Vol. 5, 1085-1091, October 1994 Cell Growth & Differentiation 1085

Expression of Insulin-like Growth Factor (IGF)-Il and IGF-IReceptor during Proliferation and Differentiation of

. 1CaCo-2 Human Colon Carcinoma Cells

Raffaele Zarrilli,2 Sandro Pignata,3 Marco Romano,Adriano Gravina, Stefano Casola, Carmelo B. Bruni,and Angela M. Acquaviva

Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R.,Dipartimenlo di Biologia e Palologia Cellulare e Molecolare “L. Califano”

IR. Z., A. G., S. C., C. B. B., A. M. Al; Cattedra di GastroenterologiaIS. P.], Facolt#{224}di Medicina e Chirurgia, Universit#{224} di Napoli “FedericoII,” Dipartimento di Internistica Clinica-Cattedra di Gastroenterologia,

Secondo Ateneo di Napoli IM. RI, Napoli, Italy

Abstract

We have studied the expression of insulin-like growthfador type II (IGF-ll) and its autocrine role during theproliferation and differentiation of the CaCo-2 coloncarcinoma cell line. ICE-Il RNA levels were high inproliferating cells and decreased by more than 10-foldwhen cells ceased to proliferate and differentiated.Immunoreadive ICE-Il protein was high in theconditioned media of proliferating cells and decreased20-fold in the media of differentiated cells. ReducedICE-Il expression was associated with a decrease inICE-I receptor number that was high in proliferatingcells (approximately 80,000 binding sites/cell) andreduced by 4-fold in differentiated cells. Exogenouslyadded ICE-Il was able to stimulate proliferation ofserum-deprived cells in a dose-dependent fashion. ICE-Iladed through the ICE-I receptor, since both basal andICE-Il-stimulated cell proliferation was inhibited by themonoclonal antibody a-lR3, which blocks the bindingsites of the ICE-I receptor. The inhibition of CaCo-2basal cell growth by the a-lR3 antibody suggests thatICE-lI may ad as an autocrine growth fador for thesecells.

Introdudion

The turnover of the gastrointestinal epithelium is very rapidand is completed in about 3 days (1). Intestinal epithelialcells arise from multipotent stem cells located in the cryptsfrom where they migrate upward along vertical axes, ac-quiring an increasingly differentiated phenotype (i). Eachvillus contains several thousands of differentiated surfaceenterocytes, whose renewal is driven by concomitant loss

Received 5/27/94; revised 7/25/94; accepted 8/8/94.1 This work was supported by grants from Associazione Italiana per Ia

Ricerca sul Cancro (to A. M. A.), Progetti Finalizzati Ingegneria Genetica,

and Applicazioni Cliniche della Ricerca Oncologica of the ConsiglioNazionale delle Ricerche (to C. B. B.).2 To whom requests for reprints should be addressed, at Dipartimento di

Biologia e Patologia Cellulare e Molecolare “1. Califano”, Centro di Endo-crinologia ed Oncologia Sperimentale del C.N.R., Universit#{224} di Napoli

“Federico II”, Via S. Pansini, 5, Napoli 80131, Italy.5 Present address: Divisione di Oncologia Medica B, Istituto Nazionale dei

Tumori, Fondazione “G. Pascale,” Napoli, Italy.

and proliferation of the cells. The regulation of the balancebetween the proliferative and differentiative compartmentsof intestinal epithelium is complex and not completelyclear.

Several studies have focused on the role of polypeptidegrowth factors in the regulation of intestinal cell growth(2-6). In particular, insulin-like growth factors (IGF4-Iand IGF-ll) regulate proliferation of intestinal mucosa (4-6).IGFs are secreted as small peptides that are structurallyrelated to insulin and display multiform effects on cellgrowth and metabolism (7). IGF-I and IGF-II both exert theirmitogenic activity through the type I receptor (IGF-I recep-tor) (7). IGF-II is expressed at high levels in the fetus and atlower levels in adult life and functions as a prenatal growthregulator in mammals (7, 8). In fact, lGFs and/or IGF-Ireceptor knock-out mice show growth retardation andimpaired organ formation (8, 9).

