a soluble insulin-like growth factor i receptor that ... · (3 1), as 486/stop, whereas the...

CANCER RESEARCH56. 4013â€”4020.September I. 19961

ABSTRACT

By a frame-shift mutation, we have engineered a human IGF-l receptor(IGF-IR) eDNA that produces a receptor 486 amino acids long (plus the 30amino acids of the signal peptide). This receptor, which we have designated as 486/STOP, is partially secreted into the medium ofcells in cultureand markedly inhibits the autophosphorylation of the endogenous IGFIRs as well as the activation of the signaling pathway. The 486/STOPreceptor acts as a strong dominant negative for several growth functions:(a) it inhibits the growth of cells in monolayers; (b) it inhibits the growth

of transformed cells in soft agar; (c) it induces extensive apoptosis in vivo;and (d) it inhibits tumorigenesis in syngeneic rats. This is the first demonstration that a dominant negative of the IGF-IR can induce massiveapoptosis of tumor cells in vivo.

INTRODUCTION

The IGF-1R3 belongs to the family of tyrosine kinase growth factorreceptors (I). Although it is 70% homologous to the IR (2), theIGF-IR has clearly distinct biological actions. In recent years, it hasbecome clear that the IGF-IR activated by its ligands (IGF-I, IGF-II,and insulin at supraphysiological concentrations) regulates the proliferation of cells, either in vivo or in s'itro, by three different mechanisms: (a) it is mitogenic; (b) it plays a crucial role in the establishment and maintenance of the transformed phenotype; and (c) itprotects cells from apoptosis (3). The IGF-IR has been known toinduce a mitogenic response in cells in culture, ordinarily in cooper

ation with other growth factors such as platelet-derived growth factorand epidermal growth factor (4â€”6).The importance of the IGF-IR incell growth in vivo was demonstrated by the fact that mouse embryos

with a targeted disruption of the IGF-IR and IGF-II genes have a sizeat birth that is only 30% the size of wild-type littermates (7, 8).3T3-like cells derived from mouse embryos devoid of IGF-IRs (Rcells) grow in 10% serum, albeit more slowly than other 3T3-like cellswith a physiological number of IGF-IRs, but do not grow at all in

SFM supplemented by a variety of growth factors, which can sustainthe growth of cells derived from wild-type littermate embryos (Wcells) or of other 3T3 cells (9).

The importance of the IGF-IR in transformation was dramaticallyunderscored by the finding that R cells are refractory to transformation by SV4O large T antigen, by an activated ras, or a combination ofboth (9, 10); by the ES protein of the bovine papilloma virus (1 1); orby overexpressed growth factor receptors such as the epidermalgrowth factor receptor ( I2), the platelet-derived growth factor /3receptor (13), and the IR (14); all conditions that readily transformcells from wild-type littermate embryos or other 3T3-like cells. Con

Received 3/14/96; accepted 6/28/96.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 Supported by Grants CA 53484 and GM 33694 from the NIH.

2 To whom requests for reprints should be addressed, at Kimmel Cancer Center.

Thomas Jefferson University. 624 Bluemle Life Sciences Building, 233 South 10th Street,Philadelphia. PA 19107. Phone: (215) 503-4507; Fax: (215) 923-0249: E-mail:Baserga [email protected].

3 The abbreviations used are: IGF-IR. insulin-like growth factor I receptor; IGF-l,

insulin-like growth factor I; IGF-II. intulin-like growth factor II; IR, insulin receptor;SCM, serum-free medium: FBS. fetal bovine serum; CM, conditioned medium; MAP,mitogen activated protein; IRS- I . insulin receptor substrate I.

versely, overexpression and/or constitutive activation of the IGF-IR in

a variety of cell types leads to ligand-dependent growth in SFM and

the establishment of a transformed phenotype (10, 12, 15â€”20).Otherfindings support the notion that the IGF-IR plays an important role in

the establishment and maintenance of the transformed phenotype.Thus, antisense expression plasmids or antisense oligodeoxynucleotides against either IGF-II (21, 22), IGF-I (23), or the IGF-IR (9,

24â€”28),antibodies to the IGF-IR (29, 30), and dominant negativemutants of the IGF-IR (31â€”33)can all reverse the transformed phenotype or inhibit tumorigenesis and induce loss of the metastaticphenotype (34).

There are also several reports that IGF-I and/or the activatedIGF-IR can protect cells from apoptosis (16, 35â€”37).This is also trueof cells overexpressing the proto-oncogene c-myc (38) and of severalhuman and rodent tumor cell lines, both in vitro (39) and in vivo (26,27). Thus, a variety of methods, ranging from gene deletion todominant negatives and from antibodies to antisense strategies, support the conclusion that the IGF-IR plays an important role in cellularproliferation, transformation, and apoptosis.

In view of the importance of the IGF-IR in cell proliferation, wehave been searching for ways of inhibiting its functions, in addition tothose mentioned above. A reasonable approach is the development of

dominant negatives; i.e., the use of mutant IGF-IRs to inhibit thefunction of wild-type endogenous receptors. One such dominant neg

ative, 952/STOP (truncated at residue 952), has been described by

Prager et a!. (31). We were particularly interested in dominant negatives that could induce apoptosis and therefore abrogate tumorigene

sis. In a previous paper, Burgaud et a!. (33) reported that severalmutants of the IGF-IR could act as dominant negatives for growth inmonolayers and soft agar but could not induce apoptosis of tumorcells in vivo. In this paper, we describe the construction of an IGF-IR

truncated at residue 486 that is partially secreted into the medium,inhibits IGF-IR signaling and the growth ofcells in monolayers and in

soft agar, induces apoptosis in vivo, and abrogates tumorigenesiswhen expressed in rat glioblastoma cells.

MATERIALS AND METHODS

Strategy for the Construction of the IGF-IR Truncated at Residue 486.The CVN-IGF-IR plasmid (2) containing the full-length coding sequenceeDNA of the human IGF-IR under the control of the SV4O promoter wasdigested with AgeI. which cuts at nucleotide 1574 [numbering according to

Ullrich et a!. (2)]. The overhangs were filled with Klenow, and the plasmid was

re-ligated. This procedure generated a frame-shift mutation that resulted in the

creation of an early stop codon 12 nucleotides downstream from the AgeI site.The mutation was confirmed by sequencing both strands (data not shown). Thewild-type and mutant DNA sequences and their translation in the area cone

sponding to the mutation are shown below. The AgeI site is underlined. The

mutation abrogates this restriction site. Wild-type TOG C AC COG TAC

CGG CCC CCT GAC TAC and . . . . W H R Y R P P D Ymutant TGG CAC CGG CCG GTA CCG GCC CCC TGA CTA. . . . and

W H R P V P A P * where WHR are amino acids 509â€”511, if we

include the signal peptide (479â€”481 without the signal peptide). Therefore. themutant receptor is 5 16 amino acids long (or 486 amino acids long without the

signal peptide), although the correct sequence of the receptor terminates at

residue 48 1. The receptor was designated, following the nomenclature ofPrager et a!. (3 1), as 486/STOP, whereas the expression plasmid was desig

4013

A Soluble Insulin-like Growth Factor I Receptor That Induces Apoptosis of TumorCells in Vivo and Inhibits Tumorigenesis'

Consuelo D'Ambrosio, Andres Ferber, Mariana Resnicoff, and Renato Baserga2

Ki,n,nel Cancer Center. Jefferson Medical (â€˜ollege.Thomas Jefferson University, Philadelphia. Pennsylvania 19107

Research. on November 6, 2020. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

DOMINANT NEGATIVE IGF-IR

nated as pIGFIRsol. This plasmid. like the original plasmid, contains theneo-resistance gene.

