(CANCER RESEARCH56. 3891—3894,September 1, 1996]

Advances in Brief

Regulation of Vascular Endothelial Growth Factor Expression in Human ColonCarcinoma Cells by Cell Density'

Aaryan N. Koura, Wenbiao Liu, Yasuhiko Kitadai, Rakesh K. Singh, Robert Radinsky, and Lee M. Ellis2Departments of Surgical Oncology [A. N. K., L M. E.J and Cell Biology [R. R., Y. K.. W. L, R. K. S., L M. El, The University of Texas M. D. Anderson Cancer Center, Houston,Texas 77030

Abstract

To determine the effect of cell density on vascular endothelial growthfactor (VEGF) expression and the mechanism of this effect, four humancolon cancer cell lines were grown as sparse or confluent monolayers or asspheroids. VEGF mRNA increased >2-fold in cells grown as confluentmonolayers or spheroids compared with cells grown as sparse monolayers.Semiquantitative reverse transcription-PCR demonstrated a 2-fold increase in the larger VEGF mRNA isoform (189 bp) in confluent cells.Sparse cells grown in conditioned medium from confluent cells demonstrated a >2-fold increase in VEGF mRNA. These data suggest thatVEGF expression may be regulated by an unidentified soluble factor.

Introduction

Angiogenesis is essential for tumor growth beyond 1—2mm@(1).Recent evidence suggests that metastasis is also angiogenesis dependent and that tumor cells are rarely shed into the circulation before thetumor is vascularized (2). In several human solid tumors, increasedangiogenesis has been associated with a poor prognosis (3).

VEGF3 (also known as vascular permeability factor or vasculotropin), an angiogenic protein, is a Mr 34,00050,000 dimericprotein synthesized by both malignant and normal cells (4). Allexperimental data suggest that VEGF acts via a paracrine mechanismon specific receptors present on the surface of endothelial cells (5),thereby stimulating their proliferation, migration, and organization invitro (6) and hence angiogenesis in vivo (7).

The expression of numerous genes has been shown to be celldensity dependent (8), and some reports suggest that regulation ofangiogenic factors is affected by cell density and cell-to-cell contact(9). The purpose of this study was to determine the influence of celldensity on expression of steady-state VEGF mRNA in four humancolon cancer cell lines. Our data demonstrate that human coloncarcinoma cells express increased levels of specific VEGF mRNAtranscripts under confluent culture conditions and after treatment withconditioned medium from confluent cells.

Materials and Methods

Human Colon Carcinoma Cell Lines. The human colon carcinoma celllines KM2O, KMI2C, and KM12SM were a gift from Dr. I. J. Fidler (TheUniversity of Texas M. D. Anderson Cancer Center). The KM2O and KMI2C

Received 5/20/96; accepted 7/16/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by NIH T-32 Grant CA09599 (to A. N. K.),

NIH/National Cancer Institute Grant R29 CA67952 (to R. R.), the Sid RichardsonFoundation (to R. R.), and American Cancer Society Career Development Award 94-21(to L. M. E.).

2 To whom requests for reprints should be addressed, at Department of Surgical

Oncology, Box 106, University of Texas M. D. Anderson Cancer Center, 1515 HolcombeBoulevard, Houston, TX 77030. Phone: (7 13) 792-6926; Fax: (7 13) 792-0722.

3 The abbreviations used are: VEGF, vascular endothelial growth factor; TGF-@,

transforming growth factor f3; GAPDH, glyceraldehyde phosphate dehydrogenase; bFGF,basic fibroblast growth factor.

cell lines were derived from primary colon cancers (Dukes' stages D and B,respectively; Ref. 10). The KM12SM cell line is a metastatic variant derived

from a liver metastasis after injection of the KM12C cell line into the cecum

of a nude mouse (10). The SW620 cell line, obtained from the American TypeCulture Collection (Rockville, MD), was derived from a lymph node metastasis of a patient with colon carcinoma.

In Vitro Cell Culture Conditions. The KM2O, KM12C, and KM12SMcell lines were grown in DMEM supplemented with 10% fetal bovine serum,

sodium pyruvate, nonessential amino acids, L-glutamine, and 2-fold vitaminsolution (Life Technologies, Inc., Grand Island, NY). The SW620 cell line was

grown in DMEM supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamlne,and vitamin solution (Life Technologies, Inc.).

