EXPERIMENTAL CELL RESEARCH 230, 284–292 (1997)ARTICLE NO. EX963430

The IGF-I Receptor in Mitogenesis and Transformation of MouseEmbryo Cells: Role of Receptor Number

MICHELE RUBINI,*,† ATSUSHI HONGO,* CONSUELO D’AMBROSIO,* AND RENATO BASERGA*,1

*Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and †Istituto di Genetica Medica,Universita di Ferrara, Italy

growth-arrested cells (7), that they are autophosphory-The type 1 receptor for insulin-like growth factors lated by the appropriate ligands (4), and that they can

(IGF-IR) plays an important role in the growth and transmit a signal resulting in growth in size of thetransformation of several types of cells. We have inves- cells (8), and the expression of specific genes, such astigated the role of IGF-IR number in IGF-I-mediated ribosomal RNA genes (9), c-fos (10), c-jun (11), a tran-mitogenesis and transformation of mouse embryo fi- scription factor for thyroglobulin (12), and several otherbroblasts. We have used R0 cells (3T3-like cells origi- unidentified genes (13). These findings raise the ques-nating from mouse embryos with a targeted disruption tion of why the IGF-IR is nonmitogenic in certain quies-of the IGF-IR genes) transfected with a plasmid ex- cent cells, unless the cells are also incubated withpressing the human IGF-IR cDNA to generate clones PDGF.with receptor numbers ranging from zero to 106 recep- The simplest explanation is that both the PDGF andtors per cell. In this model, between 15,000 and 22,000 IGF receptors activate separate and independent path-receptors per cell are sufficient to render mouse em- ways, both necessary but not sufficient for mitogenesis.bryo cells competent to grow in serum-free medium

A second possibility, which, in reality, is a modificationsupplemented solely with IGF-I. For growth in softof the first, is that PDGF could mobilize a specific IGF-agar, 30,000 receptors per cell seem to be the minimumIR substrate, not available in quiescent cells, whichrequirement. These experiments indicate that a smallcould then transmit the mitogenic signal. A third alter-increment in the number of receptors per cell, wellnative is that the determinant factor is the number ofwithin the physiological range, can modulate the mito-IGF-IRs, a possibility receiving some support from thegenic and transforming activities of the IGF-IR in 3T3-several reports that PDGF (and EGF) increase thelike cells. q 1997 Academic Press

number of IGF-I binding sites, as well as the secretionof the ligand [14–16] and that PDGF and EGF alsoactivate transcription from the IGF-IR promoter [17],INTRODUCTIONleading to an increase in IGF-IR number. The pros andcons of these three hypotheses have been discussed inMouse embryo fibroblasts, like 3T3 cells, requirea recent review by Baserga [18].more than one growth factor for optimal growth in

The purpose of the present investigation is to exploreSFM2 [1–3], usually PDGF and IGF-I, either of thesethe third possibility, by determining the role of IGF-IRtwo growth factors being incapable, singly, of stimulat-number in the mitogenic response to IGF-I. It is alreadying growth. Thus, addition of IGF-I alone to quiescentknown that gross overexpression of the IGF-IR (50–3T3 cells fails to elicit a mitogenic response [1–4]. But100 times the number of wild-type cells) renders cellsif the quiescent cells are previously or simultaneouslycapable to grow in SFM supplemented solely with IGF-incubated with PDGF (and/or EGF [5]), then the addi-I [4, 16, 19, 20], but this gross overexpression may acti-tion of IGF-I results in a substantial stimulation ofvate pathways that are not usually activated by IGF-Icellular proliferation. Although IGF-I, by itself, cannot[21]. In these experiments, we wished to investigateinduce proliferation of mouse embryo fibroblasts, orthe effect of receptor levels closer to physiological lev-other growth-regulated cells like human diploid fibro-els, and, for this purpose, we selected R0 cells, devel-blasts (6), it is known that IGF-IRs are present inoped from mouse embryos with a targeted disruptionof the IGF-IR genes [22, 23]. These cells, designated as

1 To whom correspondence and reprint requests should be ad- R0 cells, and developed by a protocol similar to the onedressed. Fax: (215) 923-0249. E-mail: baserga1@jeflin.tju.edu. used to generate 3T3 cells, are totally devoid of IGF-2 Abbreviations used: IGF-I, insulin-like growth factor I; IGF-IR,

IRs, grow in 10% serum (albeit at a lower rate thanIGF-I receptor; PDGF, platelet-derived growth factor; EGF, epider-mal growth factor; SFM, serum-free medium. wild-type cells), and do not grow at all in SFM supple-

2840014-4827/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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285IGF-I RECEPTOR NUMBER