Transcription and Translation of pIGFIRsoL The wild-type and 486/

STOP receptors' cDNAs were subcloned into pcDNA3 vector under thecontrol of SP6 promoter to obtain a construct suitable for in vitro transcription

with mMESSAGE mMACHINE@kit (Ambion). Synthesis of capped mRNAwas performed according to the manufacturer's instructions. The templatefrom Xenopus elongation factor 1 (Xef-l, provided by the company) was usedas a control for mRNA synthesis, leading to a product of 1.9 kb. Roughly 10

ILg of mRNA (4.2 kb) were obtained from the wild-type IGF-IR and IGFIRsoI

cDNAs. Five hundred ng were used to perform the in vitro translational assayby using the Retic Lysate IVT@kit (Ambion). Xef-l mRNA, encoding for a Mr50,200 protein and provided by the company, was used as a control. Proteinswere labeled with [35S]methionineand resolved on 4â€”15%SDS-PAGE. Gelwas dried and then exposed to Kodak X-OMAT AR film at -80Â°C.

RNA Extraction and Northern Blots. Total RNA was extracted fromcells according to the procedure described by Chomczynski and Sacchi (40).The Northern blot was carried out by standard procedure. The probe used wasthe SphI fragment of the wild-type IGF-IR cDNA (2.2 kb).

Generation of Cell Lines Expressing the 486/STOP IGF-IR. The parental cell lines used have been described in detail in previous papers: C6 cells arerat glioblastoma cells that grow even in SFM and form colonies in soft agar(24, 26); R cells (9, 10) are 3T3-like cells derived from mouse embryos witha targeted disruption of the IGF-IR genes (7, 8); BALB/c 3T3 cells are standard3T3 cells that have been passaged in our laboratory for several years; and p6cells (17) are BALB/c 3T3 cells stably transfected and overexpressing a humanIGF-IRcDNA.

C6/IGFIRSo1 cells were developed by cotransfection of the IGFIRSo1

plasmid and pPDV6+ plasmid containing the puromycin resistance gene (41).Cells were selected in 2.5 p@g/mIof puromycin; the resulting clones were thenswitched to medium containing 1400 pg/mi of G4l8 to keep them under strictselection (the pIGFIRso1plasmid also contains the neo-resistance gene). Weused this double selection method because selection in only G418 does notalways kill all untransfected C6 cells. Other C6 cells were transfected by thesame procedure with an empty vector or the same vector containing unrelated

inserts. Ba1bIIGFIRSo1were obtained by stable transfection with the IGFIRSo1plasmid and subsequent selection in 1400 @g/ml of G418. R cells were

cotransfected with pIGFIRs0I and pdeltaSUpac (also containing the puromycinresistance gene), and the cells were selected in 2.5 pg/mi of puromycin.

Growth Curves. Two different types of experiments were performed onC6 and C6IIGFIRSo1cells. In the first one C6 and C6IIGFIRSo1cells wereseeded in 10% serum at a density of 3 X l0@cells/35-mm plate and switchedto SFM (DMEM + 0.5 mg/mI BSA + 50 ,.tg/ml transferrin) or SFM + IGF-I

(20 ng/ml) after 24 h. Cell number was determined after 96 h. In the secondexperiment, the same cells were plated at a density of 8 X 104/35-mm dish in

10% serum, and the number of cells was counted after 48 h.Soft Agar Assay. C6 and C6IIGFIRSo1 cells were seeded at 5 X l0@/

35-mm plate in DMEM containing 10% FBS and 0.2% agarose (with 0.4%agarose underlay). C6 cells were also plated at the same density in CM fromC6 or C6/IGFIRSo1clones with 10% FBS and 0.2% agarose. Colonies largerthan 125 @mwere scored after 2 weeks and 1 week, respectively. p6 or T/Wcells were plated at a density of 1 X l03/35-mm dish in CM from R/IGFIRSoIor Balb/IGFIRSo1cells with 10%FBS and 0.2% agarose (with 0.4%agarose underlay). Colonies larger than 125 @.tmin diameter were scored after2 (p6 cells) or 3 (T/W cells) weeks.

Determination of Apoptosis in Vivo. This has been described by Resnicoff et a!. (26). Briefly, we used a diffusion chamber implanted into the s.c.tissue of rats or mice. This diffusion chamber is a lucite ring closed at bothends by a Millipore filter, with pores 0.1 @.tmin diameter. The pore size allowsthe passage of proteins, nutrients, antibodies, and so forth, but not of cells. Thecells to be studied were placed in the previously sterilized diffusion chamber,which was then inserted into the s.c. tissue of rats or mice under anesthesia.The diffusion chambers can be removed at the desired intervals after insertion,and the cells therein can be counted or otherwise examined. Cells in thediffusion chamber behave essentially as cells injected into the s.c. tissue ofanimals. Thus, cells from several transplantable tumors of human or rodentorigin placed in diffusion chambers double in number in 24 h during in vivoincubation (26, 27). indicating that conditions are optimal for their survival and

proliferation. At the times indicated, the cells recovered from the chambers

were examined for the presence of apoptotic cells. This was done by severalmethods, including cytofluorimetric analysis, DNA laddering, and others (26,27).

Tumorigenesis. To determine the ability of C6 cells and derivative celllines to produce tumors, syngeneic BD IX rats were injected s.c. with 1 X 10@cells, as described previously (24). Wild-type C6 cells at this concentrationgive palpable tumors in 4 days, and the animals usually die or have to be killedafter 20â€”25days. Rats that did not develop tumors were kept under observation for as long as 180 days. The rats used in the diffusion chamber experiments were then used to determine the host response induced in rats by cellswith decreased numbers of IGF-IRs (24, 26).

Autophosphorylatlon of IGF.IR. This was carriedout as described indetail in several papers from this laboratory (9, 12, 14, 24, 42).

Tyrosyl Phosphorylation of IRS-i. Lysates from IGF-I-stimulated andunstimulated cells were prepared (see â€œAutophosphorylation of IGF-IRâ€•).

Protein lysates (1500 @.tg)were immunoprecipitated overnight at 4Â°Cwithanti-IRS-l polyclonal antibody (UBI) and protein A Agarose (Oncogene

Science). SDS-PAGE, transfer, hybridization with anti-phosphotyrosine antibody, and detection were perfonned as described previously.

MAP Kiaase Activation Assay. This assay was carriedout on C6 andC6/IGFIRSoI cells as described by Cook et a!. (43). Cells were grown until

80% confluence and incubated in SFM for 18 h at 37Â°C.They were thenwashed three times with Hank' s solution, and fresh SFM was added to eachplate for an additional 15-mmincubation. Cells were stimulated with IGF-I (20ng/ml) for 15 mm and 6 h. The plates were washed with cold PBS, then lysed

on ice in 1 ml of lysis buffer [10 inst Tris-HCI (pH 7.4), 1% Triton X-lOO,0.5% NP4O, 150 msi NaC1, 20 mist sodium fluoride, 0.2 mM sodium orthovana

date, 1 mM EDTA, 1 mist EGTA, and 0.2 nmi phenylmethylsulfonyl fluoride].Unstimulated cell lysates were prepared as well. Protein lysates (150 @g)wereimmunoprecipitated overnight at 4Â°Cwith anti-ERK2 (C-l4) AC antibody(Santa Cruz Biotechnology). Pellets were washed and resuspended in 15 pAofkinase buffer [10 mistTris-HC1(pH 7.4), 150mistNaC1,10 mrviMgC12,and 0.5mM DIT). Five @tlofATP mix [41.7 @dkinase buffer, 1.2 @tlof25 mitt AlP

(pH 7), 2 @lof 2 MMgCl2, and 2.2 jd of [32P]ATP]and 7 @dof 10 mg/ml ofmyelin basic protein were added to each sample, and the kinase reaction wasallowed to proceed for 20 mm at 30Â°C.After resolution on 4â€”15% SDSPAGE, the gel was dried and exposed to Kodak X-OMAT AR film. Foldincrease in MAP kinase activity was determined by laser densitometry.