Cells were plated as sparse (15—20%confluence; 4 X l0@cells/cm2) orconfluent (90—100%confluence; 4 X 106cells/cm2)monolayers. Conditionedmedium from confluent cultures was harvested after 3 days, centrifuged at lowspeed to remove cellular debris, and used immediately or stored at —20°C.Forspheroid formation, tumor cells (2 X l0@)were seeded onto 1%agarose-coatedplastic plates and grown for 3—4days (1 1). The resulting spheroids were snapfrozen in liquid nitrogen and then processed for mRNA extraction.

Cells grown under confluent conditions were trypsinized and grown assparse monolayers with enriched DMEM or conditioned medium from con

fluent cells. To test the effects of TGF-@, a growth factor that has beendemonstrated to up-regulate VEGF, we grew KM12SM cells as sparse andconfluent monolayers in enriched DMEM supplemented with 4 ng/ml ofTGF-@l (Santa Cruz Biotechnology, Santa Cruz, CA) for 72 h.

Northern Blot Analysis. Polyadenylated mRNA was extracted using theFast Track mRNA isolation kit (Invitrogen, San Diego, CA) and quantitated.

mRNA was loaded (2—3@xgIlane)and electrophoresed on a 1% denaturing

formaldehyde-agarose gel, electrotransferred at 0.6 A to a GeneScreen nylonmembrane (DuPont NEN, Boston, MA), and UV cross-linked at 120,000p.1/cm2.Hybridizations were performed as described previously (12). Nylonmembranes were washed at 65°Cwith 30 m@isodium chloride, 3 mistsodiumcitrate, and 0. 15% (w/v) SDS solution, and autoradiograms were obtained.Densitometric analysis (Molecular Dynamics, Sunnyvale, CA) was used toquantitate the VEGF mRNA expression using GAPDH mRNA transcripts asinternal controls for possible loading differences.

cDNA Probes. A human VEGF-specific 204-bp cDNA probe was a gift ofDr. Brygida Berse (Harvard Medical School, Boston, MA; Ref. 4). This probeidentifies all alternatively spliced forms of VEGF mRNA transcripts. A 1.3-kbPst-1 cDNA fragment corresponding to rat GAPDH mRNA was used as an

internal control (13).Semiquantitative Reverse Transcription.PCR. cDNA was synthesized

from total RNA extracted from KML2SMcells grown as sparse and confluentmonolayers by reverse transcription in a 20-pJ reaction containing 0.5 @xgofrandom primers (Life Technologies, Inc.), 200 units of SuperScript RNaseHRT (Life Technologies, Inc.), 0.1 @gof mRNA, 4 jxlof 5X RT buffer [375 mMKC1, 250 mM Tris-HC1 (pH 8.3 at room temperature), 15 mM MgCl2], 5 mMDTT, 0.1 mM of each deoxynucleotide triphosphate, 20 units of RNasin (LifeTechnologies, Inc.), and diethyl pyrocarbonate-treated water added to 20 p1

Each mixture was incubated at 37°Cfor 1 h and then quick-chilled on ice.Each PCR was performed in a 50-pi reaction mixture containing 5 xl of RT

reaction mixture, 1X PCR buffer [50 mistKCI, 10 mMTris-HC1 (pH 9.0 atroom temperature), and 1% Triton X-l00J, 0.2 mM concentration of eachdeoxynucleotide triphosphate, 1.5 mM MgCl2, 2.5 units of Taq polymerase(Promega Corp., Madison, WI), 50 pmol/pi VEGF primers, and 50 pmolipi

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CELL DENSITY REGULATION OF VEGF

SW620 KM20 KM12C KMI2SM

@@ @o @2

VEGF ::@ ihi.v@;iI

GAPDH1.3kb-0. a@@@ . * * . _

Densitometry 1.0 10 1.0 2.0 3.0 1.0 2.0 2.5 1.0 2.2 4.3

Fig. 1. Northern blot analysis of four colon carcinoma cell lines demonstrating VEGFand GAPDH mRNA expression in sparse and confluent monolayers and spheroid cultures.VEGF mRNA transcripts are detected as distinct bands measuring 4.4, 2.0, and 1.6 kb.The values shown at the bottom of the lanes are the ratio between VEGF-specifictranscripts and GAPDH-specific transcripts (1.3 kb). VEGF mRNA levels are increasedmore than 2-fold in cells grown under confluent conditions and as spheroids comparedwith cells grown under sparse conditions.