DMEM) containing 0.5 ng/ml of 125I-IGF-I (NEN DuPont) and in-mented with the purified growth factors, which sustaincreasing amounts of unlabeled IGF-I, ranging from 0 to 40 ng/ml.the growth of other 3T3-like cells [24, 25]. In addition,Binding was carried out at 47C for 6 h. Cells were then washedR0 cells cannot be transformed by oncogenes such as three times in ice-cold Hank’s solution and lysed with 0.03% sodium

the SV40 large T antigen, an activated and overex- dodecyl sulfate. Nonspecific binding was defined as the binding ob-served in presence of excess (500 ng/ml) unlabeled IGF-I. Lysatespressed ras, or the E5 protein of the bovine papillomawere counted in a gamma-counter. Cell numbers were determinedvirus [24–26], all of which can readily transform mouseon wells which were treated simultaneously to the experimentalembryo cells with a wild-type number of IGF-IRs. Thewells. Data were converted to Scatchard plots, assuming that 125I-

growth deficits of R0 cells are corrected by the stable labeled- and unlabeled-IGF-I have the same affinity for the IGF-IR.transfection of a wild-type human IGF-IR cDNA. We The computer softwares used for drawing a line for the plots were

the commercially available Microsoft Excel and CricketGraph; bothtransfected R0 cells with a plasmid expressing the hu-methods gave the same lines. All binding data represent averageman IGF-IR cDNA under the control of a rat IGF-IRcounts from duplicate wells.promoter [27, 28]. Many clones were generated, with

Cell growth. All cell lines were maintained in Dulbecco’s Modifieddifferent numbers of receptors, and these are the objectEagle Medium (DMEM) containing 5% fetal bovine serum, 5% calf

of this communication. Clearly, the results obtained serum and 200 mg/ml of hygromycin B. Incubations were at 377C inwith R0 cells do not necessarily apply literally to other a 9.6% CO2 atmosphere. To make cells quiescent, they were seeded

at 2.51 103/cm2 in DMEM supplemented with 5% fetal bovine serumcell lines, with different backgrounds. Cell lines varyand 5% calf serum. After 24 h, the cells were washed three times withgreatly in the number of IGF-IRs they display (see forHank’s solution and medium was replaced with serum-free mediuminstance [29]), and probably each cell line has its own (SFM) (DMEM supplemented with 50 mg/ml transferrin and 0.1%

physiological level. With these experiments, we only bovine serum albumin). Fresh SFM was renewed every 24 h. Cellswanted to determine the effect of IGF-IR number on were completely quiescent after 2 days culture in SFM. IGF-I growth

response was tested by stimulating quiescent cells with concentra-mitogenesis and transformation in cells derived fromtions of IGF-I (Bachem) ranging from 0 to 100 ng/ml. In other experi-a single parental cell line, which is a 3T3-like cell linements, cells were stimulated either with PDGF-BB only (Gibco, 5[24] and therefore a reasonable representative of ng/ml) or IGF-I only (20 ng/ml) or both. Cell growth was evaluated

mouse embryo fibroblasts. Although we have also de- by cell counting after 2 days stimulation.termined the effect of PDGF on receptor number, these Anchorage-independent growth assay. Cell growth in soft agarexperiments were not designed to determine the role was assayed by scoring the number of colonies formed in medium

(DMEM supplemented with 5% fetal bovine serum and 5% calf se-of PDGF in the growth process.rum) containing 0.2% agarose, with a 0.4% agarose medium under-Despite the fact that the numbers are not absolutelay. Cells were seeded at a density of 103 per 35-mm diameter dishesnumbers, strictly applicable to other cell lines, there is in triplicate. IGF-I (50 ng/ml) was added to some cultures. The num-

no a priori reason, on the other hand, why the biological ber of colonies ú125 mm in diameter was counted after 14 days.effects of relative increases in IGF-I receptor number Tyrosine phosphorylation assay. Cells were seeded in 100-mmmay not be cautiously extrapolated to other types of dishes and made quiescent as described above. After two days culture

in SFM, cells were stimulated with 20 ng/ml IGF-I at 377C for 5 min.growth-regulated cells.Culture dishes were then placed on ice and cells were rinsed threetimes in cold Hank’s. Cells were lysed in lysis buffer (50 mM Hepes,

MATERIALS AND METHODS pH 7.5; 150 mM NaCl; 1.5 mM MgCl2; 1 mM EGTA; 10% glycerol;1% Triton X-100; 100 mM NaF; 0.2 mM Na3VO4; 10 mM Na4P2O7;