Preparation of CM. CM from cell lines expressing pIGFIRsol was prepared as follows. Cells were grown until 80â€”90%confluence, washed carefully with Hank's solution, and incubated in SFM for 72 h. CM was thencollected and centrifuged at 3000 rpm for 5 mm to discard dead cells. FreshCM was used to test its effect on cell growth.

[email protected](20).Briefly. cells were plated at 2 X 106cells/lOO-mmplate in DMEM supplemented with 10% PBS. After 24 h, the cells were placed in methionine-freeDMEM (Life Technologies, Inc.) containing 0.1 mCi/ml of L-[35S]methionine(Amersham) and were incubated overnight. The cells were then lysed, and 600

I.Lg of proteins were immunoprecipitated with the anti-IGF-IR antibody (Santa

Cruz Biotechnology). Immunoprecipitation with anti-IGF-IR monoclonal antibody (Oncogene Science) was also carried out on R@ cells as a control.

Immunoprecipitation was also performed on the CM after concentration withCentricon columns with a M30,000 cutoff. The 35Slabeling of the IGF-IR orthe 486/STOP receptor was visualized by autoradiography.

RESULTS

Generation of Cell Lines Expressing the Soluble IGF-IR. Thecell lines expressing the soluble IGF-IR were generated as describedin â€œMaterialsand Methods.â€•After selection and screening, we obtamed several clones from three different original cell lines: BALB/c3T3 cells, R cells, and C6 rat glioblastoma cells. The BALB/c 3T3-and R -derived clones were used almost exclusively to prepare CM tobe tested on other cell types. C6 cells, wild-type or stably transfectedwith the plasmid pIGFIRsol, were used for most of the experimentsdescribed below. C6 cells transfected with an empty vector or withvectors expressing unrelated cDNA gave rise to clones that wereindistinguishable from wild-type cells. For this reason, in the rest of

4014



Cell lineNo. of colonies in softagarC6301.317C6

+ CM from wild-typeC6>1.000C6/SRcloneI417Clone4136Clone

S167.148Clone6100Clone

729,122


the paper, we will refer to only C6 wild-type cells as controls,although they may also include C6 cells transfected with the vector orother plasmids.

Characterization of the Soluble IGF-IR. Accordingto the way inwhich the expression plasmid was engineered, the 486/STOP IGF-IRshould have a size of about Mr 52,000S5,000 (516 amino acidsbefore cleavage of the signal peptide) but an mRNA of the samelength as the mRNA of the wild-type human IGF-IR cDNA fromwhich it originated. We determined the sizes of the RNA and proteinof both the wild-type and soluble receptors by an in vitro transcription-translation system (Ambion; see â€œMaterials and Methodsâ€•). Fig.

1A shows the RNAs transcribedfrom the two cDNAs: Lane 1 is thecontrol provided by the manufacturer; Lanes 2 and 3 are the RNAstranscribed from the wild-type cDNA; and Lanes 4 and 5 are theRNAs from the mutant receptor. Both RNAs have the exact same size(â€”4000 bp). Fig. lB shows the translation products of these twocDNAs: Lane I is the marker provided by the manufacturer (given asMr 50,000) Lane 2 is the translation product of the mutant cDNA; andLane 3 is the translation product of the wild-type cDNA. The latterone has the largest band of the expected size of about Mr 150,000 (topband in Lane 3), whereas the mutant cDNA gives a translation productof a maximum size of M@ @S0,0O0.In all three lanes there are smallerbands, which (according to the kit manufacturer) are premature termination products not infrequent with this system, but the maximumsize products are quite different. The sizes of the two RNAs were alsodetermined by Northern blots of various cell lines, in which total RNAwas hybridized to an IGF-IR probe (see â€œMaterialsand Methodsâ€•).Asa control we used HL6O cells, in which the IGF-IR RNA is vigorouslyexpressed (44). C6 cells stably transfected with either the wild-type orthe mutant IGF-IRs expressed the 4.2-kb RNA that is the size of theinserted cDNA (2, 17), whereas the IGF-IR RNA of the HL-60 cellswas 7 kb (data not shown).

Growth Characteristics of C6 Cells Expressing the 486/STOPIGF-IR. Several clones of C6 rat glioblastoma cells stably transfected with plasmid pIGFIRsol were selected and screened. Most of

AI 2 3 4 5 kb

â€” â€”4.2@@it@:.;@..@ .@@

B

I 23kDa

204â€”

121â€” â€” â€”9782â€” p

â€” @50

Fig. 1. Sizes of the transcription and translation products of the IGF-IR cDNAs. Thewild-type and mutant IGF-IR cDNAs were transcribed and translated in vitro as describedin â€œMaterialsand Methods.â€•A, RNA transcripts. Lane 1. control included in the kit; Lanes2 and 3, wild-type IGF-IR; Lanes 4 and 5. 486/STOP receptor. The expected size of bothRNAs is 4.2 kb. B. translation products. Lane I. control product from kit; Lane 2.486/STOP; Lane 3. wild-type receptor. The markers give the expected sizes of thewild-type and mutant receptors.

12 010%FBS@ SFM

10@ IGFI

w * not done@ 8

.E 6V

:@

Cell lines

Fig. 2. Growth of C6 cells and derived clones expressing the 486/STOP IGF-IR. Cellswere plated at a density of 3 X [email protected] number of cells was determined after 96 h ofgrowth in the designated medium. Abscissa, cell clones used; ordinate. the fold increaseover plating density.

Table 1 Anchorage-independent growth of C6 rat glioblastorna cells expressing the486/STOP IGF-IR

C6 cells, wild-type or stably transfected with plasmid pIGFlRsoI, were seeded in softagar and supplemented with 10% serum at a density of S X io@. Colonies >125 sm indiameter were scored after 2 weeks, with the exception of the wild-type C6 incubated in10% serum plus their own CM, which had to be counted after only I week.

the clones grew significantly more slowly in 10% serum, and three ofthese clones are shown in Fig. 2, in which the growth of wild-type C6

cells is also shown. These clones were then tested for their ability to

grow in SFM with or without IGF-I (20 ng/ml). C6 cells at 37Â°Cgrew

in SFM, or in SFM supplemented with IGF-I; the difference is not

significant because these cells are known to produce IGF-I as well as

other growth factors (Ref. 24, and see below). Clones 5, 6, and 7 (allexpressing the 486/STOP receptor) were markedly inhibited under thesame conditions (Fig. 2). These are essentially the same results as weobtained with C6 cells stably transfected with a plasmid expressing anantisense RNA to the IGF-IR RNA (24).

These and other clones were then tested for their ability to formcolonies in soft agar. Wild-type C6 cells form colonies in soft agar(24), but (with the sole exception of clone 1) the clones expressing the486/STOP receptor were markedly impaired in their anchorage independence (Table 1). Inhibition ranged from 46â€”60%. This is quiteremarkable because C6 cells have abundant IGF-IRs and also produceabundant IGF-I (24, 26). Indeed, when wild-type C6 cells are seededwith the addition of their own CM, the number of colonies more thandoubles (second row of Table 1. Note also that in this case the numberof colonies was determined at 1 week), indicating that these cellsproduce growth factors that favor colony formation in soft agar.

