www.elsevier.com/locate/ygyno

94 (2004) 125–133

Gynecologic Oncology

Vascular endothelial growth factor (VEGF) and ovarian carcinoma cell

supernatant activate signal transducers and activators of transcription

(STATs) via VEGF receptor-2 (KDR) in human

hemopoietic progenitor cells

Feng Ye,a,1 Huai-Zeng Chen,b,1 Xing Xie,a,* Da-Feng Ye,a,1

Wei-Guo Lu,a,1 and Zhi-Ming Dinga,1

aDepartment of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, ChinabCentral Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China

Received 26 November 2003

Available online 6 May 2004

Abstract

Objective. To investigate the STATs signaling pathway activated by VEGF in human hemopoietic progenitor cells.

Methods. CD34+ hemopoietic progenitor cells, which isolated from umbilical cord blood, were treated with VEGF or culture supernatant

of ovarian carcinoma cell line which could secrete large amount of VEGF, phosphorylation and nuclear translocation of STAT3 and STAT5

were then detected by Western Blot and immunocytochemistry. Expression of VEGFR2/KDR on CD34+ cells was studied by

immunocytochemistry. The specific VEGFR2/KDR heptapeptide antagonist ATWLPPR was used to identify whether the activation of STATs

signaling pathway was specifically mediated by VEGFR2/KDR.

Results. The concentration of VEGF in SKOV3-supernatant was 4024.84 F 505.59 pg/ml. CD34+ progenitor cells could express

VEGFR2/KDR. When CD34+ cells were stimulated by VEGF and SKOV3-supernatant, STAT3 appeared tyrosine-phosphorylation and

nuclear translocation, but STAT5 was only phosphorylated, and not translocated. When ATWLPPR was used to block the binding of VEGF

to KDR, VEGF and the SKOV3-supernatant failed to activate the phosphorylation of STAT3 and STAT5.

Conclusions. STAT3 may participate in the signal transduction pathways activated by VEGF specifically mediated by VEGFR2/KDR in

human hemopoietic progenitor cells, and the aforementioned signaling pathway participated in the interaction of ovarian carcinoma cells and

progenitor cells.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Vascular endothelial growth factor; Vascular endothelial growth factor receptor-2; CD34+ hemopoietic progenitor cells; Signal transducers and

activators of transcription; Ovarian neoplasms

Introduction molecule and endothelial cell-specific mitogen. VEGF plays

Vascular endothelial growth factor (VEGF), also known

as vascular permeability factor, is a potent angiogenesis

0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygyno.2004.03.038

* Corresponding author. Department of Gynecologic Oncology,

Women’s Hospital, School of Medicine, Zhejiang University, Xueshi Road

#2, Hangzhou 310006, China. Fax: +86-571-87061878.

E-mail addresses: yefyef@sohu.com (F. Ye), boychen888@sohu.com

(H.-Z. Chen), xiex@mail.hz.zj.cn (X. Xie), Lyahai@mail.hz.zj.cn

(D.-F. Ye), Lwg@hzcnc.com (W.-G. Lu).1 Fax: +86-571-87061878.

a major role in both embryonic hematopoiesis and tumor

angiogenesis. VEGF is 34- to 42-kDa protein produced by

almost all tumor cells, and its increasing level is closely

associated with a poor prognosis [1,2]. Tumor angiogenesis

and subsequent tumor growth can be inhibited by antibodies

not only directed against VEGF [3], but against soluble

VEGF receptors [4] or by expression of dominant-negative

VEGF receptors [5]. Although the role of this multifunc-

tional cytokine in angiogenesis was well established, recent

study indicated that VEGF was also a potent regulator of

immunological function. For example, CD34+ cells cultured

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133126

in serum-free medium supplemented with VEGF increased

the expansion of total cells, especially CFU-GM, HPP-CFC,

and CD34+KDR+CD38� cells, which represented the more

primitive stem cell subset [6]. VEGF could also promoted

the mobilization and recruitment of hematopoietic stem cells

into tumor sites, injured tissues, and other specific organs in

vivo [7,8].

On the other hand, VEGF may dramatically affect the

differentiation of multiple hematopoietic lineages [9,10]. It

has been demonstrated that VEGF signaling could inhibit

NF-nB activation in hemopoietic progenitor cells and lead

to defective maturation of dendritic cells (DCs) [11].