Cell lines. R0 cells are cells originated from mouse embryos with 10 mM phenilmethylsulphonyl fluoride; 0.1 mg/ml aprotinin) for 4a targeted disruption of the IGF-IR genes [22, 23]. They were devel- min at 47C. Lysates were collected and cleared from nuclei by centrif-oped by a protocol similar to the one used to generate other 3T3 cell ugation. Lysates (400 mg for Shc or IGF-IR precipitation, or 50 mglines, and can therefore be considered as 3T3 cells. W cells are similar for IRS-I precipitation) were diluted with HNTG (20 mM Hepes, pHbut originated from wild-type littermates. These cells have been 7.5; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 100 mM NaF;characterized in detail in previous reports [24, 25, 30–32]. R/ cells 0.2 mM Na3VO4; 10 mM phenylmethylsulphonyl fluoride; 0.1 mg/mlare R0 cells stably transfected with the plasmid Cvn-IGF-IR [4, 33], aprotinin) containing 10 ml of either IGF-IR monoclonal antibodyexpressing the human IGF-IR cDNA under control of an SV40 pro- (Oncogene Science), or anti-Shc polyclonal antibody (Transductionmoter. All other clones are R0 cells stably transfected with Laboratories) or 5 ml of anti-IRS-I polyclonal antibody (UBI) and 20pMRIGF1R12, a derivative of Cvn-IGF-IR, that expresses both the ml of agarose-conjugated protein A (Oncogene Science). Antibody–hygromycin B phosphotransferase gene of Escherichia coli [34], and antigen complexes were allowed to form for 4 h at 47C and thenthe human IGF-IR cDNA under control of the rat (02350 / I640) collected by centrifugation at 47C for 4 min. ImmunoprecipitatedIGF-IR promoter [17, 27, 28]. Transfectants were selected in medium complexes were washed three times in HNTG and resuspended incontaining 200 mg/ml of hygromycin B. 20 ml of Laemli buffer (20% glycerol; 3% sodium dodecyl sulfate; 3% b-

mercaptoethanol; 10 mM EDTA, 0.05% bromophenol blue). SamplesDetermination of IGF-I binding sites. The number of IGF-I bind-ing sites per cell was determined by Scatchard analysis. The ligand were boiled for 4 min and proteins were separated on 4–20% gradient

polyacrylamide gel (Bio-Rad). Proteins were electroblotted onto areplacement protocol described by Miura et al. [31] was used. Briefly,cells were seeded at a density of 104/cm2 in 6-well tissue culture nitrocellulose filter. Phosphorylated proteins were detected by West-

ern immunoblot analysis with an anti-phosphotyrosine antibodyplates and made quiescent as described below. After 2 days, mediumwas replaced with SFM containing 5 ng/ml PDGF-BB (Gibco-BRL) (PY20), conjugated with horseradish peroxidase (Transduction Labo-

ratories), and visualized by the enhanced chemiluminescence (ECL)or with SFM alone and then incubated for another 16 h. Cells werewashed three times with ice-cold Hank’s solution and incubated in detection system (Amersham). Shc and IRS-1 proteins were identi-

fied after stripping the filter and rehybridizing with an anti-SHCbinding buffer (25 mM N-2-hydroxyethylpiperazine-N *-2-ethane sul-fonic acid (Hepes), pH 7.4, and 1 mg/ml bovine serum albumin in monoclonal antibody (Transduction Laboratories) or an anti-IRS-1

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286 RUBINI ET AL.

monoclonal antibody (Transduction Laboratories), followed by blot- point to clarify our approach to this assay: (1) absoluteting with an anti-mouse IgG-horseradish peroxydase-conjugated an- counts in assays for IGF-I binding sites vary, becausetibody (Sigma) and ECL detection.

the batches of radioactive IGF-I provided by the manu-facturer also vary; (2) the relative proportions of IGF-

RESULTS I binding sites among the selected clones remained con-stant, and still remain constant, throughout more thanCell lines. Clones of R0 cells transfected with plas-3 years of experimentation; (3) this reproducibility ismid pMRIGF1R12 were selected as described undervalid only if the number of IGF-I binding sites is deter-Materials and Methods. A large number of clones weremined on monolayers that are only 40–50% confluent.obtained that expressed variable levels of IGF-IRs.If the monolayers are confluent, the results are erratic;Some, but by no means all, of the clones obtained are(4) the number of receptors is a calculation made fromshown in Table 1. The number of receptors in cellsScatchard plots, which requires several assumptions.transfected with the IGF-IR cDNA under the control ofHowever, if the same calculations are carried out, thethe rat IGF-IR promoter [27, 28] never exceeded 60,000receptor number is highly reproducible; for instance,receptors per cell. When the IGF-IR cDNA under thewith Balb/c 3T3 (our reference cell line), receptor num-control of a viral promoter was transfected under theber has remained constant since 1990. With all cellsame conditions, the number of receptors was almostlines, under the conditions just enumerated, variationsinvariably in excess of 100,000 per cell, and often moreranged between 10 and 15%.than 500,000 [4, 25, 30]. At the present moment, we

cannot say whether these differences are due exclu- Characterization of cell lines. In terms of their mi-sively to the intrinsic properties of the promoters, or togenic response to IGF-I, the different clones (Tableif they indicate a tight feedback regulation of IGF-IR 1) could be classified into 3 groups: (1) clones with fewerexpression, which would be absent in a viral promoter. than 15 1 103 IGF-IRs per cell did not grow in SFM

supplemented solely with IGF-I. These clones were alsoDetermination of receptor numbers. The number ofunresponsive to a combination of PDGF plus IGF-I. Wereceptors was determined by Scatchard analysis (seehave chosen the clone designated R12 as prototype ofMaterials and Methods). Since this determination isthis group; (2) clones with 30 1 103 receptors per cellscrucial to the whole investigation, it is necessary at this(or more), again in terms of response to growth factors,grew in IGF-I only as well as they did in IGF-I plusPDGF. Clone R600 was chosen as representative of thisTABLE 1class; and (3) the two clones R508 and R503, which

Classification of R0 Transfectants have an intermediate number of receptors and will bediscussed in more detail below.