C6 Cells Expressing the 486/STOP Receptor Undergo Apoptosis in Vivo. We have previously shown that the IGF-IR protects C6rat glioblastoma cells from apoptosis in vivo (26, 27). Antisensestrategies against the IGF-IR RNA caused C6 cells to undergo mas

4015



Table 2 C6 rat glioblastoma cells expressing the soluble IGF-IRundergoapoptosisinvivoApoptosis

was determined in vivo as described by Resnicoff et al. (26) and inâ€œMaterialsandMethods.â€•Ineachcase,5 X 10' cellswereinoculatedintothediffusionchamber,

which was then inserted into the s.c. tissue of BD IX rats. Cell numbersweredeterminedafter 24 h and are expressed as percentage of cells originallyinoculated.Clones

4â€”7were all stable transfectants of C6 cells, expressing the 486/STOPIGF-IR.Cell

line PercentrecoveryWild-type

C6 cells215Clone44Clone68Clone50.1Clonel

2

Jd b@@ OB 120 160 196 664 256

FL2

@T0 40 60 160 160 600 640 200

FL2

900@ *[email protected]?14600

700

600

500

400

300

200

100

CD

.

. Y

,u â€¢uiouiououuaio@uoFL2

. .- - -C700600500400300600

to:t


fusion chambers at 90 mm, 2 h, and 4 h after implantation in the s.c.

tissue of a rat. However, cells were also collected from diffusionchambers at 15, 30, 45, 60, and 90 mm and at 2, 4, 6, 8, 20, and 24 hafter insertion into the s.c. tissue of rats, so that Figs. 3B-D are simplyrepresentative of several determinations in time, each requiring 20single chambers (because of the small number of cells that wererecovered). A peak of apoptotic cells (Figs. 3B-D, to the reader's farleft) is clearly visible in cells recovered from the diffusion chamber,whereas cells in vitro do not show any detectable fraction of apoptoticcells (Fig. 3A). In this respect, too, C6 cells expressing the 486/STOPIGF-IR behave like C6 cells expressing an antisense RNA to theIGF-IR RNA (24, 26, 27). There was, however, a subtle difference inthe kinetics: with the antisense plasmid, the cells died massively in thefirst 3 h (26); whereas with the 486/STOP receptor, the cells diedmore slowly, giving rise to smaller apoptotic peaks at all timesexamined, although by 24 h, the results were essentially the same (seeTable 2, in which the extent of apoptosis is in the same range withcells expressing an antisense RNA to the IGF-IR RNA).

C6 Cells Expressing the 486/STOP IGF-IR Are No LongerTumorigenic. We reported previously that C6 cells stably transfectedwith a plasmid expressing an antisense RNA to the IGF-IR RNA donot form tumors when injected s.c. into syngeneic BD IX rats (24).Furthermore, rats injected with the antisense C6 cells become immuneto a subsequent challenge with wild-type C6 cells. Similar results havebeen obtained when C6 cells are treated with antisense ODN to theIGF-IR RNA (27). Wild-type C6 cells (or sense cells) form tumorspalpable within 4â€”5days after s.c. injection in naive syngeneic ratsand do not protect the animals from a subsequent challenge with otherwild-type C6 cells (24, 27).

Four clones of C6 cells expressing the soluble IGF-IR were injecteds.c. into BD IX rats (three animals/clone) and none of the animals

4,.0Ez

4,

0

C-4,.0E

z

4,0

sive apoptosis in vivo, whereas sense RNA or random oligodeoxynucleotides had no effect. We asked whether the expression of the486/STOP IGF-IR had any effect on the extent of apoptosis of C6cells placed in the diffusion chamber described by Resnicoff et a!. (26,27). The results are summarized in Table 2. As usual, wild-type C6cells grow very well in the diffusion chamber, more than doublingtheir number after 24 h in vivo. Four clones of C6 cells expressing thesoluble receptor were also tested in the diffusion chamber. All of themunderwent apoptosis, with the recovery ofcells ranging from 0.1â€”8%.As in previous reports, we observed the high sensitivity of this test,the extent of apoptosis in vivo being more dramatic than the inhibitionof growth in soft agar (26, 27). Repeated experiments gave essentiallythe same results.

Although apoptosis in C6 cells was demonstrated by several methods, we elected to show the results of cytofluorimetric analysis herebecause they show some intriguing differences from the results obtamed with cells stably transfected with a plasmid expressing anantisense RNA to the IGF-IR RNA (26). Fig. 3 gives an analysis of C6cells expressing the 486/STOP receptor and recovered from the dif

I..wE

z

w0

C..4,

E

z

4,0

1800 [email protected] VU I %[email protected] ?LZ 7

1600

I 400

1600

1000

600

600

400

600

A

Fig. 3. Detection of apoptosis by cytofluorimcoy. C6 cells stablytransfectedwiththeplasmidexpressing the 486/STOP IGF-IR. A, cells growingin monolayers before inoculation into the diffusionchambers. B. C, and D, cells after 90 mm, 2 h, and4 h. respectively, in the diffusion chamber insertedinto the s.c. tissue of a rat.

40 .o tan s.o 200 24@

FL2

1@Z4.@T FLZ 72*

4016



Cell type injectedChallengeTumors (no. ofanimals)Wild-type

486/STOP (4 clones)Wild-type486/STOP receptorNone

NoneWild-typeWild-type12/12

0/1214/140/I 2


developed tumors, whereas control animals injected with wild-typeC6 cells promptly developed tumors that brought about the death ofthe animals in 20â€”25days (Table 3). The animals injected with the C6cells expressing the 486/STOP receptor remained free of tumors for

more than 8 months. To test for the immune response, we had to goback to the animals of Table 2 (who were implanted with diffusionchambers loaded with either wild-type C6 cells or C6 cells expressingthe soluble receptor). All animals were tumor-free (because the chambers had been removed to count the surviving cells), and they werechallenged 2 weeks later with wild-type C6 cells. Of the animalsimplanted with chambers containing the C6/IGFIRSo1 cells, nonedeveloped tumors when challenged with wild-type C6 cells. Ratsimplanted with diffusion chambers containing wild-type C6 cellsdeveloped fatal tumors after s.c. injection with wild-type C6 cells(Table 3).

Autophosphorylation of IGF-IRs and Tyrosyl Phosphorylationof IRS-i in C6 Cells. We then determined the effect of 486/STOPreceptor expression on the autophosphorylation of the endogenousIGF-IRs in C6 cells. The results are shown in Fig. 4, where the gelswere purposely overexposed to detect the autophosphorylation of thereceptors in cells expressing the 486/STOP receptor (Lanes 1â€”4).Thus, the control lanes (Lanes 5 and 6) are grossly overexposed; witha shorter exposure, no bands at all would have been visible in Lanes1â€”4 (cells expressing the 486/STOP receptor). Clearly, the presence

of the soluble receptor caused a marked decrease in the extent ofautophosphorylation of the endogenous IGF-IRs of C6 cells. In allcases, the receptor was autophosphorylated, even in cells not incubated with IGF-I, again confirming that C6 cells produce substantialamounts of IGF-I (24). Similar results were obtained with IRS-i, amajor substrate of the IGF-IR (45â€”47),whose tyrosyl phosphorylation was decreased in clones expressing the 486/STOP receptor (datanot shown).

