p e en ed VEGF-C induced ERK1/2 phospho yla ion as shown by Wes e n blo analysis. Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 5 o 10 Howe e , besides epigene ic mechanisms, also he p e- sence o absence o ans-ac ing ac o s may go e n he exp ession o KDR and FLT4. Thus, i has been shown ha ansc ip ion ac o binding si es (Sp1, AP-2 and NFB) a e essen ial o he base-line ac i i y o he KDR p omo e [27]. He e, we se ou o ind whe he NFB also plays a ole o he exp ession o FLT4. In luence o ansac i a ing ac o s Du ing in lamma ion, new lympha ic essels a e o med. NF-B is a key media o o in lamma o y p ocesses and has ecen ly been iden i ied as induce o FLT4 on lym- pha ic endo helial cells [13]. To es whe he NF-B con ibu es o FLT4 exp ession in leukemic cells, we s i- mula ed he FLT4 nega i e cell line EM-2 and he FLT4 posi i e cell line OCI-AML1 wi h syn he ic MALP-2. MALP-2 binds o oll-like ecep o s-2 and -6 [28]. MALP-2 igge s he NF-B pa hway [29] which leads o he exp ession o NF-B a ge s like TNFa[30]. Acco dingly, MALP-2 (100 ng/ml, 7 min) induced phospho yla ion and deg ada ion o he NF-B inhibi o IB and s imula ed phospho yla ion o p38 in cell lines EM-2andOCI-AML1(Figu e6).MALP-2(100ng/ml, 1 h) igge ed exp ession o he NK-B a ge sTNFa (80× in EM-2, 1000× in OCI-AML1) and IP-10 (600× in EM-2, > 1000× in OCI-AML1) in bo h cell lines. How- e e , he exp ession o FLT4 was no a ec ed, nei he in he FLT4 posi i e cell line OCI-AML1 no in he FLT4 nega i e (me hyla ed) cell line EM-2. MALP-2 did also no inc ease he FLT4 s imula ing e ec o 5-Aza-dC on EM-2 cells (da a no shown). Thus, ou esul s do no suppo he iew ha NF-B is a ansac i a o o FLT4. We obse ed a 10- old inc ease in KDR in cell lines OCI-AML1 and EM-2 ( he la e p e ea ed wi h 5-Aza- dC). Howe e , as he le el eached a e s imula ion was s ill ex emely low, i appea s unlikey ha NF-Bisan impo an egula o o KDR ei he . Figu e 3 Bisul i e sequencing o he KDR p omo e . A CpG island is loca ed be ween -1231 and 1125 ela i e o he ATG codon o KDR. The 3’pa o he KDR p omo e egion and exon 1 (612 bp, 53 CpG si es) we e sequenced a e bisul i e con e sion o DNA om cell lines DOHH-2 (KDR nega i e) and HEL (KDR posi i e) as well as om HDMECs and HUVECs. Each line depic s a sequenced clone ep esen ing he me hyla ion s a us o an indi idual allele. CpGs a e ep esen ed as open do s (i unme hyla ed) o illed do s (i me hyla ed). Resul s o qRT-PCR and me hyla ion speci ic PCR (MSP) a e shown on he le hand side. M: signal in M-PCR; U: signal in U-PCR. Figu e 4 Me hyla ion s a us o KDR and FLT4 in cell lines and p ima y cells. The me hyla ion s a us o KDR and FLT4 in leukemia cell lines and in HDMECs and HUVECs was analyzed by MSP a e bisul i e con e sion o he DNA. Aga ose gels o KDR and FLT4 M- and U-PCR a e shown. A comple e lis o esul s is shown in Table 2. NTC, non empla e con ol. Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 6 o 10 KDR and FLT4 in HDMECs and HUVECs HDMECs, HUVECs and KDR posi i e leukemia cell lines exhibi ed deme hyla ed KDR p omo e s (Figu e 3, Table 2). Howe e , mRNA and p o ein le els we e dis- inc ly highe in he p ima y cells han in he leukemia cell lines (Figu e 1). Resul s o Wes e n blo analysis we e con i med by low cy ome y (Figu e 7). HDMECs and HUVECs exp essed he pan-endo helial ma ke CD31 (Figu e 7). HDMECs, p ima ily consis ing o lym- pha ic endo helial cells, we e also posi i e o he Figu e 5 Bisul i e sequencing o he FLT4 p omo e . A CpG island is loca ed be ween -1231 and 769 ela i e o he ATG codon o FLT4. Pa o he p omo e egion (337 bp, 20 CpG si es) was sequenced om cell lines EM-2 (FLT4 nega i e) and SUP-B15 (FLT4 posi i e) as well as om HDEMCs and HUVECs. Each line depic s a sequenced clone ep esen ing