No. ofResponse

The number of IGF-IRs increases after stimulation ofreceptors

to IGF-I IGF-I / PDGFquiescent cells with PDGF, confirming previous results

Clones (1103) (mitogenesis) that PDGF increases the number of IGF-I binding sites[4, 15, 35], and activates expression from the IGF-IR405 2 0 0promoter [17]. Table 2 gives the number of IGF-IRs504 8 0 0

507 4 0 0 (before and after PDGF) in a typical experiment, in the509 5 0 0 cell lines described below; 10% serum has the same510 10 0 0 effect as PDGF on receptor number (not shown, but see512 (R12) 3 0 0

[17]). Clone R508 had 15 1 103 receptors per cell that506-94 3 0 0were increased to 23 1 103 by PDGF treatment. Clone507-94 5 0 0

508 (R508) 15 0 / R503 had 22 1 103 receptors per cell, increased to 30503 (R503) 22 / / 1 103 by PDGF. However, the increase in receptor num-R600 30 / /

ber caused by PDGF varied considerably in differentS 32 / /experiments; there was always an increase, but it wasTC2 58 / /

TC/r 58 / / not highly reproducible (see Discussion). While almosthT 160 / / every clone responded to PDGF with an increase inR/ 900 / / receptor number, there was an occasional exception.

For instance, clone S, with 32 1 103 IGF-IRs per cell,Note. Number of receptors per cell was determined as describedunder Materials and Methods. Mitogenic response is defined as abil- remained unchanged by PDGF stimulation. While itity to grow in monolayer in serum-free medium supplemented solely certainly would be interesting to know why in this clonewith IGF-I or IGF-I plus PDGF (see text). With this methodology, PDGF does not increase the number of receptors, weBalb/c 3T3 cells have 15,000 receptors per cell, and do not grow in

are not considering it further in this paper, since it isIGF-I only, but do grow in IGF-I plus PDGF. The table lists onlyabout one-third of the clones originally selected. responsive to stimulation by IGF-I only (Table 1). The

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TABLE 2 an IGF-IR to grow in monolayer in serum-supple-mented media.Summary of Representative R0 Transfectants Clones

Effect of increasing receptor number with PDGF onIGF-I receptors number 1103

cell growth. The R0 derived cell lines were then testedfor their ability to grow in SFM supplemented withCell line No. PDGF /PDGFboth IGF-I (20 ng/ml) and PDGF (5 ng/ml), which in-

R0 0 0 creases IGF-IR number (see Table 2). The results of aR12 3 7 typical experiment are shown in Fig. 2. The four cellR508 15 23 lines do not grow appreciably in PDGF only, with theR503 22 30

possible exception of R600. Since R600 cells grow inR600 30 58IGF-I only, and since it is well-established that PDGFS 32 31

R/ 900 900 increases the production and secretion of IGF-I (seeabove), it is possible that this modest stimulation of

Note. The cell lines were derived from R0 cells stably transfected growth may be attributable to the secretion of moder-with a human IGF-IR cDNA under the control of a rat IGF-IR pro-ate amounts of IGF-I. In IGF-I plus PDGF, R12 cellsmoter (02350//640), with the exception of R/ cells, in which thestill grow poorly, R503 grow somewhat better, andIGF-IR cDNA was under the control of an SV40 promoter (24, 25).

The determination of the number of receptors was highly reproduc- R600 grow equally well in IGF-I only or the combinedible, with variations of õ15% in separate experiments, over a period growth factors. But the most interesting result is thatof at least 1 year. For this reason, standard deviations are omitted. obtained with clone R508. Clone R508 grows well in

PDGF / IGF-I, whereas the two growth factors, singly,have little or no stimulatory effect: this is the cell linewith 15 1 103 receptors per cell, which increase to 23 1number of receptors did not increase in cell lines hT103 after treatment with PDGF. This is in remarkableand R/, expressing an IGF-IR cDNA under the controlagreement with clone R503, which has 22 1 103 recep-of a viral promoter [25]. We will use the receptor num-tors per cell and grows in IGF-I only. It confirms that,ber obtained before PDGF stimulation as the numberat approximately 22,000 receptors per cell, the trans-characteristic of a given cell line, unless otherwise