IGFs are abundantly expressed in several human coloncarcinoma cell lines (6) and are overexpressed in 30% ofcolorectal tumors compared to normal colon mucosa (10,ii). High levels of IGF-l and IGF-lI receptors have beenreported in normal adult human colonic epithelium (1 2).Furthermore, tumor tissues and tumor cell lines show higherIGF-l receptor number compared to normal colon mucosa(13). IGF-lI, as well as IGF-l and IGF-II receptors, are ex-pressed in IEC-6 rat intestinal epithelial cells (4, 5), andIEC-6 cell growth is stimulated by IGFs (4). Autocrine actionof IGF-I or IGF-ll on cell proliferation has been demon-strated in other experimental systems (i 4-i 7). Therefore,the overexpression of lGFs may also stimulate intestinalepithelial cell growth through an autocrine mechanism.

In the present study, IGF-Il expression was evaluated inthe enterocyte-like cell line CaCo-2, a human colon carci-noma cell line that spontaneously differentiates, showingfeatures of small bowel enterocytes and of fetal intestinalcells of the 15th week (1 8-20). This cell line represents aunique opportunity to study the regulation by growth fac-torsof intestinalcellproliferationwith respect to the degreeof cell differentiation. IGF-Il and IGF-l receptor expressionwas high during proliferation and decreased during differ-entiation of CaCo-2 cells. These data suggest that IGF-Ilmight be an autocrine growth factor for these cells.

Results

ICE-Il Expression during Differentiation of CaCo-2 Cells.IGF-lI RNA levels were high in proliferating CaCo-2 cellscultured in complete medium. IGF-Il RNA levels decreasedby more than i 0-fold at day 14 of culture, when the cells

4 The abbreviations used are: IGF, insulin-like growth factor; kb, kilobase(s);

ApoA-l, apolipoprotein A-I; SI, sucrase-isomaltase; FCS, fetal calf serum;Mab, monoclonal antibody; BSA, bovine serum albumin; bp, base pair(s);

cDNA, complementary DNA; PBS, phosphate-buffered saline.

1086 IGF-lI in CaCo-2 Cells

IGF-lI

:::�:.1 S.

SI

6.0 kb -

APOAl

1.0kb- �, U

28S�O#{216}

1 2 3

- 28S

- 18S

- 28S

- 18S

Fig. 1. Northern blot analysis oftGF-lI, SI, and ApoA-l transcripts in CaCo-2

cells at day 3 (Lane 1), 8 (Lane 2), and 14 (Lane 3) of culture. Twenty pg(Lanes 1 and 2) and 1 5 pg (Lane 3) oftotal RNA were loaded. The same filler

was sequentially hybridized to human IGF-Il, human SI, human ApoA-l, andhuman 285 rRNA probes. The sizes of the transcripts in kb are indicated onthe lett. The migration of 28S and 1 8S rilxsomal RNA species, as sizemarkers, is indicated on the right.

Table 1 IGF-ll, ApoA-l, and SI mRNA levels during differentiation of

CaCo-2 cells”

Days of culture IGF-ll SI ApoA-I

3 100 2.8±1.9 8.3±3.5

8 84±7.9 12.1 ±4.5 32±7.1

14 8±3.i 100 iOO

., The mean densitometric scan values and SEs of at least three independent

experiments are presented. mRNA levels were normalized to those obtained

after hybridization of the same filters with a human 285 rRNA probe. mRNAlevels on day 3 of culture for IGF-lI and on day i4 for SI and ApoA-l.respectively, were arbitrarily taken as 100. IGF-II mRNA levels were oh-tamed by analysing both 6.0 and 4.8 kb transcripts.

were fully differentiated (Fig. i; Table i). Northern blotanalysis identified two major specific IGF-II transcripts, 6.0and 4.8 kb, derived from P� and P4 human promoters,respectively (6, 7). IGF-ll transcripts were differentially ex-pressed. The 6.0-kb transcript was more abundant, ac-counting for 85% of all IGF-hl mRNAs at day 3 of culture