Meanwhile, in the presence of VEGF, CD34+ precursor

cells predominantly developed into endothelial-like cells

by increased expression of endothelial cell markers and

decreased expression of CD1a and CD83 molecules,

which represented an intermediate phenotype in the path-

way that led to mature endothelial cells instead of den-

dritic cells [12,13]. As a result, the number of mature

tumor-infiltrating DCs, which is inversely correlated with

the expression of VEGF, was markedly reduced in tumor

sites [14,15]. Thus, VEGF may be proposed as one of the

major factors responsible for immunosuppression in ma-

lignancies including ovarian carcinoma. The inhibition of

the CD34+ cells to mature DC cells by VEGF could

explain the paradoxical phenomenon, an increased number

of mobilized CD34+ progenitor cells while a decreased

number of mature DCs within peripheral blood and tumor

tissue of ovarian cancer.

Although the fact that VEGF could regulate differen-

tiation of multiple hemopoietic lineages has been well

understood, the exact mechanism of its regulation, espe-

cially the post-receptor signaling pathways underlying

VEGF actions on hemopoietic progenitor cells, remains

unclear. Two high-affinity VEGF receptors, VEGFR1

(Flt-1) and VEGFR2 (Flk-1/KDR), have been identified

in CD34+ progenitor cells [16,17], suggesting that VEGF

can directly effect on these cells. VEGF receptors have

tyrosine kinases activity and phosphorylate on specific

tyrosine residues of SH2 domain-containing signaling

molecules [18]. It has been recently discovered that a

new class of SH2 domain-containing signal molecules

belongs to the family of latent cytoplasmic transcription

factors known as signal transducers and activators of

transcription (STATs) [19]. STATs are the only known

intracellular phosphotyrosine-dependent transcription fac-

tors and employed in many mammalian cytokine

responses, for example, prolactin, erythropoietin, granulo-

cyte-macrophage colony-stimulating factor, interleukin-3,

and growth hormone for specific transcriptional activation

of genes [20]. STATs proteins can regulate cell survival,

apoptosis [21,22], proliferation [23], and the differentia-

tion of developing human bone marrow stem cells and

hemopoietic progenitor cells [24,25]. As conserved tran-

scription factors that transduce high-fidelity signals for

the cytokine family of ligands and receptors, STATs are

most frequently recognized for their role in regulating

both innate and acquired immunity [26].

More recently, it has been proved that STATs is involved in

VEGF/VEGFR2 signaling transduction in cardiac myocytes,

aortic endothelial cells and microvascular endothelial cells

[27–29]. On the basis of the above findings, we hypothesize

that STATs also participate in the VEGF signaling transduc-

tion pathway in human CD34+ cells, which is probably

associated with tumor-induced chemoattraction and defective

differentiation of hemopoietic progenitor cells. We treated

CD34+ progenitor cells with recombinant human VEGF165

(rhVEGF165) and the culture supernatant of ovarian carci-

noma cells. Tyrosine-phosphorylated STAT3 (p-STAT3) and

STAT5 (p-STAT5) were then detected byWestern blot and the

nuclear translocation was monitored by immunocytochemis-

try following VEGF and the supernatant stimulation.

We demonstrate, for the first time, that STATs participate

in the VEGF/VEGFR2 signaling transduction pathways in

human hemopoietic progenitor cells, and we also provide

evidence for the existence of above-mentioned signaling in

the interaction between ovarian carcinoma and hemopoietic

progenitor cells.

Materials and methods

Preparation of ovarian carcinoma cells culture supernatant

Cell culture supernatant from three ovarian epithelial

carcinoma cell lines (SKOV-3, Caov-3, and 3AO) was

employed in the studies. SKOV-3 and Caov-3 were pur-

chased from American Type Culture Collection; 3AO was

purchased from cell bank of Shanghai Institute of Bioche-

mistry and Cell Biology of Chinese Academic of Science.

Briefly, seeding 25-cm2 flasks with 1 � 106 tumor cells in

10 ml of complete RPMI 1640 medium (Gibco BRL, USA)

supplemented with 10% heat-inactivated FCS (Gibco BRL).

The supernatant was collected after 24 h, centrifuged at

4000 rpm for 5 min to remove cells and debris, aliquoted per

250-Al, and stored at �20jC for further VEGF ELISA

assay, and then screened a cell line with the highest

secretion of VEGF.