MAP Kinase Activity. Because MAP kinase is activated by eitherinsulin or IGF-I (48, 49), we measured MAP kinase activity in C6 and

A

â€˜50UC

â€˜50IL

: C6â€”0-â€” C6/SR#5

â€”â€˜-â€”C6/SR#63

2

Time after stimulation

B

lGFi0 15m 6h

C6@Ul@

C6/SR#5

C6/SR#6@

Fig. 5. Effect of IGF-I on MAP kinase activity of C6 cells and derived clones. MAPkinase activity was measured as described in â€œMaterialsand Methodsâ€•.B. MAP kinaseactivity of C6 cells and two clones expressing the 486/STOP receptor. A gives the dataobtained by densitometry measurements of the bands in B.

C6/IGFIRSoI cells. The results are shown in Fig. SB, in which theactivities shown are from cells in SFM and 15 mm or 6 h after

stimulation with IGF-I. The bands were also subjected to densitometry

measurement, and the results are given in Fig. 5A. All cell linesshowed a modest increase in MAP kinase activity at 15 mm, but only

in wild-type C6 cells did the activity continue to increase between 15mm and 6 h.

Effect of CM from R Cells on the Growth of p6 Cells. In thenext experiments, we wished to determine whether the biologicalactivity of the 486/STOP soluble receptor could be detected in the CM

of cells expressing it. We did not examine the CM of C6 cells because,as already mentioned, these cells produce many growth factors thatwould complicate any assay. We therefore transfected both R andBALB/c 3T3 cells with pIGFIRsol and determined the effect of themedium conditioned by these cells on the growth of p6 cells, which

are 3T3 cells overexpressing the wild-type human IGF-IR ( 17). CMfrom R or BALB/c 3T3 cells stably transfected with plasmid pIGFIRsol was collected as described in â€œMaterialsand Methods.â€•TheCM from both sources inhibited the growth of p6 cells in monolayers(data not shown).

The CM from the same sources was then used to study its effect oncolony formation in soft agar. When CM from several clones of R or

BALB/c 3T3 cells stably transfected with plasmid pIGFIRsol wasadded to the assay, it markedly inhibited colony formation, with theinhibition ranging from 47â€”86%(Table 4). CM from R cells (untransfected) had no inhibitory effect; in fact, if anything, it increasedthe number of colonies in soft agar. These experiments were repeatedseveral times, even using different cell types (data not shown).

Table 3 Expression of the soluble IGF-!R abrogates turnorigenesis by C@6cells

BD IX rats were injected s.c. with either wild-type C6 cells or C6 cells expressing the486/STOP IGF-IR. The latter animals were kept for 180 days without the appearance oftumors. In the second half of the experiment, we used rats that had been implanted witha diffusion chamber containing either wild-type C6 cells or C6 cells expressing the486/STOP receptor for 24 h (see Table 3). Two weeks after removal of the diffusionchamber, the rats were challenged with I X l0@ wild-type C6 cells.

123456kDa

199 â€”

120â€”@87â€”@ â€”3 subunit

48Fig. 4. Autophosphorylation of the IGF-IR in C6 cells and C6 cells expressing the

486/STOP receptor. Lysates from C6 cells and derived clones were immunoprecipitatedwith an antibody to the IGF-IR. and the gels were blotted with an anti-phosphotyrosineantibody (see â€˜Materialsand Methodsâ€•).Lanes 1 and 2, clone C6/SR6; Lanes 3 and 4,clone C6/SR5; Lanes 5 and 6, wild-type C6 cells. In each case, the second lane is a lysatefrom unstimulated cells, the first lane is from cells 5 mm after stimulation with IGF-I.

4017



Table 4 Effect of CMfrom R or Balb/c 3T3 cells expressing the soluble IGF-IRonsoftagar growth of transformedcellsp6

cells were seeded at a density of 1 X iO@cells/plate in 10% serum, without orwiththeCM from several clones of R cells or Balb/c 3T3 cells stably transfectedwithplasmid

pIGFIRsol. Colonies > 125 @xmin diameter were scored after 2weeks.Treatmentâ€•

No. of colonies in softagarNone

382, 350,281CMfromR

cells468CMfromClones

1, 5, 8, 10, 14 38, 21, 78, 38,56CMfromClones

A, B, C, D 84, 94. 90.78ClonesE, G, H, I 57, 68, 72,90ClonesM, 0, P 124, 60,59a

The numbered clones are R cells expressing the 486/STOP receptor; theletteredclones

are Balb/c 3T3 cells also expressing the 486/STOP receptor.


tion in the a domain, is at least in part secreted and can be found inthe medium, in which it could also act on neighboring cells. AlthoughPrager et a!. (31) showed that their truncated receptor inhibited

tumorigenesis in nude mice, they did not test for apoptosis, nor couldthey test for the host response elicited in animals when the IGF-IRfunction is targeted.

Our soluble receptor and the 952-truncated receptor of Prager et a!.(31) are presently the only dominant negatives of the IGF-IR that areeffective in inhibiting tumorigenesis; ours by inducing apoptosis,theirs presumably for the same reason. Several other mutants of theIGF-IR have been tested: the ATP-binding site mutant (12, 50), theY950F mutant (42), the triple tyrosine mutant (32), and the Y125IFand Y1250F mutants (14). All of them partially inhibit growth in bothmonolayer and soft agar, thus acting as dominant negatives, but noneof them induced apoptosis (33). A possible explanation for thisdifference is that the other receptors (mutant but of correct size) canform hybrids with endogenous receptors and, especially, can transactivate or be transactivated intermolecularly or intramolecularly (51â€”55). Indeed, Burgaud and Baserga (56) have shown that even inactive

IGF-IRs can be transactivated intermolecularly by other growth factorreceptors, which thus mimick the effect of the IGF-IR. The size of the486/STOP receptor does not preclude, theoretically, some hybridformation with the a domain of the IGF-IR, but clearly it could nottransactivate or be transactivated because it completely lacks theintracellular signaling domains.

C6/IGFIRSo1 cells have a markedly decreased autophosphorylationof their endogenous receptors and a decreased tyrosyl phosphorylationof IRS-i , and IGF-I-mediated stimulation of MAP kinase activity isalso impaired. These findings are not unexpected because both IRS- 1and the MAP kinases are required for fibroblast proliferation (57â€”59)and transformation (60, 61), which are inhibited by 486/STOP. It isalso interesting that it is the delayed increase in MAP kinase activitythat is impaired in cells expressing the 486/STOP receptor, becausethis delayed increase has been more specifically associated with

mitogenesis than the early time increase (see review by Marshall, Ref.62). Because MAP kinase activity is toward the distal end of the ras

pathway (63, 64), we assume that the intermediate steps between thereceptor itself and MAP kinase are also inhibited. Thus, it seems thatthe presence of the soluble receptor effectively inhibits most, if not all,biological functions of the endogenous IGF-IRs as well as its signaling. A reasonable interpretation is that the 486/STOP receptor acts asa dominant negative in a classic way; i.e., by competing for thebinding of IGF-I to the endogenous receptors. The 481 wild-typeamino acids of the soluble receptor comprise what is generally believed to be the binding domains of the IGF-IR for IGF-I (65, 66).According to Schumacher et a!. (66), the IGF-I binding domain in thea subunit of the human IGF-IR roughly localizes between residues

131 and 315 (corresponding to the cysteine-rich region), well withinthe sequence of our receptor. We suspect that some of the receptor is

kDa 1 2 3

5Oâ€”@

We then tested for the presence of the soluble receptor in themedium by labeling the cells with [35S]methionine (see â€œMaterialsand Methodsâ€•)and immunoprecipitating the CM with an antibody tothe a subunit of the IGF-IR. The results from a typical experiment areshown in Fig. 6, in which a band is visible in the CM of cellsexpressing the 486/STOP receptor (Lane 1), which has the expectedsize, and is absent from the CM of cells not expressing the 486/STOPreceptor (Lane 3). A faint band is also visible in Lane 2, which is CMfrom parental cells. Repeated attempts to coprecipitate the 486/STOPreceptor with the endogenous receptors of cells with a physiologicalnumber of IGF-IRs have failed (data not shown). We cannot saywhether this failure means that the 486/STOP receptor does not makehybrids with the endogenous receptors or if it may be due simply tothe sensitivity of the assay.