(Fig. i , Lane 1), and was not detectable at day i 4 of culture(Fig. 1 , Lane 3). The 4.8-kb transcript was less abundant atday 3 ofculture (Fig. i, Lane 1), was still present, albeit atreduced levels, at day 8 (Fig. i, Lane 2), and was notdetectable at day i4 of culture (Fig. 1, Lane 3). A longerexposure of the autoradiograph showed that the 4.8-kbIGF-ll transcript was barely detectable at day 14 of culture(data not shown). In the same experiments, we evaluatedthe expression of ApoA-l and of the brush border-associatedhydrolase sucrase-isomaltase (SI) as markers of enterocytedifferentiation. ApoA-l and SI RNA levels increased in atime-dependent fashion during differentiation of the cells (Fig.i). Densitometric scan analysis of the experiments showedthat ApoA-l and SI RNA levels were i 0-fold and 30-fold higherat day i 4 than at day 3 of culture, respectively (Table 1).CaCo-2 cells, irrespective of their proliferating/differentiatingstates, did not express IGF-l. In fact, no specific IGF-l signalwas detected by Northern analysis (data not shown).

IGF-II Synthesis and Secretion in CaCo-2 Cells at Dif-ferent Days of Culture. IGF-ll mRNA levels and proteinsecreted into the medium were measured in CaCo-2 cells,either at the proliferative state or at confluency. The cellswere grown in complete medium for 4, 6, 8, i2, and i4days (Fig. 2) and then switched to serum-free medium. Foreach time point, conditioned media and cells for RNAextraction were harvested after 48 h in serum-free medium(see “Materials and Methods”). IGF-ll niRNA levels de-creased over the time of culture (Fig. 2A, compare Lanes1-3 to Lanes 4 and 5), with the 6.0-kb transcript being theone most affected. Densitometric scan analysis showed a2.5- and 1 0-fold lower amount of the 6.0-kb transcript atdays i 2 and i 4 compared to day 4 of culture, respectively;the expression of the 4.8-kb transcript did not vary signifi-cantly at the different time points (Fig. 2A). IGF-Il peptidelevels were analyzed either by Western blot (Fig. 2B) or byimmunoenzymatic analysis (Table 2) of conditioned mediafrom cells at different days of culture. Western blot analysisof conditioned media identified two reactive peptides of Mr7,500 and Mr i 5,000 that comigrated with CF-Il hormoneand prohormone molecules, respectively (Fig. 2B, compareLanes 1-6 to Lane 7). IGF-ll peptides were secreted at highlevels in the medium collected from cells at day 4 of cultureand then progressively decreased during the subsequentdays not being measurable at days i2 and 14 (Fig. 28).lmmunoenzymatic analysis of conditioned media showedthat the process of differentiation was associated with a20-fold decrease in IGF-ll protein secretion by CaCo-2cells, from 232 ng/i 0#{176}cells at day 4 of culture to i 1 ngJi 0#{176}cells at day 14 of culture (Table 2).

IGF-I Receptor Expression during Differentiation ofCaCo-2 Cells. Since the mitogenic effects of IGF-ll are me-diated through the interaction with the IGF-l receptor (7, 9),

we studied the expression of IGF-l receptor during CaCo-2cell differentiation. A competitive binding assay (Fig. 3A)showed that ‘25l-IGF-l binding was displaced in a dose-dependent manner by both IGF-l and IGF-ll cold higandswith the expected relative potency (7). When the specificbinding was evaluated by Scatchard plot analysis, a singleclass of high affinity binding sites for IGF-I was identified(Fig. 3B). The binding affinityof IGF-I was calculated interms of the equilibrium dissociation constant (K,1), and thetotal number of specific binding sites � for IGF-l wascalculated from the Scatchard analysis of specific bindingdata. The K,1 estimated values were approximately 1 .6 nMand did not change at different days of culture (Table 3). On

