Proc. Nati. Acad. Sci. USAVol. 86, pp. 9294-9298, December 1989Cell Biology

The insulin receptor as a transmitter of a mitogenic signal inChinese hamster ovary CHO-Ki cells

(growth control/insulin-like growth factor I/insulin-like growth factor H)

MICHAEL MAMOUNAS*, DENNIS GERVIN*, AND ELLIS ENGLESBERGtt*Cell and Physiology Section, and tBiochemistry and Molecular Biology Section, Department of Biological Sciences, University of California,Santa Barbara, CA 93106

Contributed by Ellis Englesberg, August 15, 1989

ABSTRACT Insulin is the only hormone required forcontinued growth of Chinese hamster ovary CHO-K1 cells inthe defmed medium M-F12. When CHO-K1 cells are incubatedin M-F12 without insulin for 48-72 hr, the cells accumulate inG1. In response to physiological concentrations of insulin an18-fold increase in rate of DNA synthesis occurs due to cellsentering S phase after an 8- to 10-hr lag; cell division beginsafter 24 hr. The inhibitory effect of actinomycin D and 5,6-dichlorobenzimidazole riboside indicates that RNA synthesis isrequired for progression to S phase. CHO-K1 cells possessinsulin receptors, and the insulin effect results from insulinbinding to its own receptor: (a) Binding occurs at physiologicalinsulin concentrations with a half-maximal stimulation at 14ng/ml. (it) At insulin concentrations used, insulin-like growthfactor I and II (IGF-I and IGF-H) have little or no effect. (i)Scatchard analysis of 12I-labeled insulin binding shows thecurvilinear response typical of insulin. (iv) The Kd for theso-called high-affmity binding site and the K. are characteristicof the insulin receptor. (v) At the minimal insulin concentra-tions that stimulate growth, IGF-I and IGF-ll compete poorlywith insulin for insulin binding, insulin competes poorly withIGF-I for IGF-I binding, and affinity labeling with 12'I-labeledinsulin identifies a polypeptide (Mr = 125,000) typical of the asubunit of the insulin receptor.

The insulin family of hormones, including insulin and insulin-like growth factor I and II (IGF-I and IGF-II), play anessential role in human growth and development. Thesehormones generate transmembrane signals that regulate car-bohydrate, lipid, and protein metabolism; glucose and aminoacid transport; DNA synthesis; and cell division and mor-phological transformation. Because of the degree of similar-ity of structure and apparent function of these hormones andtheir receptors (insulin and IGF-I) and the cross-reactivitywith regard to receptor binding, much effort has been ex-pended to determine the actual mode of action of the indi-vidual hormones vis-a-vis their unique receptors. Both theinsulin and IGF-I receptors seem to mediate some ofthe sameacute responses that have usually been thought reserved forthe insulin receptor (1). Both receptors possess ligand-dependent, tyrosine-specific protein kinase activity similar toreceptors of other hormones and proteins of several onco-genes (2). The broad response elicited by IGF-II has usuallybeen found to be mediated by the IGF-I and the insulinreceptors (1). Recently, the receptor for IGF-II has also beenfound to be the receptor for mannose-6-phosphate (3). TheIGF-I receptor is "normally" thought responsible for growthstimulation (1). The insulin stimulation of cell growth inculture has been assumed to be from insulin binding to theIGF-I receptor as a result of the use of insulin at greater thanphysiological insulin concentrations (4-8); however, this

conclusion is open to question (9-15). We have developed amodified F12-defined medium (M-F12) for CHO-K1 cells inwhich insulin is the only hormone required for growth (16).On the basis that insulin stimulated growth in this medium atthe nanogram range (1-10 ng/ml) characteristic of the levelsfound at normal physiological concentrations (17), we pro-posed that insulin at these concentrations is acting as its ownreceptor (16).

In this paper we characterize the insulin receptor ofCHO-K1 cells and present evidence that the insulin stimu-lation of DNA synthesis and cell division in this cell lineresults from insulin binding to its own receptor. In addition,we show that when CHO-K1 cells are starved for insulin,growth is arrested in G1 phase and that the insulin stimulationof DNA synthesis and cell division of these quiescent cellsrequires RNA synthesis.