noted.Growth in IGF-I. Up to this point, the clones listed

in Table 1 had been tested for their response to IGF-Ionly, or to a combination of PDGF and IGF-I. The fol-lowing experiments were carried out only on represen-tative cell lines. The ability of these representativeclones to grow in SFM supplemented solely with in-creasing concentrations of IGF-I is shown in Fig. 1. Forclarity, we have omitted from Fig. 1 R0 cells (no growthat all) and the R/ cell line that grows vigorously inIGF-I even at the lowest concentrations. These two celllines have been characterized repeatedly in previouspapers [24, 25, 30, 36] and still behaved as reported.The R12 clone (3,000 receptors per cell) and the R508clone (15,000 receptors per cell) do not grow at all inSFM supplemented only with IGF-I, even at a concen-tration of 100 ng/ml of IGF-I. The other two clones,R503 and R600, behave essentially in the same man-ner, growing when IGF-I reaches a concentration of 2–5 ng/ml. These experiments, that were repeated, andthe results with other clones discussed above, indicatethat: (1) at receptor levels of 15 1 103 receptors per cellor below, IGF-I cannot sustain the growth of trans-fected R0 cells; and (2) at a level of approximately FIG. 1. Dose response of cell lines to stimulation by IGF-I. Cells

were plated at a density of 2.5 1 103 cells/cm2, made quiescent as22,000 receptors per cell, our 3T3-like cells grow indescribed under Materials and Methods and then IGF-I was addedIGF-I only, without a need for other growth factors. Allat the indicated concentrations. Cell counting was done on triplicatecell lines in Table 1, including R/ and R0 cells, grow cultures after 48 h further incubation at 377C. Open diamonds, R600

well in 10% serum, confirming our findings with R0cells; closed diamonds, R503 cells; open squares, R508 cells; closedsquares, R12 cells.cells [25, 30] that mouse embryo fibroblasts do not need

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288 RUBINI ET AL.

FIG. 2. Growth of transfected R0 cells in serum-free medium supplemented with growth factors. Cells were plated in triplicates at adensity of 2.5 1 103 cells/cm2, made quiescent, and then stimulated with either IGF-I (20 ng/ml), or PDGF (5 ng/ml) or both. Cell countingwas done on triplicate cultures after 48 h incubation at 377C. The ordinate gives the fold increase in cell number (1.0, no increase). Barsrepresent standard deviations.

fected R0 cells become responsive to the mitogenic stimu- soft agar is lower, but still evident without supplemen-tation with IGF-I [37].lus of IGF-I. These experiments indicate that, in mouse

embryo fibroblasts derived from mice with a targeted dis- Receptor phosphorylation. Since these cells are ex-ruption of the IGF-IR genes, the threshold for stimulation pressing wild-type receptors, and the only difference isby IGF-I only can be localized between 15,000 and 22,000 in their numbers, there is no a priori reason why theyreceptors per cell. should be defective in autophosphorylation. Figure 3

shows that, indeed, in the four clones tested, receptorGrowth in soft agar. Cells overexpressing the IGF-IR assume a transformed phenotype, which is some- autophosphorylation is detectable after stimulation

with IGF-I, the intensity of the band correlatingtimes ligand-dependent (see Discussion). We thereforetested the above cell lines for their ability to form colo- roughly with the number of receptors. The methodol-

ogy, including the time of exposure of the blots, wasnies in soft agar (Table 3). It is important to rememberthat soft agar assays are done in 10% serum, and underthese conditions the number of receptors is increasedabove the base value, as when the cells are stimulated TABLE 3by PDGF [17]. We therefore use here the augmented

Colony Formation in Soft Agar of the Several Cell Linesreceptor number. At about 7–22,000 receptors per cell,the clones are essentially incapable of forming colonies Number of coloniesin soft agar (Balb/c 3T3 cells, R12 and R508 cells).

Cell line 0IGF-I /IGF-IWhen the number of receptors is about 30,000 per cell,or more (in 10% serum), then the cells begin to form

R0 0 0colonies in soft agar, which are ligand-dependent (R503 R/ 213, 221, 218 247, 283, 265and R600 clones). None of the clones, though, was as R12 0, 2, 0 5, 3, 1effective as R/ cells (106 receptors per cell) in forming R508 0, 0, 1 3, 2, 4

R503 5, 1, 4 19, 13, 18colonies in soft agar. In previous experiments, we re-R600 2, 1, 0 15, 14, 17ported that R0 derived cells with wild-type receptor

levels between 500,000 and 106 per cell, all give large Note. Cells were plated at 1000 cells/35 mm dish; the number ofnumbers of colonies in soft agar [25, 30]. At 160,000 colonies was determined after 14 days. The cell lines are described

in the text and in Table 1.wild-type IGF-I receptors per cell, colony formation in

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289IGF-I RECEPTOR NUMBER

an 8-fold difference in the extent of autophosphoryla-tion. Indeed, the level of IRS-1 tyrosyl phosphorylationhad already reached the maximum value in R508 cells,that do not respond to IGF-I only with mitogenesis. It isalso interesting that even clone R12 shows a significantextent of IRS-1 tyrosyl phosphorylation.