A

IGF-II

�

28S �I 2345

B

1 5 kDa -

7.5 kDa - #{149}‘44��I �‘r-

1 234567

- 45 kDa

- 29

. - 18

,. � -14

-3

Cell Growth & tJifferentiation 1087

Fig. 2. Time course of IGF-lt expression and synthesis in CaCo-2 cells. A, Northern blot analysis ofthe RNA from cells at day 4 (Lane 1), day 6 (Lane 2), clay8 (Lane fl, clay 12 (Lane4), and day i4 (Lane5)ofculture. Twenty pgoftotal RNAwere loaded on each lane. The saniefilterwas sequentially hybridized to

the ProI)es shown in the figure. The sizes of the transcripts are indicated on the left. B, immunoblot of media from cells at clay 4 (Lanes 1 and 2), clay 6 (Lane0. clay B (Lane 4(, clay 1 2 (Lane 5(, and clay 14 (Lane 6( of culture and c)f purified human IGF-ll (Lane 7) with a mouse monoclonal antibody to rat IGF-ll (36(.

Conditioned medium, 100 and 200 pl. corresponding to 1 5 and 30 pg of protein, were examined in Lanes 1 and 2, respectively. The amount of media loadedin !�afl(’c l�- (, w,ls alculate(I by normalizing to Protein content of the cells at different clays of culture. Human recombinant IGF-ll (20 ng( was lo,icled in Lane

7. The nhigration of fllole( ular weight markers is indicated on the right.

Table 2 IGF-tl pepti(Ie le� ‘els in conditioned media of CaCo-2 cells

Da ys of c ulture Immunoreactive IGF-ll (ng/i 0” cells”

4

6

8

2

14

232 ± 10

78±8

25±5

18±9

11±6

., Reported values were obtained by semiquantitative measurements of serialdilutions of (Ondlitiofle(I n�’dia directly spc)ttedl on t3A85 nitrocellulose filters

(see “Materials and Methods” for details). Results are the mean ± SD of threeindependent (‘xI)erinlents.

the other hand, the B,,,,\ values of 12l-IGF-l binding at day4 of culture were 1 .5- and 3.5-fold higher when comparedto cells at days 8 and 1 2 of culture, respectively (Table 3).Proliferating CaCo-2 cells showed a high number of surfaceIGF-l receptors (approximately 80,000 binding sites/cell).Since the K,� values for cells at different days of culture weresimilar, the decrease in � values for confluent CaCo-2cells could be explained by a reduction in surface IGF-lreceptor nuniber.

Effect of Exogenously Added IGF-lI on CaCo-2 Cell Pro-liferation. To evaluate whether IGF-Il regulates the growthof CaCo-2 cells, we tested the effect of exogenously addedIGFs on proliferation of preconfluent CaCo-2 cells. Asshown in Fig. 4A, IGF-lI stimulated proliferation ofthe cellsin a dose-dependent fashion. At 1 0 nvt concentration, IGF-land IGF-ll were equally effective in stimulating cell growth,and the magnitude of the effect was similar to that obtainedby 10% FCS (Fig. 48). In the same experiments, a smallincrease it3 the cell number was also observed when cellswere grown in serum-free mediuni (Fig. 4, A and B).

Effect of the Blocking Antibody a-lR3 on Basal andIGF-II-stimulated Cell Growth. To demonstrate that theeffect of exogenously added IGF-ll on cell growth was

mediated by the IGF-l receptor, we evaluated whether IGF-Il-induced stimulation of CaCo-2 cell proliferation was pre-vented by the monoclonal antibody cs-lR3, which blocksthe binding sites of this receptor (21). As shown in Fig. 5A,

cr-1R3 Mab inhibited IGF-ll-mediated growth of CaCo-2cells in a dose-dependent fashion. At 5 pg/mI, cs-1R3 Mabreduced IGF-ll-mediated cell proliferation to 30% of max-imal growth; the same concentration of the isotypic controlantibody MOPC-21 had no inhibitory effect (Fig. 5A).

To verify if the observed basal level of proliferation of

CaCo-2 cells in serum-free medium (Fig. 4) was contributedto by secreted IGF-ll functioning in an autocrine loop, weevaluated whether c�-lR3 Mab inhibited basal proliferationofthe cells. Addition of cs-1R3 Mab significantly (P< 0.001)reduced cell growth in serum-free medium, whileMOPC-21 Mab had no effect (Fig. 58).