MATERIALS AND METHODSCell Line and Culture Conditions. The Chinese hamster

ovary cell line CHO-K1 and its growth characteristics havebeen described (16). CHO-K1 was routinely maintained inMEMCHO-4 medium in monolayers. For certain experi-ments, these cells were transferred to a defined, modified F12medium, (M-F12). This medium contains insulin as the solehormone. M-F12-I is the above medium minus insulin (16).Assay for DNA Synthesis. Cells were grown in MEMCHO-4

medium and harvested (16) when theyjust became confluent.They were transferred to M-F12 containing 1 ,ug of insulin perml at 2 x 106 cells per 150-mm dish. After incubation for 72hr fresh medium was added, and the plates were incubated foranother 24 hr. The cells were subsequently washed 5 timeswith 10 ml each of phosphate-buffered saline, harvested asdescribed (16), resuspended in M-F12-I, and transferred to35-mm dishes at 4 x 104 cells per dish in 2 ml of the samemedium. The cells were then incubated for 48-72 hr until theywere generally in the fibroblastic state (16). (In recent ex-periments cells harvested from MEMCHO-4 medium weretransferred directly to 35-mm dishes containing M-F12-I plus5 or 20 ng of insulin per ml, incubated for 48 hr, washed withM-F12-I, and incubated with M-F12-I for 48 or 72 hr.) Growthfactors were then added from a concentrated stock solution,followed by 0.4 jCi of [3H]thymidine per ml, specific activ-ity, 18.2 Ci/mmol (1 Ci = 37 GBq) at the time intervalsindicated. The medium was then aspirated, and cells werewashed with ice-cold M-F12-I. One milliliter of ice-cold 5%(vol/vol) trichloroacetic acid was then added for 30 min, andthe cells were washed two additional times with 2 ml ofice-cold 5% trichloroacetic acid. Subsequently 0.8 ml of 0.1M NaOH was added, and radioactivity was determined with

Abbreviations: IGF-I and -II, insulin-like growth factor I and II,

respectively.tTo whom reprint requests should be addressed.

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a Beckman liquid scintillation counter. The values given areaverages of triplicate determinations with SDs.

Labeling of Nuclei. Cells were plated in 16-mm multiwelltrays containing 1 ml of M-F12-I plus insulin at 5 ng/ml at adensity of 9 x 103 cells per well for 48 hr, washed withM-F12-I, and incubated for 48 hr in M-F12-I. Insulin was thenadded at 20 ng/ml, and at various times 2 ,Ci of [3H]thymi-dine (18.2 Ci/mmol) was added for 2 hr. Labeled nuclei werethen determined by a described procedure (18).

Binding of '2SI-Labeled Insulin to Cells. Cells were grown asdescribed in AssayforDNA Synthesis, except that cells wereinoculated into M-F12-I in 60-mm dishes at an initial con-centration of 1 x 106 or 5 x 105 cells per dish for 2 or 10 days,respectively. The cells were then washed 2 times with 5 ml ofwashing buffer composed of 100 mM Hepes, 120 mM NaCl,1.2 mM MgSO4, 5 mM KCl, 10 mM glucose, 1 mM EDTA,and 15 mM sodium acetate (final pH 7.9). One milliliterbinding buffer, composed of washing buffer plus bacitracin at2 mg/ml, 1% bovine serum albumin, and 6.7 x 10-11 M1251I-labeled insulin (1.5 x 109 cpm/nmol), and various con-centrations of cold insulin, was added in triplicate. The cellswere then incubated at 220C for 100 min, washed 5 times with5 ml each of ice-cold washing buffer, and solubilized with 1ml of 0.1 M NaOH; radioactivity was assayed as describedabove. Nonspecific binding was determined with the additionof unlabeled insulin at 100 kug/ml. Data were analyzed by themethods of Scatchard (19). A computer program (LIGAND)was used to analyze the data, assuming two independent sitesfor insulin binding. The data were also analyzed on the basisof a negative cooperativity model (20-22).

