A definite correlation was found between Ang-1 mRNA expression and clinical stage of cervical cancer (p<0.05)

(angiogenesis)

1-3

, ,

, .⁴

5 ,⁶ ,

, (atherosclerosis),

, ⁷

, .⁸

.⁹

3,10,11

Department of Obstetrics and Gynecology, College of Medicine, Ewha Womans University, College of Medicine, Seoul, Korea

Objective : The angiopoietins (Ang) are recently described as one of growth factors for vascular endothelium. This study was aimed to investigate the Ang-1 and Ang-2 mRNA expression between in cervical cancer and in normal cervix and to assess the relationships among their expression and other prognostic factors of cervical cancer.

Patients and Methods : Ang-1 and Ang-2 mRNA expression were investigated by quantative competitive PCR between in cervical cancer of 34 cases and in normal cervix of 14 cases at Ewha Womans University Mokdong Hospital from March 1998 to June 2000.

Results : Both in cervical cancer and normal cervical tissue, 612 bp sized Ang-1 mRNA and 518 bp sized Ang-2 mRNA were expressed. In comparison to normal cervix, their expression was significantly elevated in cervical cancer (p<0.05). A definite correlation was found between Ang-1 mRNA expression and clinical stage of cervical cancer (p<0.05). There was significant correlation among Ang-2 mRNA expression and clinical stage, tumor sizes, SCC-Ag levels, lymph node metastasis of cervical cancer (p<0.05).

Conclusion : The expression of Ang-1 & -2 mRNA could be associated with the progression and metastasis of cervical cancer and might have a role as a prognostic parameter in cervical cancer.

Key Words : Cervical cancer, Angiopoietin

(02-PJI-PG3-20599-0009).

. .

(solid tumor) ,

, .¹²

vascular endothelial

growth factor (VEGF), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming

growth factor-beta (TGF- ) ,

Angiopoietin( Ang )

.¹² Ang

(vascular endothelial growth factor) . Ang-1 ,

(hypervascularization) , Ang-2 Ang-1

.

Ang Ang-1, -2, -3 -4

,¹³ Ang-1 -2 Tie2 .¹⁴ Ang-1 -2

60% Tie2

.¹⁵ Ang/Tie system

.^16,17

Ang-1 amino-terminal secretory signal sequence

498 ,

Tie2 tyrosine phosphorylation . Ang-2 Ang-1 (antagonist) 496

, Ang-1

Tie2 phosphorylation .¹⁸ Ang-1

Ang-1

.¹² Ang-2 Ang-1 VEGF

, VEGF

,^14,19,20 Ang-2

.^12,14

Ang-2 mRNA glioblas-

toma, Kaposi , , , ,

, ,12,17,18,21-24 Ang-2

.¹⁸ Ang-2

. Ang-1

. (xenograft)

Ang-1 antisense oligonucleotide nude mice

,²⁵ Ang-1 . Ang-1

.²⁶

Ang-1 -2 mRNA Ang-1 -2 mRNA

Ang-1 -2 mRNA ,

, , SCC-Ag

.

1998 3 2000 6

34

. 14

. .

1) RNA

total RNA .

phosphate buffered saline( PBS)

100 mg 1 ml RNA-STAT-60 (Tel-Test “B”

Inc., Frendswood, TX) . RNA-

STAT-601 ml 500 µl chloroform isopropanol 500 µl 75% ethanol

diethylpycocarbonate (DEPC) . RNA

.

2) (RT-PCR)

(ologonucleotide primer) Ang-1 -2 National Institute of Health

National Center for Biotechnology Information Gene Bank Database OLIGO 5.0 primer analysis software (National Bioscience, Plymouth, MN)

(oligonucleotide primer) . mRNA

Table 1 .