DiscussionIn humans, IGF-ll is highly expressed during fetal life, andits synthesis has been demonstrated in adult liver and adultextrahepatic tissues (7). However, the role of IGF-ll duringdifferentiation is still unclear. Gene disruption of the activeIGF-ll allele in the mouse embryo results in growth retar-

dation hut normal organ formation (8). On the other hand,IGF-l/IGF-ll or IGF-ll/IGF-l receptor double mutants exhibita more severe dwarfism and impaired organ formation,suggesting that IGF-l more than IGF-ll is essential for correctembryonic development (9). IGF-lI expression in other ex-perimental systems directly correlates with cell differentia-tion (7, 22, 23). In fact, an increased expression of IGF-lloccurs during terminal stages of muscle development (22),and differentiation of myoblasts to myotubes is dependenton autocrine secretion of IGF-ll (23). On the contrary,down-regulation of IGF-lI expression has been reportedduring cAMP analogue-mediated differentiation of SaOS-2human osteosarcoma cells (24).

i012 10�8 10.6

PEPTIDE CONCENTRATION (M)

A

120

100

80

60

40

20

0

B0.12

0.1

w�0.08

�0.06

0

0.04

0.02

0

0 5.0 10 � 1.0 10 �‘#{176} 1.5 10

BOUND (M)

Fig. 3. A, log-dose competitive inhibition ofspecific binding of � 251-IGF-l to

CaCo-2 cell monolayers at 1 2 days after plating by various concentrations ofIGF-l (#{149})and IGF-ll (U. Each point is the mean of two observations from asingle experiment and is representative of three separate experiments. B,representative Scatchard plot of 125I-IGF-l binding to CaCo-2 cells 12 days

after plating. Each point is the mean of duplicate determinations from arepresentative experiment of a total of three experiments.

.

0 12 24 36 48

hours

A 200

100

10

B 200

�1000

S.0E3C

0

Fig. 4. A, effect of increasing IGF-II concentrations on CaCo-2 cell prolif-eration. 0, 10 nM IGF-II; U, 2.5 n�s IGF-It; *, 0.625 nxi IGF-II; C, 0.1 56 nxi

IGF-II; 0, 0.039 nM IGF-ll; #{149},serum-free medium. 8, effect of serum, IGF-l,and IGF-ll on CaCo-2 cell proliferation. *, 1 0% FCS; � , 1 0 flM IGF-lI; 0, 10nM IGF-l; #{149},serum-free medium. Hemocytometric cell counts presented are

from a representative experiment in which each point is the mean of tripli-

cate determinations; bars, SD, which are not shown if too small for visualdisplay.

.

1012 24 36 48

1088 IGF-ll in CaCo-2 Cells

0z0z

LL0Ill

Table 3 IGF-l receptor expression in CaCo-2 cells on different days ofculture

Days of culture KD (nmol/Liler)a Bmax (fCflOl/106 cells)a

4 1.66±0.31 130± 17

8 1.74±0.28 98±9

12 1.59±0.22 38±3

a Values are the mean ± SD of at least three independent experiments. K0

and Bma. values were obtained using the EBDWLIGAND program (46, 47).

Little is known about the physiological role of IGFs duringintestinal cell growth and differentiation. IGF-lI ligand andIGF-l receptor are expressed in IEC-6 rat intestinal epithelialcells (4, 5). IGF-Il expression and regulation have been studiedpreviously in a human intestinal epithelial cell model, theHT-29 colon carcinoma cell line (25). These cells secrete lowamounts of IGF-ll peptides (25) and possess high affinity IGF-lreceptors (26). However, exogenously added IGFs fail to stim-ulate their growth (25). Preliminary data by Singh et a!. (27)show that IGF-lI RNA and protein levels are reduced duringCaCo-2 cell differentiation.