Binding of 125I-Labeled IGF-I and IGF-ll to Cells. Similarexperiments, as described above for insulin binding, weredone with 1251I-labeled IGF-I and IGF-IT. IGF-I binding wasmeasured with cells grown in MEMCHO-4 and M-F12-Imedia, whereas MEMCHO-4-grown cells were used in mea-suring IGF-II binding. For growth in MEMCHO-4 medium,cells were inoculated into 60-mm dishes containing 5 ml ofMEMCHO-4 medium at a density of 5 x 105 cells per dish.The cells were allowed to grow until they were confluent(= 4 x 106 cells per dish) and processed as described above.Specific activity of iodinated IGF-I and IGF-II used was 4.4x 109 cpm/nmol for IGF-I and 3 x 107 cpm/nmol for IGF-II.

Affinity Labeling of the Insulin Receptor. The cells weregrown in 150-mm dishes containing MEMCHO-4 mediumuntil confluent, washed with phosphate-buffered saline, har-vested by scraping, and membranes were then prepared (23).Membranes from 6 x 107 cells were brought to a vol of 1 mlin binding buffer plus 1 mM phenylmethylsulfonyl fluoride.12I1-labeled insulin was then added to each oftwo tubes at 8.3x 10-10 M (1.5 x 106 cpm/ml). To one tube, unlabeled insulinwas added to 1.67 x 10-6 M. The tubes were incubated at22°C for 100 min. Subsequently the membranes were pelletedby centrifugation for 5 min at 15,000 g and then washed oncewith 1 ml of binding buffer. The pellet was then resuspendedin binding buffer without bovine serum albumin and insulinwas covalently linked by the disuccinimidyl suberate proce-dure (24). The membranes were then collected by centrifu-gation for 5 min at 15,000 x g, and the pellet was resuspendedin 100 ,u of gel sample buffer containing 100 mM dithiothrei-tol. Electrophoresis was carried out on 7% polyacrylamidegel (25) along with appropriate molecular weight markers.The gel was stained with Coomassie blue and was then driedand subjected to autoradiography that used Kodak X-Omatfilm with an enhancing screen.

Reagents. IGF-I was from A. Pardee (Dana-Farber CancerInstitute) and L. Wudl (Amgen Biologicals). IGF-II was a giftof M. P. Czech (University of Massachusetts MedicalSchool). Insulin was a gift from Eli Lilly. Initial bindingexperiments were done with 'l25-labeled insulin and IGF-Ipurchased from NEN and Amersham, respectively. Subse-

quently '25I-labeled insulin and IGF-II were prepared by thechloramine-T method.

RESULTSInsulin Stimulation of the DNA Synthesis Rate and Cell

Division. We measured the rate of DNA synthesis and celldivision of CHO-K1 cells subsequent to adding insulin (finalconcentration, 20 ng/ml) to cells that were previously incu-bated in M-F12-I medium for 72 hr. There is an 8- to 9-hrlag before the cells respond to insulin-induced stimulation ofDNA synthesis (Fig. 1). After the lag the rate of the DNAsynthesis rapidly increased, reaching a 16-fold increase overbasal level in 20 hr. Cell division was not detectable until 24hr after insulin addition, and in 76 hr the cells completed twodivisions. We next measured the effect of insulin on DNAsynthesis by measuring the incorporation of labeled thymi-dine into nuclei, as well as into total DNA, in an experimentsimilar to that described above. We found that nuclei of 11%of the cells were labeled at the initiation of the experiment,and this percentage remained the same for -8 hr. At 19 hr70% of the nuclei were labeled (Fig. 1, Inset). The rate ofincrease in labeled nuclei corresponds to the increase in totalDNA synthesis. These results thus confirm that cells starvedfor insulin are almost totally blocked in G1 stage, and theincrease in rate of DNA synthesis is due to recruitment ofadditional cells into the S phase of the growth cycle.