DISCUSSION

By using a frame-shift mutation, we have engineered an IGF-IRcDNA that produces a full-length mRNA but a truncated receptor,which is (after cleavage of the signal peptide) 486 amino acids long.We adopted this strategy because truncated mRNAs are often highlyunstable. This truncated receptor is designated here as the 486/STOPreceptor, although the correct sequence of the human IGF-IR actuallystops at residue 481 . The size of the receptor has been ascertained byin vitro translation and was confirmed by a pGEX construct (data notshown). This receptor is, at least partially, secreted into the medium(Fig. 6), in which its biological activity can be detected. Attempts todetermine whether the soluble receptor may form hybrids with theendogenous receptor have failed, without ruling out the possibility.

The 486/STOP IGF-IR has a dramatic effect on the cells thatexpress it. C6 cells stably expressing the 486/STOP IGF-IR changemorphology, grow slowly even in 10% serum, and are stronglyinhibited in an assay testing for IGF-I-mediated mitogenesis. TheseC6 cells also form fewer colonies in soft agar, undergo massiveapoptosis in vivo, and are no longer tumorigenic in syngeneic rats. Inthis respect, C6 cells expressing the 486/STOP IGF-IR behave like C6cells expressing an antisense RNA to the IGF-IR RNA (24, 26).Another characteristic of cells expressing an antisense RNA to theIGF-IR and undergoing apoptosis in vivo is to elicit a host responsethat protects the animals from subsequent challenge with wild-type C6cells. The C6IIGFIRSo1 cells also protect syngeneic rats from subsequent challenge with wild-type C6 cells. Thus, biologically, the 486/STOP IGF-IR acts as a strong dominant negative, as effective as anantisense expression plasmid. A similar dominant negative of the

IGF-IR was described by Prager et al. (31). However, their receptorwas truncated at residue 952 (iuxtamembrane domain) and was therefore anchored to the cellular membrane. Our receptor, with a trunca

Fig. 6. Detection of the 486/STOP receptor in CM. The preparation of CM from 3T3cells labeled with [35Slmethionine is described in â€œMaterialsand Methods.â€•The concentrated CM was precipitated with an antibody to the a subunit of the IGF-IR. Lane 1, CMfrom cells expressing the 486/STOP receptor; Lanes 2 and 3, CM from parental cells. Themolecular weight of the 486/STOP receptor is indicated in the figure.

4018




secreted free into the medium, but some may somehow remain firmlyattached to the membrane.

There are, however, alternative explanations. One can speculate thatthe 486/STOP receptor may somehow change the conformational statusof the endogenous IGF-IRs and thus render them insensitive to stimulation by IGF-I. The transforming receptor tyrosine kinase AxI can apparently modulate its own activity by proteolytic cleavage of the wild-typereceptor that generates a soluble extracellular domain (67).

Although an intact, soluble IR has been described (68), the 486/STOP receptor is an artificial receptor that offers some interestingfeatures. It inhibits growth but causes extensive apoptosis of tumorcells, therefore inhibiting tumorigenesis. If the hypothesis put forwardby Baserga (3) that targeting of the IGF-IR only inhibits normal cellsbut causes apoptosis of tumor cells is correct, our soluble receptorwould be extremely useful because it would not have to be deliveredto all cells â€”not only because, being secreted, it could exert its effecton neighboring cells (bystander effect), but also because of the peculiarity of cells with functionally impaired IGF-IRs to elicit a hostresponse that seemingly takes care of the survivors (24, 27). This hostresponse, which is being characterized, is rather striking because itconfers complete immunity to the subsequent challenge with wildtype C6 cells, and it occurs every time the IGF-IR function is targeted,regardless of whether we use an antisense expression plasmid, anantisense oligodeoxynucleotide, or a dominant negative, as in thepresent investigation.

In conclusion, we have engineered an IGF-IR that is secreted insufficient amounts to exert remarkable biological activities that indude the inhibition of signaling and cell growth, reversal of thetransformed phenotype (colony formation in soft agar), induction ofmassive apoptosis in vivo, and inhibition of tumorigenesis in rats. Itsuse for basic studies should not be underestimated either, because itoffers some advantages over antibodies to the receptor, which sometimes inhibit and sometimes stimulate, and antisense strategies, whichsometimes are not target-specific.

REFERENCES

1. Ullrich, A., and Schlessinger. J. Signal transduction by receptors with tyrosine kinaseactivity. Cell, 61: 203-212, 1990.

2. Ullrich, A., Gray, A., Tam, A. W., Yang-Feng, T., Tsubokawa, M., Collins,C., Henzel, W., Le Bon, T., Kahuria, S., Chen, E., Jakobs, S., Francke, U.,Ramachandran, J., and Fujita-Yamaguchi, Y. Insulin-like growth factor I receptorprimary structure: comparison with insulin receptor suggests structural determinantsthat define functional specificity. EMBO J., 5: 2503â€”2512, 1986.

3. Baserga, R. The insulin-like growth factor I receptor: a key to tumor growth? CancerRes., 55: 249â€”252, 1995.

4. Scher, C. D., Shephard, R. C., Antoniades, H. N., and Stiles, C. D. Platelet-derivedgrowth factor and the regulation of the mammalian fibroblasts cell cycles. Biochim.Biophys. Acta, 560: 217â€”241,1979.

5. Stiles, C. D., Capone, G. T., Scher, C. D., Antoniades, N. H., Van Wyk, J. J., andPledger, W. J. Dual control of cell growth by somatomedins and platelet-derivedgrowth factor. Proc. Natl. Acad. Sci. USA, 76: 1279â€”1283, 1979.

6. Leof,E.B.,Wharton,W.,VanWyk,J. J.,andPledger,W.J. Epidermalgrowthfactor(EGF) and somatomedin C-regulated G1 progression in competent BALB/c3T3 cells.Exp. Cell Res., 141: 107â€”115,1982.

7. Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J., and Efstratiadis, A. Mice carryingnull mutations of the genes encoding insulin-like growth factor I (Igf-1) and type IIGF receptor (Igflr). Cell, 75: 59â€”72,1993.

8. Baker, J., Liu, J. P., Robertson, E. J., and Efstratiadis, A. Role of insulin-like growthfactors in embryonic and postnatal growth. Cell, 75: 73â€”82,1993.

9. Sell, C., Dumenil, G., Deveaud, C., Miura, M., Coppola, D., DeAngelis, T., Rubin, R.,Efstratiadis, A., and Baserga, R. Effect of a null mutation of the type 1 IGF receptorgene on growth and transformation of mouse embryo fibroblasts. Mol. Cell. Biol., 14:3604â€”3612,1994.

10. Sell, C., Rubini, M., Rubin, R., Liu, J. P., Efstratiadis, A., and Baserga, R. Simianvirus 40 large tumor antigen is unable to transform mouse embryonic fibroblastslacking type-1 IGF receptor. Proc. Natl. Acad. Sci. USA, 90: 11217â€”11221,1993.

