Proc. Natl. Acad. Sci. USAVol. 85, pp. 8032-8036, November 1988Cell Biology

Replacement of insulin receptor tyrosine residues 1162 and 1163does not alter the mitogenic effect of the hormone

(DNA synthesis/autophosphorylation/tyrosine kinase/S6 kinase activation)

ANNE DEBANT*, ERIC CLAUSERt, GILLES PONZIO*, CHANTAL FILLOUX*, COLETTE AUZANt,JEAN-OLIVIER CONTRERES*, AND BERNARD Rossi**Hormones Polypeptidiques et Physiopathologie Endocrinienne, Institut National de la Sante et de la Recherche Mddicale U145, Facult6 de Mddecine, Avenuede Valombrose 06034, Nice CUdex, France; and tPathologie Vasculaire et Endocrinologie Rdnale, Institut National de la Sante et de la Recherche MddicaleU36, 17 rue du Fer-A-Moulin 75005, Paris, France

Communicated by Alfred Jost, May 31, 1988

ABSTRACT Chinese hamster ovary transfectants thatexpress insulin receptors in which tyrosine residues 1162 and1163 were replaced by phenylalanine exhibit a total inhibitionof the insulin-mediated tyrosine kinase activity toward exoge-nous substrates [histone, casein, and poly(Glu/Tyr)]; thislatter activity is associated with total inhibition of the hyper-sensitivity reported for insulin in promoting 2-deoxyglucoseuptake. We now present evidence that the twin tyrosines alsocontrol the insulin-mediated stimulation of glycogen synthesis.Surprisingly, this type of Chinese hamster ovary transfectantis as hypersensitive to insulin for its mitogenic effect as areChinese hamster ovary cells expressing many intact insulinreceptors. Such data suggest that (i) the insulin mitogenic effectroutes through a different pathway than insulin uses to activatethe transport and metabolism of glucose and (ii) the mitogeniceffect of insulin is not controlled by the twin tyrosines. At themolecular level, the solubilized mutated receptor has noinsulin-dependent tyrosine kinase activity, whereas this recep-tor displays measurable insulin-stimulated phosphorylation ofits /3 subunit in 32P-labeled cells. We therefore propose that theautocatalytic phosphorylating activity of the receptor reports acryptic tyrosine kinase activity that cannot be visualized by theuse of classical exogenous substrates.

Binding of insulin to its receptor induces the autophospho-rylation of the 03 subunit predominantly at tyrosine residuesin solubilized receptor and at tyrosine, threonine, and serineresidues in intact cells (1-3). Autophosphorylation of the ,3subunit simultaneously activates tyrosine kinase activitytoward exogenous (1-3) and endogenous substrates (3). Thetyrosine kinase activity in several protooncogenes and incertain receptors of growth factors such as epidermal growthfactor or platelet-derived growth factor (4) suggests that thistype of enzymatic activity is involved in the control of cellgrowth. Moreover, Livneh et al. (5) showed recently that amutated form of epidermal growth factor receptor devoid ofkinase activity could not generate a mitogenic growth factorresponse. Sequencing of the human insulin receptor (hIR)cDNA has provided information about the receptor primarystructure (6, 7). Notably, the tyrosine residues in positions1162 and 1163 behave as a major site of phosphorylation,probably owing to their conserved position among variousoncogene-encoded protein and/or growth factor receptorscontaining a tyrosine kinase domain (8). Using site-directedmutagenesis, Ellis et al. (9) established the pivotail role oftyrosines 1162 and 1163 in the control of kinase activityrelated to the stimulation ofdeoxyglucose transport. We usedthe same kind of Chinese hamster ovary (CHO) transfectantsin this study to determine the effects of replacing the twin

tyrosines on the insulin mitogenic effect. Concomitantly, weinvestigated the alterations induced by this mutation inreceptor autophosphorylation and consecutive tyrosine ki-nase activation in response to insulin.