0

x

E3

U

In this study, we showed that proliferating CaCo-2 cellsexpressed high levels of IGF-lI RNA. As demonstrated inother human tumor cells, IGF-ll transcripts were derivedfrom the two main fetal promoters P3 and P4 (6, 7). IGF-lIRNA expression was cell growth and/or differentiation de-pendent, since it was maximal during the proliferative,undifferentiated state and decreased when the cells stop toproliferate and differentiate. The decrease in IGF-II RNAlevels was more evident in serum-fed cells, where both 6.0-and 4.8-kb transcripts were decreased, than in serum-starved cells, where only the 6.0-kb transcript was regulatedto the same extent (Figs. i and 2). It has been reported thatIGF-Il expression is induced by serum and that the effectrelies on the transcription factors that bind the early growthresponse elements (EGR-i) of P3 and P4 promoters (28, 29).Differential regulation of the two IGF-Il promoters at theEGR-i sites or differences in the relative stability of the twomRNA species might explain their differential expression inserum-starved cells.

We also showed that proliferating CaCo-2 cells secretedabundant amounts of IGF-Il protein into the medium (Fig. 2;Table 2). Two IGF-ll forms were secreted, the mature Mr

200

150

0

x

�5 100.0E

a)0

50

0

A B

Cell Growth & Differentiation 1089

Fig. 5. Inhibition of IGF-lt stimu-

lated(A) and basal (B) cell growthby cr-lR3 l)lmkiflg antibody. Cellswere incul)ated with a-tR3 or

MOPC-2 I Mabs in the presence

of 10 riu IGF-Il (A) or in serum-

free medium (B). �, no Mabs; �,

0.2 ps/nil a-IR I; TI, 1 ps/nil cx-1R3;LI, 5 ps/mI (�-lR3; �. 5 ps/mIMOPC-2 1 . Columns, mean cell

counts for triplicate wells; bars,SD.

7,500 hormone and the Mr 1 5,000 prohormone. The Mr

1 5,000 kDa form has been found in several human normaland tumor tissues and has been demonstrated to be biolog-ically active (30, 31). The secretion of IGF-ll peptides wasreduced during differentiation of CaCo-2 cells. This findingcorrelated with the decrease in both IGF-lI mRNA species inserum-fed cells. On the contrary, in serum-starved cells,only the 6.0-kb transcript was reduced to the same extent.These data suggest a differential translation efficiency of thetwo IGF-ll transcripts. A differential translational regulationfor IGF-II mRNAs has been reported in another experimen-

tal system (32). The possibility of such a regulation in theCaCo-2 cells has to be further investigated.

There appears to be an inverse correlation betweendown-regulation of IGF-ll expression and up-regulation ofthe differentiation markers in CaCo-2 cells (Fig. 1). IGF-llRNA levels started to decrease when cells began to differ-entiate and became barely detectable when cells were fullydifferentiated. Conversely, partial and full expression of theApoA-l and SI differentiation markers was observed in par-allel with the decrease in IGF-ll expression. However, it isnot possible to conclude that a cause-effect relationship

exists between decreased IGF-ll expression and celldifferentiation.

Intestinal mucosa possesses abundant and high affinityreceptors for IGF-l and IGF-ll (1 2, 1 3). IGF-l and IGF-Ilreceptors are present in a decreasing gradient along thecrypt-villus axis in the epithelium of rats, with the crypt cellsshowing the highest concentration (33). IGF-I receptor ex-pression in the rat intestinal epithelial cell line IEC-6 isenhanced in all conditions of increased cell proliferation(34). The data presented here showed that CaCo-2 cells,like other intestinal tumor cells (1 3), expressed an abundantamount of IGF-l receptors (80,000 binding sites/cell) duringthe proliferative state, and this number decreased after dif-ferentiation (Table 3). Reduced IGF-lI synthesis paralleledthe decrease in IGF-l receptor expression. This coordinateregulation has not been observed in other tissues. In musclecells, for instance, IGF-l receptor down-regulation is asso-

ciated with increased IGF-l and IGF-Il synthesis duringmyotube formation (35).

