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
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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
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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
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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.
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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.
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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.
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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.
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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
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