L32-microglobulinprimers (Clontech Laboratories, Palo Alto, CA). The mixturewas overlaid with mineral oil and then amplified with a Temp.Tronic DNAamplification system by using the following amplification profile: an initialdenaturation at 94°Cfor 5 mm; then denaturation at 93°Cfor 1 mm andannealing at 59°Cfor 1 mm; extension at 72°C for varying cycle numbers; and

a final elongation step at 72°Cfor 10 mm. The PCR reaction products werethen electrophoresed in a 2% agarose gel and stained with 0.5 @xg/mlethidiumbromide, visualized under UV light, and photographed with Polaroid Type 55positive/negative film (Polaroid Co., Cambridge, MA). Specific amplification

was determined by the size of the product on gel relative to known DNAmolecular weight marker V (Boehringer Mannheim, Indianapolis, IN). Theamount of PCR product was quantitatively determined by measuring thedensity of the specific bands on the negative film with a densitometer.

RT products were amplified by the PCR using specific primers for (32-microglobulin (Clontech Laboratories; Sequence; Sense, 5'- CTCGCGCTACTCTCTC1TFCTGG-3'; Antisense, 5'-GCUACATGTCTCGATCCCACTTAA-3') and VEGF (Ref. 14; Sequence; Sense, 5'-CACATAGGAGAGATGAGCTFC-3'; Antisense, 5'-CCGCCTCGGCTTGTC ACAT-3') in

separate reactions for 18 —28cycles under the above mentioned conditions and

the products quantitated by densitometry. Curves depicting product quantityversus number of PCR cycles were generated, and the PCR cycle number that

generated product quantities representing the upslope of the curve was selectedfor quantitative PCR for each specific set of primers. Quantitative PCR wasperformed for (32-microglobulin(22 cycles) and VEGF (25 cycles); the products were quantitated by densitometry using (32-microglobulinas an internalcontrol.

Cell Cycle and VEGF Expression. SW620 colon carcinoma cells weretrypsinized, fixed with 1% paraformaldehyde, and made permeable with 2 nil

of 70% ethanol. The cells were successively incubated with 2 xg of rabbitpolyclonal VEGF antibody in 100 ml of PBS (Santa Cruz Biotechnology), 5 p.1of goat antirabbit FITC-labeled second antibody in 100 p.1of PBS (SigmaChemical Co., St. Louis, MO) and then for 30 mm at 37°Cwith 1 ml ofpropidium iodide (50 ng/ml) and I ml of RNAse A (20 p.g/ml). The cells were

analyzed using an EPICS Profile flow cytometer (Coulter Corp., Hialeah, FL).Western Blot Hybridization. Cells were lysed with protein lysis buffer

[20 mMTris-HCI (pH 8.0), 137 mMNaC1, 10%Glycerol, 1%Triton X-l00, 1mM Na3PO4, 2 mM EDTA, 100 p.g/ml phenylmethylsulfonyl fluoride, 5 p.g/ml

leupeptin, and 10 p.g/ml aprotinin], and protein was quantitated spectrophotometrically. For cell supernatants, the protein was concentrated using a 10,000

MW concentration column (Amicon, Beverly, MA). Fifty-p.g aliquots of theprotein were separated by electrophoresis on a 12.5%polyacrylamide gel. Theprotein was transferred to Immobilon-P membrane (Millipore Corp., Bedford,MA) by electrotransfer. Following blocking with 5% milk in PBS-T (0.1%Tween 20 concentration in PBS), the membrane was probed with the primaryantibody (1:200 dilution of polyclonal rabbit antihuman VEGF; Santa CruzBiochemicals, Santa Cruz, CA). The membrane was washed, and the second

ary antibody was labeled with horseradish peroxidase (rabbit immunoglobulin

from donkey 1:2000 dilution; Amersham Corp., Arlington Heights, IL) wasapplied. Using a commercial chemoluminesence kit (Amersham Corp.), protein bands were visualized, and densitometry was performed.

Results

Cell Density-dependent Expression of VEGF mRNA. Northernblot analysis demonstrated a greater than 2-fold increase in steadystate VEGF mRNA transcripts in the colon cancer cells grown underconfluent conditions compared with cells grown under sparse conditions (Fig. 1). A 2.5-fold or greater increase in the level of expressionof VEGF mRNA was observed in cells grown as spheroids comparedwith cells grown under sparse conditions (Fig. I).