The finding of high IGF-ll ligand and IGF-I receptor ex-pression in the proliferative phase of CaCo-2 cells is in

agreement with several reports, suggesting that IGF-ll is amitogen for cultured normal and tumor cell lines actingthrough an autocrine mechanism (4-7, i4-17). Accord-ingly, we found that IGF-Il stimulated proliferation of pre-confluent CaCo-2 cells. The effect was mediated by theIGF-l receptor, since a-lR3 blocking antibody to this recep-tor inhibited cell growth induced by exogenously addedIGF-II. Also, a-1R3 Mab reduced CaCo-2 cell proliferationin the absence of exogenously added growth factors. Thesefindings imply that the secreted IGF-ll is an autocrinegrowth factor for the cells.

Our data suggest an important role for IGF-II in the regula-tion of enterocyte proliferation. Therefore, CaCo-2 cells rep-resent an excellent model to study the effects of IGFs on cell

growth and differentiation of human intestinal epithelia.

Materials and Methods

Materials. Ia- 2PJdATP (3000 Ci/mmol), a- 32PIdGTP(3000 Ci/mmol), ly- �2PIATP (3000 Ci/mmol), and I’ 25IIIGF-I (2000 Ci/mmol) were from Amersham (Buckinghamshire,England). Guanidinium thiocyanate was purchased fromFluka (Buchs, Switzerland). Restriction endonucleases werefrom Boehringer (Mannheim, Germany). Radioimmunoas-say-grade BSA was from Sigma Chemical Co. (Milano,Italy). Recombinant human IGF-I and IGF-ll were obtainedfrom Sigma (Milano, Italy) and were reconstituted in 0.1 M

acetic acid and 1 mg/mI BSA (36). Mab to rat IGF-ll (36) wasobtained from Amano Pharmaceutical (Nagoya, Japan) andwas a generous gift of Dr. P. Nissley (National CancerInstitute, NIH, Bethesda, MD). a-1R3 Mab to IGF-I receptor(21) was from Oncogene Science (Manhasset, NY), andMOPC-21 (lgGK-i) control isotypic Mab was from Sigma.

Cell Culture and Cell Proliferation Assay. Passage 20 ofthe CaCo-2 cell line (18) was received from Dr. M. V.

1090 IGF-II in CaCo-2 Cells

Barone (European Molecular Biology Laboratory, Heidel-berg, Germany). Cells were grown in Dulbecco’s modifiedEagle’s medium containing 25 mr�i glucose supplementedwith iO% FCS (GIBCO, Grand Island, NY). Three X i05cells were seeded onto iO-mm tissue culture dishes andwere routinely subcultured when about 80% confluent.Monolayers became confluent 6-8 days after the inocu-lum, and the stationary phase was reached on day 10.Cultures were stopped after i 5 passages.

For proliferation assays, cells were seeded at a density of60,000 cells/35-mm dish in Dulbecco’s modified Eagle’smedium supplemented with i 0% FCS. After 3 days, cellswere washed and grown in serum-free medium. Two dayslater, cells were incubated in serum-free medium alone orwith additions of 10% FCS or 10 flM IGF-I or IGF-II at theconcentrations indicated. In the experiments evaluating theeffects of a-lR3 and MOPC-2i Mab, cells were preincu-bated for 1 h at 37#{176}Cwith Mabs and then incubated for 48h in serum-free medium alone or with additions of i 0 nt�i

IGF-ll. Cell growth was assessed at selected times by he-mocytometric cell counting following gentle trypsinization.Viability of cells was assessed by the trypan blue dyeexclusion test.

Hybridization Probes. IGF-Il probe was an 800-bpEcoRI-PstI insert that has been isolated from the humancDNA and spans exons 6-8 and the 5’-end of exon 9 (37).IGF-l probe was a 700-bp EcoRl insert corresponding to thecoding region of human IGF-I cDNA (38). SI probe was a420-bp Sa!l-EcoRl insert isolated from pHSI-i plasmid andwas kindly provided by Dr. P. G. Traber (University ofMichigan, Ann Arbor, Ml; Ref. 39). ApoA-l probe was a850-bp BamHI insert of a full-length human cDNA and waskindly provided by Dr. G. Morrone (40). 285 probe was anoligonucleotide (5’-AACGATCAGAGTAGTGGTAmCA-CC-3’) complementary to human 285 rRNA (4i).