The results obtained with Shc are shown in Fig. 5;whereas all three isoforms of Shc can be detected in allthe clones tested (Fig. 5B), tyrosyl phosphorylation ofShc is barely increased in all clones after IGF-I stimu-FIG. 3. Autophosphorylation of the IGF-I receptor in R0 derivedlation (Fig. 5A). In addition, there does not seem to becell lines. Autophosphorylation was determined as described under

Materials and Methods. Lanes: a, b, R12 cells; c, d, R508; e, f, R503; much difference between R12 and R508 or betweeng, h, R600. In each case, the first lane is without stimulation by IGF- R503 and R600.I, the second lane, 5 min after IGF-I addition.

DISCUSSION

Of the three possibilities presented in the Introduc-exactly the same for the four clones. Indeed, we havetion to explain the conversion of the IGF-IR from itsomitted R/ cells from Figure 3 because under thesenonmitogenic to its mitogenic mode, we have chosen inconditions they gave too strong a signal and we wishedthis investigation to address the third possibility, i.e.,to show that with sufficient exposure, even 3,000 recep-the role of IGF-IR number in mitogenesis and transfor-tors per cell (R12 clone), give detectable autophosphor-mation. We were especially interested in low receptorylation. By densitometry, the ratios of the four clonesnumbers, roughly within 2–3 standard deviations ofexamined, beginning with the clone with the lowestIGF-IRs number of some 3T3-like cells, such as Balb/cnumber of receptors, were 1:2.2:2.7:8.2, which do not3T3 cells, that have (with the methodologies used ingive the same ratios as the Scatchard analysesour laboratory) 15,000 receptors per cell. It was not our(1:5:7:10), especially in the middle (see Discussion).intention to define here the role of PDGF, but simplyTyrosyl phosphorylation of IRS-1 and Shc. Also im-to use it as a means to increase the number of IGF-IRsportant is to determine the tyrosyl phosphorylation ofin the same cell line. The increase in IGF-IR numbertwo major substrates of the IGF-IR, IRS-1, and Shccaused by PDGF can rightly be considered as within[38–41]. Figure 4 shows the tyrosyl phosphorylation ofthe physiological range, and it was this range of recep-IRS-1 (Fig. 4A) and the levels of IRS-1 protein (Fig.tor number that we wanted to explore in more detail.4B) in four of the cell lines. We measured the extent ofThe availability of R0 cells offered a unique opportunitytyrosyl phosphorylation of IRS-1 by densitometry: theto carry out these studies, since they start out with nochanges are much less pronounced than the variationsIGF-IRs at all; at the same time, it should be madein receptor number or the autophosphorylation of theclear that other cell lines may have and, indeed, doreceptors. For instance, the increase in IRS-1 phos-

phorylation between clones R12 and R600 is only 2.4-fold, versus a 10-fold difference in receptor number and

FIG. 4. Tyrosyl phosphorylation of IRS-1 in the same cell linesas those described in the legend of Fig. 3. Both tyrosyl phosphoryla- FIG. 5. Shc tyrosyl phosphorylation in the same cell lines. (A)

The tyrosyl phosphorylation and (B) the levels of Shc proteins (seetion (A) and levels of IRS-1 protein (B) were determined as describedunder Materials and Methods. Lanes are the same as described in Materials and Methods). Lanes are the same as described in the

legend of Figs. 3 and 4.the legend of Fig. 3.

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290 RUBINI ET AL.

have totally different numbers of IGF-IRs, and may experimental conditions and the calculations are keptconstant. Furthermore, the validity of our conclusionstherefore have different requirements. But R0 cells al-

lowed us to study the effect of receptor numbers on is not based on the fact that the number of receptorsin R508 cells is 17,000 rather than 15,000 receptors pergrowth and transformation, in the background of a sin-

gle cell line, and with a range from zero to ú106 recep- cell. The important conclusion of these investigations isthat small changes in IGF-I receptor number can altertors per cell.

Within the limits of the model, though, our results the responsiveness of mouse embryo cells to the mito-genic action of IGF-I.are clear and can be stated as follows: (1) under 15 1

103 IGF-IRs per cell, R0 transfectants do not grow in The increase caused by PDGF is less reproducible.The main reason is that the increase is always small.SFM supplemented by IGF-I only, or by a combination

of IGF-I and PDGF, and will only grow in serum, like At best, there is only a doubling in receptor number,which is probably due to the fact that the receptor levelthe parental R0 cells [25]; (2) at or above 30 1 103

receptors per cell, these 3T3-like cells grow in IGF-I may be tightly regulated by a feedback mechanism. Insupport of this explanation is the fact that we can ob-only, and PDGF will not increase their response; and

(3) the breaking point seems to occur between 15 and tain high levels of IGF-IRs only in cells transfectedwith an IGF-IR cDNA under the control of a viral pro-22 1 103 receptors per cell. At 15 1 103 receptors