11. Morrione, A., DeAngelis, T., and Baserga, R. Failure of the bovine papilloma virusto transform mouse embryo fibroblasts with a targeted disruption of the insulin-likegrowth factor I receptor genes. J. Virol., 69: 5300â€”5303,1995.

12. Coppola, D., Ferber, A., Miura, M., Sell, C., D'Ambrosio, C., Rubin, R., and Baserga,R. A functional IGF-I receptor is required for the mitogenic and transforming

activities of the epidermal growth factor receptor. Mol. Cell. Biol.. 14: 4588â€”4595,I994.

13. DeAngelis, T., Ferber, A.. and Baserga, R. Insulin-like growth factor I receptor isrequired for the mitogenic and transforming activities of the platelet-derived growthfactor receptor. J. Cell. Physiol., 164: 214â€”221, 1995.

14. Miura, M., Surmacz, E., Burgaud, J. L., and Baserga, R. Different effects of mitogenesis and transformation of a mutation at tyrosine 125 1 of the insulin-like growthfactor I receptor. J. Biol. Chem.. 270: 22639â€”22644, 1995.

15. Kaleko, M., Rutter. W. G., and Miller, A. D. Overexpression of the human insulinlike growth factor 1 receptor promotes ligand-dependent neoplastic transformation.Mol. Cell. Biol., 10: 464â€”473,1990.

16. McCubrey, J. A., Stillman, L. S., Mayhew, M. W., Algate. P. A.. Dellow, R. A.. andKaleko, M. Growth promoting effects of insulin-like growth factor I (IGF-l) onhematopoietic cells: overexpression of introduced IGF-1 receptor abrogates interleukin-3 dependency of murine factor dependent cells by ligand dependent mechanism.Blood,78:921â€”929,1991.

17. Pietrzkowski, Z., Lammers, R., Carpenter, G., Soderquist. A. M., Limardo, M.,Phillips, P. D., Ullrich, A., and Baserga, R. Constitutive expression of IGF-l andIGF-l receptor abrogates all requirements for exogenous growth factors. Cell Growth& Differ., 3: 199â€”205,1992.

18. Pietrzkowski, Z., Sell, C., Lammers, C. R., UIlrich, A., and Baserga, R. The role ofinsulin like growth factor 1 (IGF-I) and the IGF- I receptor in epidermal growthfactor-stimulated growth of 3T3 cells. Mol. Cell. Biol., 12: 3883â€”3889, 1992.

19. Liu, D., Zong, C., and Wang, L. H. Distinctive effects of the carboxyl-terminalsequence of the insulin-like growth factor I receptor on its signaling functions.J.Virol.,67:6835â€”6840,1993.

20. Surmacz, E., Sell, C., Swantek, J., Kato, H., Roberts, C. T., Jr., LeRoith, D., andBaserga. R. Dissociation of mitogenesis and transforming activity by COOH-terminaltruncation of the insulin-like growth factor I receptor. Exp. Cell Res., 218: 370â€”380,1995.

21 . Christophori, G., Naik, P., and Hanahan, D. A second signal supplied by insulin-likegrowth factor II in oncogene-induced tumorigenesis. Nature (Lond.), 369: 414â€”418.1994.

22. Rogler, C. E., Yang, D., Rossetti, L., Donohoe, J., Alt, E., Chang, C. J., Rosenfeld,R., Neely, K., and Hintz, R. Altered body composition and increased frequency ofdiverse malignancies in insulin-like growth factor II transgenic mice. J. Biol. Chem.,269:13779â€”13784,1994.

23. Trojan. J.. Johnson, T. R., Rudin, S. D., Han, J., Tykocinski. M. L.. and Ilan. I. Treatmentand prevention ofrat glioblastoma by immunogenic C6 cells expressing antisense insulinlike growth factor I RNA. Science (Washington DC), 259: 94â€”97,1993.

24. Resnicoff, M., Sell, C., Rubini, M., Coppola, D., Ambrose, D., Baserga. R., andRubin, R. Rat glioblastoma cells expressing an antisense RNA to the insulin-likegrowth factor I (IGF-t) receptor are nontumorigenic and induce regression of wildtype tumors. Cancer Res., 54: 2218â€”2222, 1994.

25. Resnicoff, M., Coppola, D., Sell, C., Rubin, R., Ferrone, S., and Baserga, R. Growthinhibition of human melanoma cells in nude mice by antisense strategies to the typeI insulin-like growth factor receptor. Cancer Res., 54: 4848â€”4850, 1994.

26. Resnicoff, M., Abraham, D., Yutanawiboonchai, W., Rotman, H. L., Kajstura, J.,Rubin, R., Zoltick, P., and Baserga, R. The insulin-like growth factor I receptorprotects tumor cells from apoptosis in vito. Cancer Res., 55: 2463â€”2469, 1995.

27. Resnicoff, M., Burgaud. J. L., Rotman, H. L., Abraham, D.. and Baserga, R.Correlation between apoptosis, tumorigenesis, and levels of insulin-like growth factorI receptors. Cancer Res., 55: 3739â€”3741, 1995.

28. Shapiro, D. N., Jones, B. G., Shapiro, L. H., Dias, P., and Houghton. P. J. Antisensemediated reduction in insulin-like growth factor I receptor expression suppresses themalignant phenotype of a human alveolar rhabdomyosarcoma. J. Clin. Invest., 94:1235â€”1242,1994.

29. Arteaga. C. L., and Osbome, C. K. Growth inhibition of human breast cancer cells invitro with an antibody against the type I somatomedin receptor. Cancer Res., 49:6237â€”6241,1989.

30. Kalebic, T., Tsokos, M., and Helman, L. J. In vivo treatment with antibody against theIGF-I receptor suppresses growth of human rhabdomyosarcoma and down-regulatesp34/cdc2.CancerRes.,54:5531â€”5534,1994.

31. Prager, D., Li, H. L., Asa, S.. and Melmed, S. Dominant negative inhibition oftumorigenesis in vis'o by human insulin-like growth factor I receptor mutant. Proc.Natl. Acad. Sci. USA, 91: 2181â€”2185, 1994.

32. Li, S., Ferber, A., Miura, M., and Baserga. R. Mitogenicity and transforming activityof the insulin-like growth factor I receptor with mutations in the tyrosine kinasedomain. J. Biol. Chem., 269: 32558â€”32564, 1994.

33. Burgaud. J. L.. Resnicoff, M., and Baserga, R. Mutant IGF-I receptors as dominantnegatives for growth and transformation. Biochem. Biophys. Res. Commun., 214:475â€”481, 1995.

34. Long, L., Rubin, R., Baserga, R., and Brodt, P. Loss of the metastatic phenotype inmurine carcinoma cells expressing an antisense RNA to the insulin-like growth factorI receptor. Cancer Res., 55: 1006â€”1009, 1995.

35. Rodriguez-Tarduchy. G.. Collins, M. K. L., Garcia, I., and Lopez-Rivas. A. Insulinlike growth factor I inhibits apoptosis in IL-3- dependent hemopoietic cells. J. Immunol., 149: 535â€”540,1992.

36. Gluckman, P., Klempt, N., Guan, J., Mallard, C., Sirimanne. E., Dragunow, M.,Klempt, M.. Singh, K.. Williams, C., and Nikolics, K. A role for IGF-I in the rescueof CNS neurons following hypoxic-ischemic injury. Biochem. Biophys. Res. Commun.,182:593â€”599,1992.