he me hyla ion s a us o an indi idual allele. CpGs a e ep esen ed as open do s (i unme hyla ed) o illed do s (i me hyla ed). Resul s o qRT-PCR and me hyla ion speci ic PCR (MSP) a e shown on he le hand side. M: signal in M-PCR; U: signal in U-PCR. Table 3 E ec o 5-Aza-dC on exp ession o KDR and FLT4 KDR FLT4 mRNA induced by Aza mRNA induced by Aza CMK pos - pos - DOHH-2 neg + neg ++ EM-2 neg (+) neg +++ HL-60 neg +++ pos - L-82 neg - neg + SC-1 neg ++ neg + Induc ion o KDR and FLT4 by 5-Aza-dC (5 μM, 3 d) when compa ed o un ea ed con ol cells: - < 2.5- old; (+) > 2.5- old; + > 10- old; ++ > 40- old; + ++ > 160- old. Exp ession le els we e de e mined by qRT-PCR. TBP exp ession was used as endogenous con ol, DMSO (0.05%) ea ed cells we e used o no maliza ion Figu e 6 Deg ada ion o IB and phospho yla ion o p38MAPK in MALP-2 esponsi e cell lines. Cell lines EM-2 (FLT4 nega i e) and OCI-AML1 (FLT4 posi i e) we e s imula ed wi h MALP-2 (100 ng/ml). The NF-B inhibi o IB was phospho yla ed (7 min) and deg aded (7 min, 21 min) in bo h cell lines. Likewise, apid phospho yla ion o p38 MAPK was obse ed in bo h cell lines by Wes e n blo analysis. GAPDH is shown as loading con ol. Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 7 o 10 lympha ic essel ma ke podoplanin, HUVECs we e podoplanin nega i e (Figu e 7). Bo h ypes o p ima y cells exp essed much highe le els o KDR han KDR- posi i e cell lines (Figu e 1). Also FLT4 exp ession le els a ied g ea ly om one cell ype o he o he : FLT4 exp ession o HUVECs was compa able o hose o FLT4 posi i e cell lines, while HDMECs showed much highe FLT4 exp ession le els (Figu es 1 and 7). These esul s a e in line wi h ou da a o MSP analyses and 5- Aza-dC expe imen s sugges ing ha DNA me hyla ion Figu e 7 Exp ession o KDR and FLT4 on HDMECs and HUVECs. Flow cy ome y analysis o KDR, FLT4, he lympha ic essel ma ke podoplanin and he panendo helial ma ke CD31. No e ha HDMECs and HUVECs show compa able KDR exp ession le els, while FLT4 is s onge in lympha ic essel cells (HDMECs) han in blood essel cells (HUCECs). The non- illed peak shows he co esponding iso ype con ol. Quen meie e al.BMC Cance 2012, 12:19 h p://www.biomedcen al.com/1471-2407/12/19 Page 8 o 10 is no he only mechanism ha con ols KDR and FLT4 gene exp ession. Conclusions Ou da a ob ained om p ima y endo helial cells and om leukemia/lymphoma cell lines show ha KDR and FLT4 a e epigene ically egula ed genes. Bo h genes can be silenced by me hyla ion. Howe e , i he p omo e s a e unme hyla ed, o he ac o s a e esponsible o he ex en o KDR and FLT4 exp ession. Fu he mo e, we show ha he KDR nega i e/FLT4 posi i e cell line OCI-AML1 is a model sys em o FLT4 signal ansduc- ion s udies. Acknowledgemen s We hank Rod AF MacLeod (DSMZ) o c i ically eading he manusc ip . Au ho de ails 1 Depa men o Human and Animal Cell Cul u es, Ge man Collec ion o Mic oo ganisms and Cell Cul u es, B aunschweig, Ge many. 2 Depa men o Hema ology and Oncology, Geo g-Augus -Uni e si y Gö ingen, Uni e si y Medical Cen e , Gö ingen, Ge many. 3 Depa men o Gene Regula ion and Di e en ia ion, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many. Au ho s’con ibu ions HQ designed he s udy, pe o med da a analyses and w o e he manusc ip . SE ca ied ou bisul i e con e sion and helped o design p ime s o M-PCR and U-PCR. JR pe o med Wes e n blo analyses. HAW supplied an ibodies, HDMECs and HUVECs and ga e good ad ice. MZ pe o med PCR and FACS analyses. HGD p o ided cell lines and good ad ice. All au ho s ead and app o ed he inal manusc ip . Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Recei ed: 18 No embe 2011 Accep ed: 17 Janua y 2012 Published: 17 Janua y 2012 Re e ences 1. 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