(R508), the cells do not grow in IGF-I or PDGF, but do moter. Other investigators [42], besides Rubini et al.[17], have recently reported an increase in IGF-IR num-grow when both growth factors are added, with the

number of IGF-IRs increased to 23 1 103 by PDGF. At ber after PDGF stimulation, which makes sense, sincecells increase in size before dividing, and if the cells22 1 103 receptors per cell (R503), IGF-I is sufficient

for a mitogenic stimulus. Thus, it seems that a small have to keep the same density and the same numberof receptors after mitosis, they must at least doubleincrement in IGF-IR number, similar to the increase

induced by stimulation with PDGF, is sufficient to ren- their numbers. We cannot offer an explanation for thevariations in the response to PDGF. Modest changesder R-transfectants responsive to the mitogenic action

of IGF-I only. Please note that there are several clones in stimulation time can change the extent of the re-sponse, but there are other causes which we have notbelow 15 1 103 receptors per cell, and several clones

with more than 30 1 103 receptors, so that this range been able to identify yet: the important point, though,is that PDGF, in most cell lines, in this as well as inis clearly defined. The breaking point is less clearly

defined. It certainly would have been desirable to have other laboratories [14–18, 42], increases the numberof IGF-IRs.more clones in the range of 15 to 22,000 receptors per

cell, but plasmid transfection does not allow the selec- Another objection is the stability of receptor number.Apart from the fact that we constantly monitored re-tion of cell lines with a predetermined number of recep-

tors, and we literally screened hundreds of clones. How- ceptor number in the selected cell lines, our generalexperience is that if the transfectant cells are kept un-ever, the behavior of more than 30 clones (at least) is

quite consistent; no clone grew in IGF-I only at 15,000 der pressure with the appropriate selecting agent, thenumber of receptors, after slight fluctuations at theor fewer receptors per cell (Table 1). At 22,000 receptors

per cell or more, all clones grew in SFM supplemented very beginning, remains remarkably constant. Thevariability in the determination of IGF-I binding sitessolely with IGF-I. This suggests that the range 15–

22,000 receptors per cell is a reasonable choice for a depends much more on the conditions used in eachsingle experiment than on fluctuations of receptorthreshold, in R0 derived cells, for response to IGF-I

only. In addition, clones R508 and R503 serve as con- number. For this reason, we have standardized ourprocedure, especially the density of the cells. If the cul-trols of each other, since R508 grow in IGF-I when

PDGF increases the receptor number, whereas R503 tures are subconfluent, or even worse, confluent, theresults can be erratic, but if the density is maintainedgrows in IGF-I only. The fact that Balb/c 3T3 cells,

unstimulated, have 15,000 receptors per cell, which in- constantly at about 40–50% confluence, the calculationof receptor number is quite reproducible. The relation-crease after stimulation (see Introduction) and do not

grow in IGF-I only but do grow in PDGF / IGF-I, may ship among the four cell lines remained the same, even3 years after the establishment of the clones (notbe a confirmation of our results or could be coincidental.

These conclusions are based on the accuracy of our shown).Leaving aside the role of PDGF in the overall process,determinations of receptor number. In this respect,

there are here two separate problems: (1) the relative we emphasize again that 3T3-like cells, like R0 cells, donot need a great increase in IGF-IR number to becomenumber of receptors required for stimulation by IGF-I

only; and (2) the increase in receptor number caused competent for growth in IGF-I only. The number ofreceptors used in this communication is given as IGF-by PDGF. As to the first problem, it has been dealt

with at the beginning of Results and we can only repeat IRs per cell in unstimulated cells in SFM. After stimu-lation with IGF-I, the number of receptors may change,here that IGF-I receptor numbers are constant, if the

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291IGF-I RECEPTOR NUMBER

because of internalization and recycling, so that one 3T3-like cells, like R0 cells. Below a certain level ofreceptors, cells will not grow in IGF-I, even after addi-should not take these numbers as invariant, but simply

as a convenient method to designate clones. The varia- tion of PDGF, nor will they form colonies in soft agar;(2) the remarkably modest increment in receptor num-tion in receptor number and function during stimula-

tion of growth has already been discussed by Zhan et ber that is sufficient to make cells grow in IGF-I onlyand the fact that, at 22,000 receptors per cell or more,al. [43] and Valgeirsdottir et al. [44]. On the other hand,

when discussing growth in soft agar, we have given no other growth factors is required, suggest that thenumber of IGF-IRs can affect the mitogenic responsethe augmented number of receptors, because soft agar

growth is tested in 10% serum, which causes an in- within the physiological range of variations. Our exper-iments do not indicate (nor were they intended to indi-crease in IGF-IR number similar to that caused by

PDGF [17]. Since this occurs in the absence of other cate) whether PDGF provides another separate func-tion; (3) it is attractive to hypothesize that an increasedstimuli, one can say that receptor number can certainly

modulate the IGF-I-mediated mitogenic stimulus. receptor number may activate another substrate (ordifferent domains in the same substrate), or that PDGFIn previous papers, we had shown that cells devoid

of IGF-IRs, R0 cells, are refractory to transformation by may make this new substrate available; and (4) 30,000receptors per cell are sufficient to make R0 transfec-certain oncogenes [24–26], by the overexpressed EGF

receptor [30], the overexpressed PDGF receptor b [36], tants form colonies in soft agar, but only after supple-mentation of IGF-I. Yet, the fact that such a smallor the overexpressed insulin receptor [32], all condi-

tions that readily transform W cells and other 3T3-like number of receptors is already sufficient to transformR0 cells confirms that the failure of certain IGF-IR mu-cells. Conversely, cells overexpressing the IGF-IR can

form colonies in soft agar [19, 24, 25, 45, 46] that are tants to transform R0 cells, although still capable ofmitogenic signaling, is based on a real qualitative dif-often increased by the addition of IGF-I to the medium.