In these cells DNA synthesis is extremely sensitive toinsulin. Significant stimulation is shown with insulin at 2ng/ml, and a direct relationship appears to exist between

Cr)co0

x

EC)

15 20 25Time (hrs)

1.2

1 tnI.U eDIA)(

0.8 X..8x

ECD0.6 E

0.4 0

.0.2

FIG. 1. Accumulation of CHO-K1 cells in G1 phase and insulinstimulation of DNA synthesis. Cells were starved for insulin inM-F12-I medium for 72 hr as described. Insulin was added to one setof plates (final concentration, 20 ng/ml), and [3H]thymidine wasadded to these plates and to plates without insulin beginning at 4 hrat 1- to 5-hr intervals for 28 hr for a 2-hr period. Plates were thenprocessed for DNA synthesis. Counts at indicated times represent[3H]thymidine uptake over the previous 2 hr-i.e., counts at 4 hrrepresent uptake from 2 to 4 hr. An additional set of plates with andwithout insulin was used for determining cell count. The experimentwas done in triplicate, and averages are plotted. *, cpm of[3H]thymidine per 105 cells plus insulin; *, cpm of [3H]thymidine per105 cells without insulin; o, total cell count plus insulin; *, total cellcount without insulin. (Inset) Nuclear labeling was done as describedexcept that cells were insulin-starved for 48 hr and also inoculatedinto multiwell trays to determine thymidine incorporation into nuclei.*, % cells that had labeled nuclei with insulin; and m, unstimulatedcells.

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54

3

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40 120 140 2 240

INSULIN ng/ml

FIG. 2. Effect of insulin concentrations on rate of DNA synthe-sis. Cells were insulin-starved for 72 hr in M-F12-I medium. Insulinwas added at the indicated concentrations, and 18 hr later[3H]thymidine was added. After an additional 12 hr cells wereprocessed for DNA synthesis. The experiment was done in triplicate,and averages are plotted as cpm per 105 cells.

insulin concentrations and DNA synthesis rate at 2-30 ng/mlwith a half-maximal stimulation at 14 ng/ml (Fig. 2).The Effect ofAntinomycin D and 5,6-Dichlorobenzimidazole

Riboside on the Insulin Stimulation of DNA Synthesis. Wefound that both inhibitors prevented insulin stimulation ofDNA synthesis when added at any time up to 7 hr after insulinaddition. Substantial DNA synthesis occurred when theinhibitors were added at 9 and 10 hr (Fig. 3).Comparison of the Effect of Insulin, IGF-I, and IGF-II on

DNA Synthesis ofCHO-K1 Cells. We tested the effect of these

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O 2 4 6 8 10

Time (hrs)

FIG. 3. Effect of actinomycin D and 5,6-dichlorobenzimidazoleriboside on insulin stimulation of DNA synthesis. After insulinstarvation for 72 hr in M-F12-1 medium, insulin was added to half theplates at 20 ng/nl, and at 1-hr intervals for 11 hr either actinomycinD or 5,6-dichlorobenzimidazole riboside in dimethyl sulfoxide was

added to final concentrations of 1 ,ug/ml and 15 ,ug/ml, respectively.As controls, to another set of plates either dimethyl sulfoxide orethanol was added at concentrations obtained with the inhibitors.After 12-hr exposure to insulin, [3H]thymidine was added to all platesfor 2 hr (i.e., 12- to 14-hr incubation), and DNA synthesis wassubsequently determined. The experiment was done in triplicate, andresults were averaged. Insulin stimulation of DNA synthesis incontrols (without inhibitor) was determined by subtracting countswithout insulin from counts with insulin. Similar determinations weremade with cells incubated with inhibitors, and % inhibition ofDNAsynthesis was calculated. Data points represent % inhibition ofDNAsynthesis seen with inhibitors (A, actinomycin D; *, 5,6-dichlorobenzimidazole) when added at the indicated times.

0.006

0.005

I-

00m

0004

0.003

0.002

0.001

0 1 2 3 4

Bound (x10 11 M / 106 cells)

FIG. 4. Scatchard plot of binding of 1251-labeled insulin to CHO-K1 cells incubated for 48 hr in M-F12-I medium. Nonspecific bindinghas been substracted.