MATERIALS AND METHODSThe wild type (peT) and the mutated (peYF3) hIR cDNAexpression plasmids were the same as those described byEllis et al. (9). Each type of plasmid was introduced into CHOcells (CHO-Kl; Flow Laboratories) by cotransfection withplasmid pSV2-neo, as described (9, 10). Single colonies ofprimary G418-resistant transformants were assayed for theexpression of the hIR using RNA blot analysis after extrac-tion of total RNA as described (11). Pure cell lines wereobtained from positive primary transformants by the limiting-dilution subcloning technique and then screened back for theexpression of hIR by RNA dot blot. Insertion of hIRmolecules into the plasma membrane was visualized byindirect immunofluorescence using a rabbit anti-insulin re-ceptor antibody (12). Studies of insulin binding to culturedcells, receptor autophosphorylation, kinase activity assays,and tryptic fragments of the hIR were done as described (12).The metabolic effects and the thymidine incorporation intoDNA were assayed as described (refs. 13 and 14, respec-tively). The phosphorylation of the ribosomal protein S6 wascarried out as described by Nilsen-Hamilton et al. (15) withslight modifications. Cells were loaded with [32P]orthophos-phate for 90 min at 37°C before 1 nM insulin was added for15 min at 37°C. Cells were then solubilized in a buffercontaining 1% Nonidet P-40/1% deoxycholate. The sampleswere subjected to a first centrifugation (3000 x g for 10 min),and then the supernatants were centrifuged at 120,000 x g for10 min at 4°C. The pellets were subjected to acid extraction4s described (16). The ribosomal proteins in the supernatantwere precipitated, and the pellets were resuspended in abuffer containing urea before being subjected to a two-dimensional electrophoresis (16).

RESULTSThree kinds of CHO cell lines were used in this study: (i) theparental cell line (CHO); (ii) the CHO cell line transfectedwith a plasmid coding for the native form ofthe hIR (CHO-R),which is similar to the CHO-T cell line described by Ellis etal. (9); and (iii) the CHO cell line expressing hIRs in whichthe twin tyrosines in position 1162 and 1163 were replacedwith phenylalanine residues by directed mutagenesis (CHO-Y2), corresponding to the cell line CHO-YF3 in the Rutter'sgroup nomenclature (9).

Abbreviations: hIR, human insulin receptor; CHO, Chinese hamsterovary; FCS, fetal calf serum.

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'2sI-Labeled Insulin Binding on CHO Transfectants. Thecharacterization of exogenous human receptors expressed intransfected CHO cells was accomplished by measuring thebinding of '25I-labeled insulin on the two transfected CHOcell lines. As indicated in Table 1, the human receptorsexpressed in CHO-R and CHO-Y2 cells bind insulin withcomparable affinities. The apparent dissociation constantsfor insulin were 0.8 nM and 0.4 nM as determined on CHO-Rand CHO-Y2, respectively. Furthermore, the two transfect-ed cells expressed approximately the same amount of high-affinity insulin receptors per cell: 8 x 105 sites (CHO-R) and5 x 105 sites (CHO-Y2) compared with 3 x 103 sitesexpressed in a parental CHO cell (17). These data are inaccord with those reported previously by Ellis et al. (9) onCHO cells transfected with identical constructions.

Effect of Twin Tyrosine Mutation on Deoxyglucose Uptakeand Glycogen Synthesis. The effect of Tyr-1162 and Tyr-1163mutation on insulin-stimulated glucose permeability wasassayed by measuring 2-[3H]deoxyglucose transport at var-ious concentrations of insulin in CHO-R or CHO-Y2 or theparental CHO cells. As shown in Fig. 1, the basal transportofglucose was similar for the three CHO cell lines. However,in the CHO-R cell line the expression of -8 x 105 humanintact receptors per cell conferred an increased sensitivity forinsulin (EC50 = 30 pM versus EC50 = 2 nM for parental CHOcells). Furthermore, the maximal stimulatory effect seen atsaturating concentrations of insulin was greater on CHO-Rcells (4-fold) than on parental CHO cells (2-fold). On the otherhand, CHO-Y2, which expressed equivalent amounts ofinsulin receptors mutated on the twin tyrosine position,presented an insulin dose-response curve for 2-[VH]deoxy-glucose transport more comparable to that of parental CHO(EC50 = 30 nM; maximal stimulation, 1.7-fold). Similar to thedata on deoxyglucose uptake, CHO-R cells were much moresensitive toward insulin stimulation of glycogen synthesis(EC50 = 0.2 nM) as compared with parental CHO or CHO-Y2cells (Fig. 2). These data indicate that mutation in the twintyrosine domain prevents the mediation of insulin-stimulatedglycogen synthesis, similar to that seen for deoxyglucoseuptake.Tyr-1162 and Tyr-1163 Do Not Control Insulin-Stimulated