Kinetics of VEGF mRNA Expression. KM12SM cells grown asconfluent monolayers, trypsinized, and then reseeded under sparseculture conditions demonstrated a 2—3-folddecrease in VEGF mRNAat 24 h and then an increase in expression over time that directlycorrelated with increasing cell density (Fig. 2). Maximum levels ofsteady-state VEGF mRNA expression were reached with 80—90%cellconfluence, and this high level of VEGF mRNA expression wasmaintained in cells analyzed for up to 28 days in culture (Fig. 2).

Effect of Conditioned Medium and TGF-@J on VEGF Expres.sion. We next tested whether a soluble factor was responsible for theobserved up-regulation of VEGF-specific transcripts in confluentcells. KM 12SM cells were grown as confluent and sparse monolayersin standard medium, in conditioned medium obtained from cellsgrown as confluent monolayers, or in standard medium supplementedwith TGF-13. The addition of conditioned medium from confluentcells to cells growing as sparse monolayers resulted in a greater than2.5-fold increase in VEGF mRNA expression at 72 h compared withcells grown without conditioned medium. However, the addition ofTGF-/3 did not affect the expression of VEGF mRNA in the tumorcells (Fig. 3).

Effect of Confluence on Expression of Larger VEGF Isoforms.Quantitative PCR analysis of RNA from KM12SM cells grown assparse and confluent monolayers demonstrated at least a 50% increasein all VEGF transcripts. There was a 2-fold increase in expression of

a transcript corresponding to the I89-bp isoform of VEGF in confluent cells as compared to sparse cells (Fig. 4).

>-I- <

D Ui..1 (I)LI.z <0 Q.0 Cl)

c@) Ifl C@l>- >- )‘.< 4 4

@ 0 0

5.5kb—@@ -.

VEGF@

GAPDH i .3kb 0 •@@ ••

Densitometry 1.0 0.3 1.0 1.0 1.0Fig. 2. Northern blot analysis demonstrating VEGF mRNA expression in KMI2SM

cells grown as confluent monolayers, trypsinized, plated as sparse monolayers, and thenfollowed in culture for up to 28 days. Values shown at the bottom of the lanes are the ratiobetween VEGF-specific transcripts and GAPDH-specific transcripts (1.3 kb). VEGFmRNA levels decrease under sparse culture conditions and demonstrate a sustainedincrease under confluent conditions.

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CELL DENSITY REGULATION OF VEGF

Effect of Cell Cycle on Regulation of VEGF. Flow cytometricanalysis demonstrated no significant variation in staining for VEGFprotein in cells at different stages of the cell cycle (data not shown).

Effect of Cell Density on VEGF Protein Levels. To determinethat the findings from the studies on VEGF mRNA correlated withVEGF protein alterations, a representative cell line (SW620) wasplated either as sparse or confluent and grown for 48 h in standardmedia. Cells were lysed, protein was extracted, and Western blotswere done for VEGF protein expression. VEGF protein was expressedat high levels in the cells grown under confluent conditions comparedto minimal expression of VEGF in cells grown under sparse conditions (Fig. 5). These findings are consistent with our observations ofthe effect of cell density on VEGF mRNA expression.

4 Unpublished data.

3893

SW620

C)0

0) Z@D 11> I-

@ CC/) m

VEGF

PROTEIN 21kD@

Fig. 5. VEGF protein from cell lysates of SW620 cells grown under sparse andconfluent conditions was analyzed by Western blot hybridization. Cells grown underconfluent conditions expressed higher levels of VEGF protein than cells grown undersparse conditions.

Discussion

A variety of positive and negative regulators of angiogenesis maybe important determinants of tumor growth, metastatic potential,

prognosis, and response to therapy. VEGF is a potent stimulator ofangiogenesis both in vitro and in vivo (15). Levels of VEGF in somehuman malignant tumors, including renal and bladder carcinomas,

have been shown to be elevated compared with those in adjacentnon-involved tissue (16). Takahashi et a!. (17) recently demonstratedthat VEGF is an important angiogenic factor associated with themetastasis of human colon carcinoma. Others have demonstrated aclose correlation between levels of expression of VEGF mRNA andVEGF protein detected by Western blot analysis in cell lysates andsupernatant and by immunohistochemistry in tissue samples (16). Theimportance of VEGF as a potential target for antineoplastic therapyhas been demonstrated in several studies in which neutralizing antibodies to VEGF inhibited tumor growth and vasculanzation in vivo