growth factors alone and in various concentrations for theirability to stimulate DNA synthesis of CHO-K1 cells starvedfor insulin for 72 hr as described above. In addition, in thisexperiment we added bovine serum albumin at 1 mg/ml toprotect the hormones from possible protease activity. Al-though insulin stimulated DNA synthesis at 4 ng/ml andreached maximal stimulation at 30 ng/ml, we found nostimulation with IGF-I at these concentrations or at concen-trations up to 225 ng/ml. There was not any stimulation withIGF-II at concentrations to 15 ng/ml or any synergistic effectof IGF-I or IGF-II with insulin at 8 or 15 ng/ml. IGF-II at 75ng/ml achieved a maximum rate of stimulation equal to whatwas obtained with insulin at 8 ng/ml (data not shown).Although we obtained no stimulation with IGF-I in theseexperiments, in other experiments we found, at most, adoubling of the rate of DNA synthesis of an IGF-I concen-tration of 225 ng/ml (data not shown). In similar experimentswe found no stimulation of DNA synthesis by platelet-derived growth factor or epidermal growth factor at concen-trations from 2 to 225 ng/ml, respectively (data not shown),as might be expected because CHO-K1 has been reported tolack receptors for these two hormones (26, 27).

Table 1. Stoichiometric binding parameters of the insulinreceptor of CHO-K1 cells

Parameters*tt§Two-independent-binding-sites model

Kdl 7.1 x 10-10Kd2t 3.0 x 10-8

S, binding sites per cell 1,926S2 binding sites per cell 31,304

Total 33,230Cooperative model

Ket 1.1 X 108 M-1Kf§ 4.x107 M-1

Binding was assayed as described in the legend for Fig. 4 and intext. Binding parameters for the two-site model were obtained by useof a computer program (LIGAND), assuming two independent bindingsites.*Kdl, affinity constant for high-affinity receptor.tKd2, affinity constant for low-affinity receptor.tK refers to the affinity constant and is expressed in M-1; Ke, affinityconstant for empty receptors.§Kf affinity constant for filled receptors; Xf/Ee is the interactionfactor (21) and equals 0.35.

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Cc0

CL01)

01)CL

2.5 5 100 2.5 100 2.5 100 # 0 5 15 100 5,15 5 15 1005 5 ,__ _ _100

Insulin IgF-I IgF-H IgF-I Insulin IgF-II

(ng/ml) (ng /ml)

FIG. 5. (A) Competition byIGF-I and IGF-II with insulin forbinding of 125I-labeled insulin. To1.67 x 10-1o M 125I-labeled insulin(1.5 x 109 cpm/nmol) the indi-cated amounts of unlabeled insu-lin, IGF-I, or IGF-II were added.(B) Competition by IGF-I, IGF-II,and insulin for binding of 1251_labeled IGF-I. To 3.2 x 1O-10 M125I-labeled IGF-I (4.4 x 109 cpm/nmol) was added to the indicatedamounts of unlabeled IGF-I, IGF-II, or insulin.

Characterization of Insulin Binding. Scatchard analysis ofinsulin binding by CHO-K1, previously incubated in M-F12-Ifor 48 hr, shows the characteristic curvilinear shape typicalof insulin binding in other systems (Fig. 4). Assuming twoindependent sites for insulin binding with different bindingaffinities (28), the number of binding sites and Kd values wereestimated by using a computer program (LIGAND). The datahave also been analyzed assuming negative cooperativity (21)(Table 1). A similar experiment was done in which the cellswere starved for insulin for 10 days with similar results asobtained for 2-day starved cells (data not shown).

Characteristics of IGF-I and IGF-ll Binding. Scatchardanalysis of IGF-I binding to CHO-K1 cells growing in MEM-CHO-4 or M-F12-I medium gave similar results. The curvesare linear with a Kd of 1.5 x 10-9 M and 50,000 binding sitesper cell. The Scatchard analysis of IGF-II binding is linearwith a Kd equal to 6.4 x 10-9 M and 28,000 binding sites percell.

Competition of Insulin, IGF-I, and IGF-ll for Binding ofLabeled Insulin and IGF-I. At an 1251-labeled insulin concen-tration of 1 ng/ml, a concentration that stimulates growth andDNA synthesis, 100 ng of IGF-I or IGF-II per ml reducesinsulin binding by 15%, whereas cold insulin at 2.5 ng/mlreduced binding of labeled insulin by 32% and at 100 ng/mlreduced binding by 88% (Fig. 5A). The latter 12% probablyrepresents nonspecific binding. These results indicate thatinsulin at a concentration that stimulates DNA synthesis is

1 2

FIG. 6. Specific cross-linkingof l25l-labeled insulin to a mem-brane preparation of CHO-K1cells. Membranes were preparedand incubated with 1251-labeled in-sulin without and with excess un-labeled insulin. Subsequently therecovered membranes were cross-linked to receptor-bound insulinby treatment with 0.2 mM disuc-cinimidyl suberate. The affinity-labeled membranes were electro-phoresed together with Mr stan-dards. Lanes: 1, membranestreated with only labeled insulin;2, membranes treated with labeledand unlabeled insulin. Position ofthe Mr X 1O-3 markers are at left.

binding to its own receptor, and that IGF-I and IGF-II havelittle affinity for this receptor.