DNA Synthesis. To determine whether the twin tyrosines alsoparticipate in the control ofthe long-term effect of insulin, wecompared the insulin response curves for DNA-synthesisreinitiation measured on parental CHO, CHO-R, and CHO-Y2 cell lines (Fig. 3). In a few cases insulin has been reportedto increase thymidine incorporation into DNA at physiolog-ical concentrations by interacting with its own receptors (18,19). This is not so for CHO cells in which the insulin EC50 forDNA synthesis occurred at 10 nM. However, introduction ofmany exogenous insulin receptors into CHO cells increasedinsulin sensitivity markedly to promote [3H]thymidine incor-poration into DNA, as indicated by an EC50 value of only 50pM in CHO-R cells. Most surprisingly, the dose-responsecurve for CHO-Y2 was superimposable to that obtained withCHO-R cells and was clearly distinct from that generatedfrom the parental CHO cell line data. These results indicatethat the insulin-stimulated incorporation of thymidine intoDNA does not depend on the presence of tyrosines 1162 and1163 on the /3 subunit.

Table 1. Insulin binding parameters on CHO-R andCHO-Y2 cells

Cell type Kd, nM Bmax, sites per cell

CHO-R 0.8 8 x 105CHO-Y2 0.4 5 x 10-

The Scatchard analysis was used to calculate these bindingparameters.

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of 2-[3H]deoxyglucose was measured in CHO-R (e), CHO-Y2 (.),and parental CHO (o) cells for 10 min after a 30-min incubation withvarious insulin concentrations.

[3H]Thymidine Incorporation Under Insulin StimulationReflects DNA Replication. To ascertain that our assay on[3H]thymidine incorporation truly reflected DNA replicationduring the S phase and not DNA repair, we checked that thisparameter was cell-cycle dependent. As shown in Fig. 4,when CHO-R and CHO-Y2 were incubated with 100 nMinsulin or 10% fetal calf serum (FCS), the two CHO-transfected cells presented the same peak in [3H]thymidineincorporation after 14 hr of incubation. This lag periodcorresponds to progression of CHO cells to S phase asobserved in the presence of 10% FCS. In addition, cellsexposed to insulin could also complete their mitotic cycle as

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FIG. 2. Insulin-stimulated [14C]glucose incorporation into glyco-gen. CHO-R (e), CHO-Y2 (.), and parental CHO (o) cells wereincubated with the indicated insulin (Ins) concentrations for 1 hr.Then, [14C]glucose was added, and the incubation was resumed for3 hr. Glucose incorporated into glycogen was measured as describedelsewhere (13).

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5-.4z0

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DNA. CHO-R (o), CHO-Y2 (.), and parental CHO (o) cells weredepleted in serum-free Ham's F-12 medium for 32 hr. Indicatedinsulin concentrations were added, and the incubation was continuedfor 14 hr before a 45-min pulse with 1 ,uCi of [3H]thymidine (1 Ci =

37 GBq).

assayed by following cell proliferation. The data presented inTable 2 show that 10% FCS promoted cell growth of CHO(generation time 13 hr) and both CHO-R and CHO-Y2(generation time 24 hr). Similarly, 1 nM insulin acted as apotent growth factor on the two CHO-transfected cell linesexpressing a large amount of intact or mutated receptors; thegeneration time was 24 hr and 30 hr for CHO-Y2 and CHO-R,respectively. In contrast, insulin hardly promoted the growthof parental CHO cells, which express few receptors (gener-ation time, 73 hr). Taken together, these data indicate that (i)thymidine incorporation actually reflected a mitogenic effectand notDNA repair, (ii) expression ofmany human receptorsgave CHO cells the capability of growing at physiologicalconcentrations of insulin with the same generation time as

that seen in 10% FCS, and (iii) mutation of Tyr-1162 andTyr-1163 did not alter, in our system, the capacity of thereceptor to mediate the mitogenic effect of insulin.