ELISA measurements of secreted VEGF

Concentration of secreted VEGF in supernatant was

quantitated by a commercial VEGF ELISA Kit (Quantikine

human VEGF immunoassay; R&D Systems) according to

the manufacturer’s protocol. The supernatant (50 Al) was

added into each well of microtiter plates, which was

precoated with anti-human VEGF polyclonal antibody,

and incubated for 2 h. Each well was washed with washing

buffer and incubated with 100 Al of enzyme-linked poly-

clonal antibody specific for human VEGF for 2 h. Fol-

lowing washes, a substrate solution was added to each

well. After incubation for 30 min at room temperature,

Table 1

VEGF Secretion of human ovarian carcinoma cell lines

Cell lines Secretion rate (pg/106/ml)

SKOV-3 4024.84 F 505.59

Caov-3 704.18 F 50.32

3AO 1047.99 F 78.84

Note. The sensitivity of the ELISA assay of VEGF was >5 pg/ml. Each data

entry constitutes four independent measurements from different cell

cultures. Data shown are mean F SD.

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133 127

enzyme reaction was stopped, and intensity was measured

at 450 nm using Universal Microplate Reader Elx800

(Biotek Instruments, Inc.).

Isolation of human cord blood CD34+ cells

Normal umbilical cord blood was immediately collected

following full-term deliveries according to the institutional

guidelines and the informed consent was obtained from all

volunteers (n = 10, data not shown). CD34+ cells were

isolated as described [30], which was modified. In brief, the

blood was diluted 1:4 with PBS and centrifuged in Ficoll-

Paque (1.077 g/ml; Amersham Pharmacia Biotech). The

peripheral blood mononuclear cells (PBMCs) were recov-

ered from the interface. Cells bearing CD34 antigen were

isolated by high-gradient magnetic cell separation (MACS)

with use of anti-CD34-mAb-conjugated immunomagnetic

beads (Miltenyi Biotec, Gladbach, Germany), and more than

95% of these isolated cells expressed CD34 when analyzed

by flow cytometry (data not shown). The enriched CD34+

cells (5 � 105/ml) were seeded and cultured in 25 cm2 tissue

culture flasks in RPMI 1640 plus 10% FCS, 5 � 10�5 M 2-

ME, and antibiotics (100 U/ml penicillin, 100 mg/ml

streptomycin) at 37jC in a humidified CO2-containing

atmosphere.

Stimulation and cell protein extraction of CD34+ cells

CD34+ cells, 5 � 106, were switched to serum-free

medium for 16–18 h at a temperature of 37jC, and then

treated with rhVEGF (50 ng/ml; R&D) for different times

(0, 15, 30, 45, 60, 90 min), or treated with ovarian

carcinoma cells supernatant (15% volume final concentra-

tion) with or without monoclonal anti-VEGF antibody

(1 Ag/ml; R&D). In some experiments, ATWLPPR, an

effective peptide screened from phage epitope library by

affinity for membrane-expressed KDR and blocking the

binding of VEGF to KDR [31] was used to identify

whether the activation of STATs pathway is specifically

mediated by KDR. When incubation was terminated by

removal of medium, cells were washed twice in ice-cold

Fig. 1. VEGFR2/KDR expression in CD34+ progenitor cells. Freshly isolated CD3

immunocytochemistry). These results are representative of four separate experime

progenitor cells; (B) negative result of control (200�).

PBS containing 2 mM Na3VO4 and 1 mM phenylmethyl-

sulfonyl fluoride (PMSF). Cell lysates were then obtained

by using ice-cold lysis buffer (20 mM Tris–HCl, pH 7.4,

1% Triton X-100, 2.5 mM EDTA, 0.1% SDS, 10%

glycerol, 2 mM Na3VO4, 50 mM NaF, 1 mM pepstatin,

1 mM PMSF, 5 mM leupeptin, and 100 U/ml aprotinin).

Lysates were centrifuged at 13,000 rpm for 30 min to

remove nuclei and cell debris and the supernatant was

retained at �80jC for Western blot.

Detection of STATs phosphorylation by Western blot

Phosphorylation of STAT -3 and -5 was detected by using

commercial goat or rabbit polyclonal primary antibodies (all

from Santa Cruz Biotech, Santa Cruz, CA): P-STAT 3

(Tyr705), P-STAT 5 (Tyr694) and Actin (C-11). Actin was

used to evaluate the relative protein amounts. Horseradish

peroxidase-conjugated donkey anti-goat IgG or goat anti-

rabbit IgG (Santa Cruz Biotech) was used as secondary

antibodies.