3) (Reverse Transcription)

Gen Amp RNA PCR

kit (Perkin-Elmer, Foster City, CA) . 5 mmol/L MgCl2, IX PCR buffer II, dNTP 1 mmol/L, 2.5 µl/L oligo deoxythimidine, 20 IU ribonuclease inhibitor, 50 IU M oloney m urine leukem ia virus reverse transcriptase( Perkin Elmer)

19 µl 1 µl 1 µg RNA 1 µl

mRNA 5’ end to 3’ end size (bp) position on mRNA

Ang-1

Ang-2

Upstream Downstream Competitor

Upstream Downstream Competitor

GCTTACCAGATTCACACTGTTCC TTGCTACCTTGCCAACAACTG

TTGCTACCTTGCCAACAACTGATTGCCTAAGGGAATG GACTG

TGGACAATTATTCAGCGACGTG GCTGGTCGGATCATCATGGTTG

GCTGGTCGGATCATCATGGTTGGTCCCTGTAAGTCCT TTAAGGTG

612

276

518

285

1771-1793 2382-2362 2025-2005, 2382-2362

1313-1334 1830-1809 1575-1553, 1830-1809

. Perkin-Eimer

DNA thermal cycler 9600 42 15 ,

99 5 4

-20 . RNA 1 µl

DEPC 1 µl .

4) Angiopoietin-1 -2 competitive target cDNA

RNA

3’-, 5’- (primer)

612 bp Ang-1, 518 bp Ang-2 target

DNA agarose gel

Promega DNA purification kit cDNA

. competitive cDNA 3’-, 5’-

3’-

floating primer 5’-

target cDNA

cDNA . competitive

cDNA Ang-1 276 bp, Ang-2 285 bp

.

5) Standard curve competitive PCR Ang-1 & -2 (standard curve)

competitive cDNA (5 f, 10 f)

target cDNA (Fig. 1).

1.9 mmol/l MgCl2, 10X PCR buffer , 0.2 mmol/l deoxy-NT, 2.5 U Taq-polymerase 100

µl PCR 0.2 µmol/L

Perkin Elmer DNA thermal cycler 9600

95 5 (denaturation)

Ang-1 94 1 , 62 45 , 72 1 , Ang-2

94 1 , 58 45 , 72 1 35

72 7 (elongation) 4

. Ethidium Bromide (ETB) 1% agarose

gel PCR 25 µl 100 bp

DNA ladder Bio-Rad

Gel-Doc 1000 system (UV) gel blot

. target

cDNA target cDNA/competitive cDNA gel blot

log .

, y=b+mx

cDNA .

RT QC PCR ±

5% .

SPSS 10.0 Man Whitney test

Spearman correlation test, logistic regression analysis , p <0.05

.

RT-PCR

612 bp Ang-1 mRNA, 518 bp

Ang-2 .

QC-PCR

target competitive band

(Fig. 1). mRNA

Ang-1

(p<0.05, Fig. 1 &

Table 2). Ang-2 target competitive

band (Fig. 2). mRNA

Ang-2 mRNA (p<0.05, Fig. 2 & Table 2).

mRNA expression (mean±S.E.) Ang-1 (pmol) Ang-2 (fmol) Normal cervix

Cervical cancer

0.48±0.24 20675.48±13080.86

0.56±0.16 333051.4±315488.5

*p value<0.05 by Man Whitney test.

Ang-1 mRNA

(p<0.05,

Table 3), , , SCC-Ag

,

(p>0.05, Table 3). Ang-2

mRNA , , SCC-Ag

,

(p<0.05, Table 3),

(p>0.05, Table 3).

Ang-1 Ang-2 mRNA

(p<0.05, data not shown).

,

, .¹⁴

, .¹

,

.²⁴ , , , ,

Clinicopathological

factors Finding Cases (n=34) Ang-1 mRNA

(p-value)

Ang-2 mRNA (p-value) Age

Stage

Pathology

Tumor size

Lymph node

SCC-Ag (ng/ml)

Squamous, Large cell keratinizing nonkeratinizing Adenocarcinoma

Small cell

<4 cm

>4 cm Negative

Positive

<2

>2

53.86±1.97 14 13 4 3 5 22 5 2 18 16 21 13 18 16

0.005

NS

NS

NS

NS

0.001

NS

0.001

0.001

0.003

*p value<0.05 by logistic regression analysis.