Kinase Activity of Solubilized Insulin Receptor. We wantedto ascertain that the mutated receptor was devoid of insulin-stimulatable kinase activity towards exogenous substrates,as described by Ellis et al. (9). Insulin receptors werepartially purified by affinity chromatography on wheat germagglutinin and tested for their ability to phosphorylate exog-enous substrates. Table 3 shows that the receptors solubi-lized from CHO-R displayed a measurable insulin-stimulatedkinase activity towards the copolymer (Glu/Tyr), histones,or casein. In contrast, receptors solubilized from CHO-Y2cells did not exhibit any insulin-stimulated kinase activity,even though a basal activity was measurable. Similarly, weverified that partially purified receptors extracted from CHOwere unable to display any insulin-stimulated protein kinaseactivity under the same conditions (data not shown). Thislatter result suggests that the kinase activity we measured onCHO-R extracts was generated from the receptors of humanorigin. These data demonstrate that the mutation of tyrosines1162 and 1163 abolishes the insulin-stimulated receptorkinase activity as measured in our cell-free assay withexogenous substrates.

Receptor Autophosphorylation in Intact Cells. CHO-R andCHO-Y2 were labeled with [32P]orthophosphate for 2 hr andthen incubated 15 min at 37°C with various insulin concen-trations before the receptors were solubilized. An equal

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FIG. 4. Time-course of [3H]thymidine incorporation into DNA.Cells were depleted as described under Fig. 3 and incubated with noeffector (v), 100 nM insulin (o), or 10%o FCS (n) for the indicatedtime. The [3H]thymidine pulse and the DNA isolation were carriedout as described for Fig. 2.

amount ofboth types of receptors (determined by 125I-labeledinsulin binding) were immunoprecipitated as described (12).The lack of tyrosines in position 1162 and 1163 affectedmoderately (20% inhibition) the total level of insulin-stimulated autophosphorylation in intact CHO-Y2 cells ascompared with CHO-R cells (Fig. 5 A and C). These resultsindicate that the mutated insulin receptor retains its capabil-ity for autophosphorylation under hormone stimulation,despite the fact that the tyrosine kinase activity towardexogenous substrates was completely abolished by the sub-stitution for the twin tyrosines. The level of 18 subunitphosphorylation was estimated by alkali treatment, which isknown to eliminate the phosphoserine residues, preserving

Table 2. Generation time for parental CHO and CHOtransfected cells

Cell type Insulin (1 nM), hr FCS (10%6), hr

CHO, parental 73 13CHO-R 30 30CHO-Y2 24 24

CHO-R, CHO-Y2, and parental CHO cells grown in 35-mm disheswere depleted for 48 hr in serum-free Ham's F-12 medium. After thisperiod, the medium was removed and replaced by Ham's F-12medium supplemented with 10% FCS or 1 nM insulin with 0.1%bovine serum albumin and transferrin (1 ,ug/ml). Cells were trypsin-ized and counted every day for 5 days.

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Table 3. Kinase activity of solubilized insulin receptor fromCHO transfected cells

Insulin 32p incorporationCells (100 nM) Histone* Casein* Poly (Glu/Tyr)t

CHO-R - 1 2 0.4+ 5.1 8.5 0.8

CHO-Y2 - 1.9 2.4 0.4+ 2 2.4 0.3

Partially purified insulin receptors from CHO-R or CHO-Y2 cells(30 fmol of insulin-binding activity) were incubated with 100 nMinsulin for 1 hr at 20'C. The kinase assay was done as described (12).*32p incorporation reported in cpm x 10-4.t32p incorporation reported as nmol/min per mg of protein.