For Western blot, 20-Ag protein samples were electro-

phoresed in 11%SDS-PAGE gel and the fractionated proteins

were transferred onto nitrocellulose membrane using stan-

dard techniques. Membranes were blocked for 30 min at

room temperature with 5% non-fat dry milk in TBS contain-

ing 0.05% Tween-20 (TBST), probed with the primary anti-

bodies at a 1:500 dilution for 1 h at room temperature and

washed four times with TBST. The horseradish peroxidase-

conjugated secondary antibody (1:2000 dilution) was added

for 1 h at room temperature. After an extensive rinsing,

immunoreactive protein bands were visualized with a chemi-

4+ cells were labeled with antibodies against specific VEGFR2/KDR (ICH,

nts. (A) Strong expression of KDR is detected on the membrane of CD34+

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133128

luminescence-based procedure using the ECLk Western

blotting Analysis System according to the instructions of

the manufacturer (Amersham Pharmacia Biotech), and sub-

sequently exposed to film. Densitometric analysis was per-

formed using Quantity one software (Bio-Rad, USA).

VEGFR-2 expression and STATs translocation detected by

immunocytochemistry

For identification of VEGFR-2 expression, freshly iso-

lated CD34+ cells were incubated with anti-VEGFR2 (Santa

Cruz Biotechnology) and immunocytochemistry was done.

In addition, CD34+ cells (5 � 105) cultured on slide glasses

stimulated by VEGF for different time were then immunos-

tained for detection of p-STAT3 and p-STAT5. After fixed

with an ice-cold acetone/methanol mixture (1:1) for 5 min at

4jC, cells were blocked with 1% BSA in PBS for 20 min,

washed with PBS and then incubated with 3% H2O2 for 15

min to remove endogenous peroxidase. Cells were probed

Fig. 2. VEGF or tumor cell culture supernatant-induced STAT3 phosphorylation. (A

min, total protein were extracted and probed with anti-p-STAT3 antibody (WB, W

(50 ng/ml), SKOV3-supernatant, SKOV3-supernatant and anti-VEGF antibody

supernatant and ATWLPPR (80 AM) for 30 min. Cell extracts were immunobl

representative of four separate experiments.

with the anti-P-STATs primary antibody (1:80 dilution) for 2

h and washed three times with PBS. This step was followed

by incubation with a biotinylated secondary anti-goat anti-

bodies (Histostaink-Plus Kit, Zhong shan Biotechnology

Co., LTD, Beijing, China) for 15 min and an additional

incubation with a streptavidine-peroxidase conjugate for 15

min. Color was developed by incubating the slides with

diaminobenzidine (DAB) for 3 min at room temperature.

Cell nuclei were identified by counterstaining with hema-

toxylin. Controls were performed by PBS instead of the first

antibodies. Photographs were recorded at a magnification of

400�.

Statistical analysis

The results are expressed as the means F SD of data

obtained from 4 or more duplicated experiments. Statistical

significance (a = 0.05) was determined using the Student’s t

test.

) CD34+ cells were treated with rhVEGF165 (50 ng/ml) for 0, 15, 30, 60, 90

estern blot). (B) CD34+ cells were respectively treated with rhVEGF165

(1 Ag/ml), rhVEGF165 (50 ng/ml) and ATWLPPR (80 AM), SKOV3-

otted with anti-p-STAT3 antibody (WB, Western blot). These results are

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133 129

Results

VEGF secretion of ovarian carcinoma cell lines

Ovarian cancer is the leading cause of death from

gynecological cancer in women. Expression of VEGF by

ovarian cancer cells directly correlates with the tumor

progression and the production of malignant ascites [32].

Significant secretion of VEGF was detectable in all culture

supernatant of SKOV-3, Caov-3 and 3AO (Table 1), where-

as VEGF secretion of SKOV-3 was higher than that of

Caov-3 and 3AO (F = 151.36, P = 0.0001).

VEGFR2 (KDR) expression in CD34+ progenitor cells

Characterization of the two VEGF receptors suggests

diverse signaling transduction properties. VEGFR2 (Flk-1/

KDR) undergoes strong ligand-dependent tyrosine phos-

phorylation, whereas VEGFR1 (Flt-1) shows a weak re-

sponse [33]. In our study, the presence of VEGFR2 protein

in cord blood CD34+ progenitor cells was confirmed using

Fig. 3. VEGF-induced nuclear translocation of p-STAT3. CD34+ cells were treated

antibodies against specific p-STAT3 (ICH, immunocytochemistry). These results

mainly in cytoplasms at 0 min; (B) p-STAT3 were in cytoplasms and perinuclear re

90 min; (E) negative result of control (200�).

anti-VEGFR2 antibody staining by immunocytochemistry,

as shown in Fig. 1 (n = 4).