(18).Following confluence, cells enter a quiescent growth phase that is

associated with alterations in expression of cell surface receptors,transcription factors, cytochemical enzymes, oncogenes, and growthfactors (9, 19, 20). Expression of another important angiogenic factor,

bFGF, has been demonstrated to be down-regulated in confluentcultures of normal fibroblasts (20) and tumor cells (9) compared withsparse cultures. One report suggested that confluent cultures producefactors that down-regulate gene expression (21 ), and expression ofone of these factors, TGF-(3, has been demonstrated to be up-regulatedin confluent colon carcinoma cells in vitro (22). Factors known to

up-regulate the expression of VEGF include hypoxia (23), which is apotent stimulant of VEGF expression both in vitro and in vivo,interleukin 1 (24), TGF-(3 (25), mutant p53 (26), epidermal growth

factor (27), platelet-derived growth factor (27), and bFGF (27). In

vitro, when cells are grown as monolayers, it is unlikely that hypoxiaplays a significant role in the regulation of VEGF, and our data showthat VEGF protein expression is independent of cell cycle regulation.Our data do demonstrate, however, that the expression of steady-stateVEGF mRNA in human colon carcinoma cells increases with increasing cell density in vitro. Preliminary studies from our laboratorysuggest that the expression of interleukin I , bFGF, and p53 is notdependent on cell density in the human colon carcinoma cell linesstudied.4 Furthermore, the addition of conditioned medium from

confluent cultures of colon carcinoma cells resulted in increasedexpression of VEGF mRNA in the same cells grown under sparse

5.5kb —@

VEGF 4.4kb—0'.-

3.7kb@

GAPDH 1.3kb—0'@

Densitometry 4.0 4.0 1.0 2.6 1.0

Fig. 3. Northern blot analysis demonstrating expression of VEGF and GAPDH mRNAin KM12SM cells grown under confluent and sparse conditions with DMEM, conditionedmedium, or TGF-j3. The values shown at the bottom of the lanes are the ratio betweenVEGF-specific transcripts and GAPDH-specific transcript (1.3 kb). VEGF mRNA isincreased 4-fold under confluent conditions and 2.5-fold in the presence of conditionedmedium. The addition of TGF-j3 does not affect expression of VEGF.

/, @.

VEGF189(300bp)@VEGF165(230bp)——0

VEGF 121 (100bp)—@

@2-MicrogIobuIin(340bp)—@ ‘@

Fig. 4. Reverse transcription-PCR products electrophoresed on a 1% agarose gelstained with ethidium bromide. PCR amplification of @2-microglobulinyields a productof 340 bp. The PCR amplification products for the 189, 165, and 121 VEGF transcriptsare 300, 230, and 100 bp, respectively. Using [32-microglobulin as internal loading control,the gel demonstrates a 50% increase in total VEGF transcripts and a 2-fold increase in the189 transcript in confluent KMI2SM cells.

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CELL DENSITY REGULATION OF VEGF

conditions. However, the addition of TGF-f3 to the medium of sparsecultures did not up-regulate expression of VEGF, suggesting thatanother factor in the conditioned medium from confluent cultures isinvolved. In addition, the effect of the conditioned medium on VEGFexpression is most likely not due to an autocrine effect of VEGFsecreted by the confluent tumor cells because most tumor cells do notexpress VEGF-specific receptors (28). The data thus support ourhypothesis that the up-regulation of VEGF in sparse cultures byconditioned medium is the result of a yet unidentified secreted factor.

The four homodimers of VEGF, containing 121, 165, 189, or 206amino acids, differ in their structural and angiogenic properties (29).The 121- and 165-bp homodimers are readily diffusable and mitogenic for endotheial cells, whereas the 185- and 206-bp homodimersare cell associated (29). This has led to the speculation that the 185-and 206-bp isoforms of VEGF may be involved in signal transductionpathways or cellular interaction (6, 29). The specific up-regulation ofthe 189-bp isoform of VEGF in confluent cultures demonstrated inour study provides further evidence that the larger isoforms of VEGFmay play a role in cellular interaction.

We suggest that increased cellular density induces secretion of afactor by tumor cells that may up-regulate expression of VEGF inadjacent tumor cells. It follows that the net result would be increasedangiogenesis, tumor growth, and possibly metastasis. By identifyingthe mechanisms by which angiogenic factors are regulated, we may beable to identify potential therapeutic targets in patients with advancedsolid malignancies.

Acknowledgments

We thank Melissa Burkett for editorial assistance.

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1996;56:3891-3894. Cancer Res   Aaryan N. Koura, Wenbiao Liu, Yasuhiko Kitadai, et al.   Human Colon Carcinoma Cells by Cell DensityRegulation of Vascular Endothelial Growth Factor Expression in

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