mainly phosphotyrosine residues (20). The mutated receptorcould still autophosphorylate, mainly on tyrosine residues(Fig. 5D). The slight decrease in the level of the hormone-stimulated autophosphorylation that we observed on themutated receptor agrees fairly well with the data previouslyreported on the same type of twin tyrosine-deficient receptor(9). Under the same conditions, we were unable to detect anyautophosphorylation of the endogenous rodent receptors(data not shown). It is therefore unlikely that the nonmutatedrodent receptor species could be responsible for the auto-phosphorylation process seen in CHO-Y2. In fact, ifCHO-Y2receptor phosphorylation were from the catalytic activity ofthe rodent receptor species toward the mutated humanreceptor through an intermolecular process, 80% of theradioactivity incorporated in the intact human receptorswould have to result from an interspecies (rodent versushuman) transphosphorylation, a condition that is very un-likely. Another possibility would be that human-rodenthybrid receptors or heteroclusters could account for theresidual autocatalytic phosphorylation seen in CHO-Y2 viaan intramolecular event. None of these hypotheses is con-sistent with the absence of transphosphorylation seen inCHO cells expressing receptors mutated at the ATP bindingsite and totally devoid of kinase activity (13).

Insulin Stimulation of S6 Protein Kinase. Phosphorylationof the S6 ribosomal protein is thought to be one of the majorsteps leading to protein synthesis. Phosphorylation of thisprotein is effected by a serine kinase, the activity of which iscontrolled by several growth factors, including insulin (21,22). We compared the stimulatory effect of the hormone onthe phosphorylation level of the S6 protein in CHO-R andCHO-Y2 cells. Fig. 6 shows that the extent of S6 kinase

CHO R

A

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C

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B D

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INSULIN(nM)

0 0.1 1 10 100 0 0.1 1 10 100

FIG. 5. Insulin receptor phosphorylation on intact cells. CHO-R(A and B) and CHO-Y2 (C and D) were loaded with [32P]ortho-phosphate (1 mCi/35-mm dish) for 2 hr and then incubated withvarious insulin concentrations (0.1-100 nM) for 15 min. The insulinreceptor was purified before being subjected to NaDodSO4/PAGE.(A and C) Autoradiograms of the labeled subunit. (B and D)Labeled ,B subunit after alkaline hydrolysis.

pH5

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activation by insulin was unaffected by the mutation intro-duced in the kinase domain. Similarly, we verified that at theinsulin concentration used (1 nM), the hormone was unableto stimulate the S6 kinase on CHO parental cells (data notshown). Consequently, the S6 kinase activation seen intransfected cells probably resulted from the tyrosine kinaseactivity generated by the exogenous hIRs and not from thekinase activity of the endogenous rodent insulin or insulin-like growth factor I receptors.

DISCUSSIONThe capability of insulin to promote cell growth by interactingwith its own receptors is still controversial (23). Neverthe-less, recent studies have proven unquestionably that, at leastin some systems, insulin behaves as a potent growth factoracting solely through its specific receptors (18, 19). On theother hand, tyrosine residues at positions 1162 and 1163,which are among the major autophosphorylation sites of theinsulin receptor (8), play a key role in control of theinsulin-mediated stimulation of glucose uptake (9). Our aimwas to better define the alterations in the insulin-stimulatedtyrosine kinase activity induced by the mutation and toexplore their putative consequences on the expression of thehormonal mitogenic effect. In a first step, we showed that themutation on the twin tyrosines at positions 1162 and 1163 alsoabolished the hypersensitivity to insulin as measured by thehormonal effect on glycogen synthesis, agreeing with previ-ous data from injecting monoclonal antibodies against thekinase domain of the insulin receptor (24). However, wefound, unexpectedly, that the CHO transfectants expressingTyr-1162- and Tyr-1163-deficient receptors presented thesame insulin concentration-response curve for DNA synthe-sis as those expressing intact human receptors. These dataindicate that this type of mutation does not affect the abilityof the insulin receptor to induce DNA synthesis. It isnoteworthy that insulin promoted cell growth by interacting