VEGF stimulates STATs tyrosine phosphorylation and

p-STATs nuclear translocation

As discussed in Introduction, it has been postulated that

phosphorylation of STATs plays an important role in differ-

entiation of hematopoietic precursor. In this study, we

examined whether VEGF stimulates the tyrosine phosphor-

ylation and nuclear translocation of p-STAT-3 and -5 in

human cord blood CD34+ cells. Furthermore, we also

analyzed whether the aforementioned signaling pathways

was involved in the interaction of ovarian carcinoma cells

and CD34+ progenitor cells.

Tyrosine phosphorylation of STAT3 was undetectable in

unstimulated CD34+ cells but detectable in CD34+ cells

stimulated by rhVEGF165 and the SKOV3-supernatant. We

noticed that VEGF stimulation resulted in a rapid and

transient STAT3 tyrosine phosphorylation and the maximal

tyrosine phosphorylation was caught at 30 min, returned to

with rhVEGF165 (50 ng/ml) for 0, 15, 30, 45, 60, 90 min, then labeled with

are representative of four separate experiments. (A) p-STAT3 were located

gion at 15 min; (C) in nucleus at 30 min; (D) mainly in cytoplasms again at

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133130

basal levels at 90 min (Fig. 2A). Blocking of the secreted

VEGF in SKOV3-supernatant by VEGF neutralizing anti-

body made the STAT3 phosphorylation significantly de-

creased (Fig. 2B). Immunocytochemistry confirmed that p-

STAT3 was mainly in cytoplasms at 15 min (Fig. 3B),

translocated into the nuclei at 30 min (Fig. 3C), and mainly

in cytoplasms again at 90 min (Fig. 3D) after VEGF stimu-

lation, but no nuclear translocation in unstimulated CD34+

cells (Fig. 3A).

VEGF-induced STAT5 tyrosine phosphorylation pattern

is presented in Fig. 4. It was evident at 15 min in response

to VEGF stimulation, reached the peak at 45 min, and

returned to basal levels at 90 min (Fig. 4A). Neutralization

of VEGF in SKOV3-supernatant also made the p-STAT5

fail to be detected (Fig. 4B). We also revealed that unlike

Fig. 4. VEGF or tumor cell supernatant-induced STAT5 tyrosine phosphorylation.

as mentioned in Fig. 2A. Cell lysates were extracted and probed with anti-p-STAT

with rhVEGF165 (50 ng/ml), SKOV3-supernatant, SKOV3-supernatant and anti-V

SKOV3-supernatant and ATWLPPR (80 AM) for 30 min. Cell extracts were imm

shown are representative of four separate experiments.

STAT3, there was only increased p-STAT5, which

appeared to be mainly in cytoplasms, but no significant

nuclear translocation of p-STAT5 after stimulation of

VEGF (Fig. 5).

ATWLPPR blocking phosphorylation of STATs induced by

VEGF

To characterize whether KDR is responsible for the

activation of STATs, the specific KDR heptapeptide antag-

onist ATWLPPR was used to bind exclusively to KDR.

Under our experimental conditions, VEGF and SKOV3-

supernatant failed to activate the phosphorylation of STAT3

(Fig. 2B) or STAT5 (Fig. 4B) when 80 AM ATWLPPR was

added into the culture medium for 30 min. It indicated that

(A) CD34+ cells were treated with rhVEGF165 (50 ng/ml) for different time

5 antibody (WB, Western blot). (B) CD34+ cells were respectively treated

EGF antibody (1 Ag/ml), rhVEGF165 (50 ng/ml) and ATWLPPR (80 AM),

unoblotted with anti-p-STAT5 antibody. (WB, Western blot). The results

Fig. 5. VEGF-induced intracellular mobilization of p-STAT5. CD34+ cells were treated as described in Fig. 3, probed with antibodies against p-STAT5 (ICH,

immunocytochemistry). These results are representative of four separate experiments. p-STAT5 were located mainly in cytoplasms at 0 min (A), 30 min (B), 45

min (C) and 90 min (D); negative result of control (E) (200�).

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133 131

the activation of STATs was specifically mediated by

VEGF/KDR.