37. D'Mello, S. R., Galli, C., Ciotti, T., and Calissano, P. Induction of apoptosis incerebellar granule neurons by low potassium: inhibition of death by insulin-likegrowth factor I and cAMP. Proc. NatI. Acad. Sci. USA, 90: 10989â€”10993. 1993.

38. Harrington, E. A., Bennett, M. R., Fanidi, A., and Evan, G. I. c-mvc-induced apoptosis

4019




in fibroblasts is inhibited by specific cytokines. EMBO J., 14: 3286â€”3295,1994.39. Sell,C.,Baserga,R.,andRubin,R.Insulin-likegrowthfactorI (IGF-I)andtheIGF-I

receptor prevent etoposide-induced apoptosis. Cancer Res., 55: 303â€”306,1995.40. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid

guanidium thiocyanate-phenol chloroform extraction. Anal. Biochem., 162: 156â€”159,1987.

41. Vara, J. A., Portela, A., and Jimenez, A. Expression in mammalian cells of a genefrom Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res.,14: 4617â€”4624, 1986.

42. Miura, M., Li, S., and Baserga, R. Effect of a mutation of tyrosine 950 of theinsulin-like growth factor I receptor on the growth and transformation of cells. CancerRes., 55: 663â€”667.1995.

43. Cook, S. J., Rubinfeld, B., Albert, I., and McCormick, F. RapVl2 antagonizesRas-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-I fibroblasts.EMBO J., 12: 3475â€”3485.1993.

44. Reiss, K., Porcu, P., Sell, C., Pietrzkowski, Z., and Baserga, R. The insulin-likegrowth factor I receptor is required for the proliferation of hemopoietic cells. Oncogene, 7: 2243â€”2248, 1992.

45. White, M. F., and Kahn, C. R. The insulin signaling system. J. Biol. Chem., 269: 1â€”4,1994.

46. Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEYmotif of the insulin receptor via a novel non-SH2 domain. Mol. Cell. Biol., 15:2500â€”2508,1995.

47. Tartare-Deckert, S., Sawka-Verhelle, D., Murdaca, J., and Van Obberghen, E. Evidence for a differential interaction of She and the insulin receptor substrate I (IRS-l)with the insulin-like growth factor I (IGF-I) receptor in the yeast two-hybrid system.J. Biol. Chem., 270: 23456â€”23460,1995.

48. Cahill, A. L., and Pearlman, R. L. Activation of a microtubule-associated protein-2kinase by insulin-like growth factor I in bovine chromaffin cells. J. Neurochem., 57:1832â€”1839,1991.

49. Webster, J., Prager, D., and Melmed, S. Insulin-like growth factor 1 activation ofextracellular signal-related kinase-l and -2 in growth hormone-secreting cells. Mol.Endocrinol., 8: 539â€”544, 1994.

50. Kato, H., Faria, T. N., Stannard, B., Roberts, C. T., Jr., and LeRoith, D. Role of thetyrosine kinase domain in signal transduction by the insulin-like growth factor 1IGF- 1 receptor. J. Biol. Chem., 268: 2655â€”2661, 1993.

51. Ballotti, R., Lammers, R., Scimeca, J. C., Dull, T., Schlessinger, J., Ullrich, A., andVan Obberghen, E. Intermolecular transphosphorylation between insulin receptorsand EGF-insulin receptor chimerae. EMBO J., 8: 3303â€”3309, 1989.

52. Lammers, R., Van Obberghen, E., Ballotti, R., Schlessinger, J., and Ullrich, A.Transphosphorylation as a possible mechanism for insulin and epidermal growthfactor receptor activation. J. Biol. Chem., 265: 16886â€”16890, 1990.

53. Taouis, M., Levy-Toledano, R., Roach, P., Taylor, S. L., and Gorden, P. Rescue and

activation of a binding-deficient insulin receptor. J. Biol. Chem., 269: 27762â€”27766,1994.

54. Takata, Y., and Kobayashi. M. Insulin-like growth factor I signaling through heterodimers of insulin and insulin-like growth factor I receptors. Diabete & Metab., 20:31â€”36,1994.

55. Chantry, A. The kinase domain and membrane localization determine intracellularinteractions between epidermal growth factor receptors. J. Biol. Chem., 270: 3068â€”3073,1995.

56. Burgaud, J. L., and Baserga. R. Intracellular transactivation of the insulin-like growthfactor I receptor by an epidermal growth factor receptor. Exp. Cell Res., in press,1996.

57. Waters, S. B., Yamauchi, K., and Pessin. J. E. Functional expression of insulinreceptor substrate- I is required for insulin-stimulated mitogenic signaling. J. Biol.Chem., 268: 22231â€”22234,1993.

58. Rose, D. W., Satiel, A. R.. Majumdar. M.. Decker, S. J., and Olefsky, J. M. Insulinreceptor substrate I is required for insulin-mediated mitogenic signal transduction.Proc. NatI. Acad. Sci. USA. 91: 797â€”801.1994.

59. Pages, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., andPouyssegur, J. Mitogen-activated protein kinases p42/mapk and p44/mpk are requiredfor fibroblast proliferation. Proc. Nail. Acad. Sci. USA, 90: 8319â€”8323,1993.

60. Cowley, S., Peterson, H., Kemp. P., and Marshall, C. J. Activation of MAP kinasekinase is necessary and sufficient for PCI2 differentiation and for transformation ofNIH 3T3 cells. Cell, 77: 841â€”852, 1994.

61. D'Ambrosio, C., Keller. S. R., Morrione. A., Lienhard. G. E., Baserga. R., andSurmacz, E. Transforming potential of the insulin receptor substrate 1. Cell Growth& Differ.,6: 557â€”562,1995.

62. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versussustained extracellular signal-regulated kinase activation. Cell, 80: 179 â€”I85,1995.

63. Blenis, J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc.Natl. Acad. Sci. USA, 90: 5889â€”5892. 1993.

64. McCormick, F. How receptors turn Ras on. Nature (Lond.), 363: 15â€”16.1993.65. Andersen, A. S., Kjeldsen, T., Wiberg, F. C., Christensen, P. M., Rasmussen, J. S., Norris,

K., Moller, K. B., and Moller, N. P. H. Changing the insulin receptor to possessinsulin-like growth factor I ligand specificity. Biochemistry, 29: 7363â€”7366.1990.

66. Schumacher, R., Soos, M. A., Schlessinger, J.. Brandenburg, D., Siddle, K., andUllrich, A. Signaling-competent receptor chimeras allow mapping of major insulinreceptor binding domain determinants. J. Biol. Chem., 268: 1087â€”1094,1993.

67. O'Bryan, J. P., Fridell, Y. W., Koski, R., Vamum, B., and Liu, E. T. The transformingreceptor tyrosine kinase, AxI, is post-translationally regulated by proteolytic cleavage.J. Biol. Chem., 270: 551â€”557,1995.

68. Papa, V., Russo, P., Gliozzo, B., Goldfine, I. D., Vigneri, R., and Pezzino, V. Anintact and functional soluble form of the insulin receptor is secreted by cultured cells.Endocrinology, 133: 1369â€”1376, 1993.

4020



1996;56:4013-4020. Cancer Res Consuelo D'Ambrosio, Andres Ferber, Mariana Resnicoff, et al.

and Inhibits Tumorigenesisin VivoApoptosis of Tumor Cells A Soluble Insulin-like Growth Factor I Receptor That Induces

Updated version

http://cancerres.aacrjournals.org/content/56/17/4013

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/56/17/4013To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



a soluble insulin-like growth factor i receptor that ... · (3 1), as 486/stop, whereas the...

Documents