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with its own receptors and not insulin-like growth factor Ireceptors because the parental CHO cells, which expresseda low level of insulin receptors, had an insulin sensitivitylower by a factor of 100. As expected, the mutation intro-duced on tyrosines 1162 and 1163 completely abolished theinsulin-dependent tyrosine kinase activity towards exoge-nous substrates, in full agreement with a previous report (9).Besides, over a long period we never observed any restitutionof the insulin-dependent tyrosine kinase activity on solubi-lized mutated receptors, suggesting that the mutation wasquite stable in transfected CHO cells. This hallmark allows usto dismiss the possibility that expression of both mitogeniceffect and receptor autophosphorylation, observed on CHO-Y2 cells, was due to a back mutation. At that point one couldconclude that the kinase activity was not essential forconveying the hormonal signal for cell growth. However,CHO cells transfected with a construction coding for humaninsulin receptors deficient in ATP-binding site lose theirability to respond with cell growth to physiological concen-trations of insulin (13). Furthermore, a dichotomy betweenthe tyrosine kinase of the insulin receptor and the mitogeniceffect of the hormone would contradict the tight correlationseen in several oncogen-encoded proteins between the func-tion of their intrinsic tyrosine kinase activity and the effectthose proteins exert on cell proliferation (25). It is generallyadmitted that kinase activity and receptor autophosphoryla-tion are two closely related events, even though recently thedegree of correlation between these two receptor functionshas been questioned (26). From a quantitative point of view,the 8 subunit of the CHO-Y2 insulin receptor can beestimated to have retained 80% of its ability to autophos-phorylate on tyrosine residues compared with that observedon the native form of the receptor, in agreement with anearlier report (9). The insulin receptor appears to be one ofthe best available substrates to test for intrinsic tyrosinekinase activity. Accordingly, the ability of the mutatedreceptor to elicit ligand-induced phosphorylation on its P3subunit is reminiscent of a cryptic tyrosine kinase activitythat could not be measured by the use of exogenous sub-strates such as casein, histones, or poly (Glu/Tyr). In anattempt to illuminate this puzzling problem, we tried tovisualize in intact cells the expression of a tyrosine kinaseactivity associated with the mutated receptors. We thusdecided to measure insulin-mediated S6 kinase activation,which appears as a good reporter of the expression of aputative cryptic tyrosine kinase. In fact, S6 phosphorylationis an essential step of the mitogenic process, which can bestimulated by the tyrosine kinase activity associated withpp60src (27) or different growth factor receptors (21, 22),including insulin. Interestingly, as presented in Fig. 6, theinsulin-mediated S6 phosphorylation level was unaffected bythe substitution of the tyrosine residues in positions 1162 and1163 by phenylalanine, strongly suggesting that a residualinsulin-dependent tyrosine kinase activity persists in themutated receptor species, which cannot be detected by theusual cell-free assays.To interpret our results in the context of presently available

data we propose the following. (i) The insulin receptor mightexert its kinase activity toward different classes of endoge-nous substrates, each kind of substrate controlling a definedpathway leading to one or more biological effects. (ii) Theaffinity and/or the efficiency of the receptor kinase activitytoward the different categories of substrates would be con-trolled by distinct and specific receptor phosphorylationsites. (iii) The kinase activity directed toward the endogenoussubstrate(s) responsible for control of insulin-stimulatedglucose metabolism could be measured using exogenoussubstrates. Conversely, the tyrosine kinase activity respon-sible for phosphorylating the substrate(s) involved in themitogenic pathway would not be measurable in this cell-free

system. In that situation mutation of tyrosines 1162 and 1163would induce a partial suppression, involving specifically thekinase activity directed toward the defined category ofsubstrates implicated in control ofthe metabolic effects ofthehormone. In contrast, the phosphorylating activity elicited bythe receptor on other classes of substrates, among which arethose mediating the mitogenic effect of insulin, like the S6kinase, would be unaffected by the mutation.

We are indebted to P. Freychet, A. Baron, M. Fehlmann, andG. W. G. Sharp for carefully reading the manuscript and for fruitfuldiscussions. We are very grateful to A. Grima and C. Minghelli forthe illustrations. This work was supported by grants from InstitutNational de la Santd et de la Recherche Mddicale (France), theUniversity of Nice, the Association pour la Recherche contre leCancer (France), the Centres de Lutte contre le Cancer (France), theFondation pour la Recherche Mddicale (France), and the Fondationdu Groupe des Populaires d' Assurances.

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