, ,

.²⁴

.

, ,

, ,

. ,²⁷

.

20

, VEGF, FGF, PDGF, TGF-

. VEGF (sVEGF)

sVEGF ,

sVEGF .²⁸

VEGF .²⁷ .

VEGF

FGF polypeptide growth factor

VEGF Ang

.

Ang Tie2

VEGF .

Ang/Tie2 system

, Ang-1 Ang-2

. Ang Tie2

, angiogenic outgrowth, (vessel remodeling), (maturation)

.

Ang Tie2 ,

.¹⁴

Ang

.²⁴

Ang-1 -2 mRNA

Ang-1 -2

mRNA . Ang-1 mRNA

, Ang-1 mRNA

,²⁹ Ang-1 mRNA

.³⁰ Ang-2 mRNA (glioblastoma), Kaposi ,

, , , ,

.12,17,18,21-24

Ang-1 -2 mRNA

Ang mRNA .

In situ hybridization

Ang-1 mRNA ,

Ang-2 mRNA

.²¹ Ang-1 mRNA

, Ang-2 mRNA

. Ang-1 Ang-2 mRNA .^15,31,32 Ang-1 -2 mRNA

, Ang-1 -2 mRNA

Ang-1 -2 mRNA . glioblastoma Ang-1 -2

, Ang-1

, Ang-2 .²¹ vascular sprouting

Ang-1 Ang-2 . Ang-1 -2 mRNA .

MCF-7 Ang-1

.²⁶ Antisense oligonucleotide

, Ang-1 mRNA Ang-1

(immunodeficient mice) ,

Ang-1

.²⁵ Ang-1

.

A431 Ang

Ang-1 Tie2

,

70% .²⁰ Ang-1

. Ang-1 mRNA

Ang-1/Tie2 .

Tie2 ,

Ang-1 .¹⁷

Tie2 Ang

Tie2 Tie2 .

Ang-2 Ang-1

VEGF , .^20,21 Ang-2 Kaposi

VEGF KDR

.¹² Ang-2

.

Ang-1 Ang-2 ,

Ang-1 Ang-2

,

.²¹ Ang-1 -2

mRNA , Ang-2 mRNA

. Ang-1

, Ang-2 Ang-1

.

Ang-2

.²⁴

Ang-2 ,

.²²

Ang-2 mRNA , ,

, SCC-Ag

. Ang-1

sprouting ,

Ang-2

. Ang-2

Ang-1 .

Ang-1 sprouting

focal adhesion kinase plasmin

.³² Ang-1 plasmin matrix metallopro-

teinase (MMP)-2 tissue inhibitors

of metalloproteinase (TIMP)-2

.^18,33 Ang MMP, VEGF

Ang-2 VEGF MMP

.²⁴ VEGF Ang-2

, .²²

VEGF,

Ang-2 MMP autocrine

Ang/Tie pathway paracrine

.²⁴ MMP, VEGF Ang-1

-2 mRNA

MMP, VEGF VEGF

Ang-1 -2 mRNA MMP

.

MMP VEGF Ang

Ang

mRNA , Ang Tie2

.

VEGF Ang

.³⁴ Antisense oligonucleotide Ang-1

(cell apoptosis) ,²⁵

anti-angiogenesis cancer therapy

.

: angiopoietin-1, 2 , Ang-1 -2 mRNA

Ang-1 -2 mRNA .

: 34 14 QT-PCR Ang-1

-2 mRNA .

: Ang-1 -2 mRNA

. Ang-1 mRNA , Ang-2 mRNA ,

, SCC-Ag .

: Ang-1 -2 mRNA , Ang-2 mRNA

. : , Angiopoietin