RNA Analysis. Total RNA was isolated from cells by theguanidinium thiocyanate acid-phenol procedure (42).Northern analysis was carried out essentially as described(43). Probes were generated by random primer extension orby 5’-end labeling (43). The relative amount of the RNAsamples was determined by hybridization of the filters with285 rRNA probe. Sizes of hybridizing RNAs were calcu-lated by reference to the rRNA bands on the filters afterethidium bromide staining. Estimated sizes for i 85 and 285rRNA transcripts were 1 .9 and 5.0 kb, respectively (4i).RNA levels were quantified by densitometric scanning ofthe autoradiographs using a Sony CCD videocamera linkedto a Macintosh llfx computer and the NIH IMAGE program(public domain).

Colledion of Conditioned Medium. Secreted IGF-lI pep-tides in conditioned media were measured as describedpreviously (45). Cells were grown on different days in se-rum-containing medium, washed three times with PBS, andincubated i 2 h in serum-free medium. The medium wasthen discarded and replaced with fresh serum-free medium(5 mI/i 0-cm2 dish). Conditioned medium was harvested 48h later and centrifuged (3000 rpm for 20 mm). Conditionedmedia were concentrated using a Centricon 3 microcon-centrator.

ICE-lI Immunological Studies. Protein analysis was per-formed using i 5% sodium dodecyl sulfate-polyacrylamidegel electrophoresis under nonreducing conditions. Afterelectrophoresis, proteins were blotted by electrotransfer tonitrocellulose filters (BA85; Schleicher and Schull; Ref. 44).Immunodetection experiments were carried out using a

mouse monoclonal antibody directed against native ratIGF-II (36) and a secondary goat anti-mouse IgG serumconjugated to horseradish peroxidase (44); the bands werevisualized using the enhanced chemiluminescencedetection system (Amersham Corp.).

Sem iquantitative measurements of immu noreactiveIGF-ll were performed by chemiluminescence on serialdilutions of conditioned media directly spotted on BA85nitrocellulose filters (45). Immunoreactive signals were an-alyzed using a Sony CCD videocamera linked to a Macin-tosh llfx computer and the NIH IMAGE program (publicdomain). Values for IGF-lI immunoreactive values wereinterpolated from standard curves constructed using serialdilutions of recombinant human IGF-II. IGF-Il concentra-tion was expressed as ng/i 06 cells.

ICE-I Receptor Assay. The cells were seeded in 1 6-mmtissue culture wells at a density of 40,000 cells/dish. At thedifferent days of culture analyzed, cells were washed threetimes with cold PBS and then incubated in a total volume of0.25 ml KRH binding medium (Krebs/Ringer/4-(2-hydroxy-ethyl)-i-piperazineethanesulfonic acid containing 0.5%BSA and adjusted to pH 7.4) for i8 h with 0.06 nmol/liter1251-IGF-l with or without various concentrations of unla-beled IGF-l or IGF-lI. At the end of incubation, cells werewashed three times with PBS and then lysed in the samebuffer containing i% Triton X-iOO and iO% glycerol; cell-associated radioactivity was counted on a BeckmanGamma 5500 counter. Nonspecific binding, determined asradioactivity bound in the presence of i06 M IGF-I orIGF-Il, was subtracted from total binding to obtain specificbinding. Nonspecific binding represented about 5% of totalbinding of 125I-IGF-I. The binding assays were also per-formed on cells in suspension after gentle trypsinization.Forty thousand cells per 0.25 ml at different days of culturewere incubated under the same experimental conditionsinto 1 .5 ml Eppendorf tubes. The binding mixture wasvortexed and incubated at 4#{176}Cfor i 8 h under gentle agita-tion. The tubes were then centrifuged; next, the pellets werewashed twice with KRH binding medium and then countedon a Beckman Gamma 5500 counter.

AcknowledgmentsWe thank F. Beguinot, M. Caraglia, L. D’Agostino, B. Daniele, and N. Perrotti

for helpful discussions and critical review of the manuscript. Special thanks

to M. Berardone, F. D’Agnello and F. Moscalo, for the artwork.

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