Using the augmented number of IGF-IRs (see above), ference. In those experiments [32, 46–48], mutant re-ceptors were used that were responsive to IGF-I-medi-we can say that, when R0 transfectants are expressing

23 1 103 IGF-IRs per cell, or less, the cells essentially ated mitogenesis, but were nontransforming at recep-tor levels that, in some cases, reached 9 1 106, or 300do not form colonies in soft agar, just like Balb/c 3T3

cells. A level of 30,000 receptors per cell is sufficient to times the levels shown in this paper to transform R0

cells.allow R0 transfectants to form IGF-I-dependent colo-nies in soft agar. The cells are transformed, by these We did not ask in this paper whether an increased

IGF-IRs number activates pathways that are not acti-criteria [19], yet, they are obviously not as transformedas cells with 500,000 receptors or more, like p6 cells vated below a certain level, but we already had evi-

dence before [21] that elevated IGF-IR number stimu-[30] or R/ cells [24, 25, and this paper], which formlarge colonies in soft agar even in the absence of added lated early gene expression. It would be interesting at

this point to investigate the signaling pathways inIGF-I. It seems, in other words, that the number ofIGF-IRs has a sort of correlation (up to a certain point) R508 and R503 clones. This is not, however, a simple

problem, as indicated already by the fact that IRS-1with the degree of transformation, at variance withIGF-I-mediated mitogenesis, where there seems to be and Shc respond with tyrosyl phosphorylation in a way

that is not strictly proportional to the number of IGF-a threshold.The receptors in the various clones examined are IRs. Indeed, preliminary experiments (not shown) indi-

cate that MAP kinase activation by IGF-I (p42/mapk)autophosphorylated by IGF-I, the extent being roughlycorrelated to the number of receptors. The lack of a was essentially the same in clones R508 and R503.

Clearly, the possibility of another, unidentified, ras-perfect correlation between receptor number (byScatchard analysis, which is accurate), and autophos- independent pathway cannot be dismissed [25, 49]. The

other intriguing question is the role of PDGF in thephorylation, may reflect the inaccuracy of the latter or,alternatively, more subtle informational contents. On overall process. Ideally, one would like to use a cell line

that does not respond to IGF-I, but responds to PDGFthe contrary, the extent of tyrosyl phosphorylation ofthe two major substrates, IRS-1 and Shc, seems to rap- and IGF-I, without an increase in IGF-R number, al-

most like the S cell line described in Table 2, which,idly reach a maximum, as if their activation did notrequire a mitogenic level of receptors per cell. This sug- unfortunately, already grew in IGF-I only. This one,

too, is not a problem easy to solve, since one can onlygests that qualitative changes in at least one of thesubstrates, or the requirement for another unknown hope in a mutant like the S cell line, but with lower

receptor number.substrate, may be important in determining the mito-genicity of the IGF-IR. While these possibilities are being explored, these

experiments show for the first time, in the context ofWe would like to draw the following conclusions fromthese experiments: (1) IGF-IR number can play a role a single cell line, that small increments in the number

of receptors per cell can change the IGF-IR from itsin IGF-I-mediated mitogenesis and transformation in

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292 RUBINI ET AL.

22. Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J., and Efs-nonmitogenic to its mitogenic mode and that a furthertratiadis, A. (1993) Cell 75, 59–72.small increase can cause the intermediate transforma-

23. Baker, J., Liu, J. P., Robertson, E. J., and Efstratiadis, A. (1993)tion of the cells.Cell 75, 73–82.

24. Sell, C., Rubini, M., Rubin, R., Liu, J. P., Efstratiadis, A., andThis work was supported by Grant CA 53484 from the National Baserga, R. (1993) Proc. Natl. Acad. Sci. USA 90, 11217–11221.

Institutes of Health and by Grants C.N.R./P.F.A.C.R.O. 94.01070; 25. Sell, C., Dumenil, G., Deveaud, C., Miura, M., Coppola, D.,C.N.R./B.P. 94,0299.CT04. M.R. was supported by the N.I.H. Short- DeAngelis, T., Rubin, R., Efstratiadis, A., and Baserga, R.Term Scientist Exchange Program and by the Associazione Italiana (1994) Mol. Cell. Biol. 14, 3604–3612.per la Ricerca sul Cancro.

26. Morrione, A., DeAngelis, T., and Baserga, R. (1995) J. Virol.69, 5300–5303.

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Received September 2, 1996Revised version received November 5, 1996

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