Discussion

The CD34+ cells are the critical developmental interme-

diate between embryonic stem (ES) cells and the lympho-

hematopoietic lineages, and also represent the B-lymphoid

potential and part of the erythro-myeloid cell potential of ES

cells as well. The development of CD34+ cells is regulated

by various hematopoietic growth factors, cytokines and

chemokines secreted in the microenvironment by accessory

cells (e.g., fibroblasts, macrophages, osteoblasts, and endo-

thelial cells)[34]. VEGF is a crucial cytokine in hematopoi-

etic microenvironment and essential for an appropriate

balance of signals that preserves the stem cell pool while

permitting controlled growth and differentiation of stem

cells [35,36]. Under physiological conditions, VEGF regu-

lates the localization, conservation, coordinated prolifera-

tion, and differentiation of primitive hematopoietic

progenitor cells. The structural and functional changes in

the microenvironment caused by abnormal expression of

VEGF are associated with immunosuppression and the

tumor progression [37]. Identification of the interaction of

VEGF and hematopoietic progenitor cells and its signaling

transduction pathway could shed more light on the regula-

tion of human immunocytes and show at a molecular level

how VEGF could affect these cells and subsequently lead to

immunosuppression in cancer patients.

The data of the present study establish a novel mecha-

nism of VEGF-mediated regulation of human hemopoietic

progenitor cells. We showed for the first time that only

STAT3 was tyrosine phosphorylated and nuclear translo-

cated by VEGF stimulation in CD34+ cells, in contrast to

STAT5, which was only phosphorylated at tyrosine residues,

but not translocated. Previous work has indicated that

tyrosine phosphorylation of STATs are necessary but not

sufficient for their transcriptional ability, which also requires

p-STATs nuclear translocation. Hence, our results suggest

that activated STAT3, rather than STAT5, participates in the

VEGF action on CD34+ progenitor cells.

It has been reported that VEGF-induced activation of

STATs was mediated by VEGFR2 (KDR) [16,17]. Coinci-

dent with previous studies, we demonstrated the presence of

KDR in human CD34+ cells. When CD34+ cells were

F. Ye et al. / Gynecologic Oncology 94 (2004) 125–133132

incubated with VEGF in the presence of specific KDR

heptapeptide antagonist ATWLPPR, STATs activation was

blocked, suggesting that activation of the STATs signaling

pathway induced by VEGF was specifically dependent on

KDR. Considering STATs function on proliferation and

differentiation of developing human stem cells and hemo-

poietic progenitor cells, our results indicate that the biologic

effects of cytokines in the microenvironment such as VEGF

depended on the intracellular balance of transcriptional

regulators.

Ovarian carcinoma is one of the malignancies producing

plenty of VEGF. It is well known that VEGF protein level

was markedly elevated in serum and malignant ascites of

patients suffering from ovarian cancer [38]. To better

understand the biological significance of our findings in

ovarian cancer, we further examined VEGF level in culture

supernatant of ovarian carcinoma cell line, and its action on

promoting tyrosine phosphorylation and translocation of

STATs in freshly isolated CD34+ cells. We found that the

concentration of VEGF in the media of SKOV3 was as high

as 4024 pg/ml, and the supernatant stimulated significant

phosphorylation of STAT3 and STAT5 in CD34+ cells.

VEGF antibody could inhibit the phosphorylation of STATs.

This could be explained by the presence of VEGF in these

media. p-STATs only appears to decrease but not disappear

completely with VEGF antibody, so we think that the

domain which VEGF binds to its receptor may be different

with the domain which VEGF immunoreacted with its

monoclonal antibody, its partial signaling activity was

reserved. Certainly, it does not exclude the possibility that

there were some other factors exist in the supernatant, which

could activate the VEGF receptor and then transduce the

message to the STATs. Taken together, the results suggested

that the ovarian carcinoma cells culture media stimulating

activation of STATs was mainly produced by VEGF al-

though various cytokines may exist in the media.

In summary, we have supplied a novel reasonable mo-

lecular mechanism of VEGF-induced activation of STATs

signaling pathway in normal CD34+ progenitor cells and the

interaction between ovarian carcinoma cells and hematopoi-

etic progenitor cells that may be associated with the immu-

nosuppression and malignant progression of ovarian

carcinoma. In contrast to other known second messenger

systems, the STATs signaling pathway consists of a chain of

specific protein–protein interactions leading to specific

protein–DNA interactions in the nucleus [39,40]. We are

in the process of examining intranuclear events after nuclear

translocation of STAT3 to clarify the biologic effects on

CD34+ progenitor cells resulting from stimulation of VEGF.

Acknowledgments

We thank the Fund support from the Department of

Science and Technology of Zhejiang Province.

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