In an experiment similar to that described above, we foundthat at 100 ng/ml, insulin had no effect on the binding of1251I-labeled IGF-I at 2.5 ng/ml, whereas IGF-II replaced only12% of IGF-I, and IGF-I replaced labeled IGF-I by 75%. Thelatter 25% probably represents nonspecific binding (Fig. SB).These results indicate that insulin has a very low affinity, ifany, for the IGF-I receptor, whereas IGF-II binds veryweakly.

Affiity Labeling of the a Subunit of the Insulin Receptor.The insulin receptor on membrane preparations preparedfrom cells grown on MEMCHO-4 medium was cross-linkedto 1251-labeled insulin (5 ng/ml), with or without unlabeledinsulin, by disuccinimidyl suberate. Electrophoresis of thelabeled membrane preparation in the presence of dithiothrei-tol shows a band with Mr, 125,000 without unlabeled insulinand no band in its presence, typical of the a subunit of theinsulin receptor (Fig. 6) (29).

DISCUSSIONInsulin has been shown to be universally required to supportmammalian cell growth in a defined medium (30). However,detailed study of this requirement has shown that, for themost part, insulin is required at concentrations greater thannormal physiological levels and that IGF-I can substitute forinsulin at physiological concentrations. These findings sup-ported the idea that IGF-I is the real growth stimulant formammalian cells and that insulin, having affinity for theIGF-I receptor, can replace IGF-I, although at much higherconcentrations (4-8). Although this explanation has pre-vailed for some time, there is growing evidence that insulin,in certain cell lines, actually stimulates growth by binding toits own receptor and, therefore, insulin in vivo may act as amitogen for certain cell types. For instance, it has beenshown that insulin growth stimulation of rat H-35 cells resultsfrom insulin binding to its own receptor (9, 10, 14). Somewhatsimilar results have been obtained with F9 embryonal carci-noma cells (11). These results are notjust due to an aberrationof some established cell lines, as evident by similar findingswith certain normal human diploid fibroblasts (12, 13, 15).We have shown that insulin is the only growth factor

required for the initiation and continued growth of CHO-K1cells in a modified F12-defined medium. Our experimentsindicate that the insulin stimulation of DNA synthesis andcell division results from insulin binding to its own receptorand not to either the IGF-I or the IGF-II receptors. We haveshown that (i) insulin stimulates DNA synthesis and celldivision at physiological concentrations (21 ng/ml); (ii) at

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these same concentrations, IGF-I and IGF-II have little or noeffect; (iii) Scatchard analysis of 125I-labeled insulin bindingshowed the curvilinear response typical of insulin binding toits own receptor (20); (iv) the Kd for the so-called high-affinitybinding site and the K,, based upon negative cooperativity,(20-22) is typical of the insulin receptor (28); (v) at minimalinsulin concentrations that stimulate growth, IGF-I or IGF-IIcompete poorly with insulin for insulin binding, insulin com-petes poorly with IGF-I for IGF-I binding, and affinitylabeling with 125I-labeled insulin identifies a polypeptide thatcorresponds in molecular weight to the a subunit of theinsulin receptor.When CHO-K1 cells are starved for insulin by incubation

in M-12-I medium for 48-72 hr, they accumulate in G1 phaseand, in response to concentrations of insulin 21 ng/ml, arestimulated to synthesize DNA and cell division, subsequentto a lag of -8-9 hr. We have demonstrated, at an insulinconcentration of 20 ng/ml, an 18-fold increase in the DNArate of synthesis and have shown that this rate increase isfrom the stimulation of cell progression through the G to theS phase of the growth cycle by nuclear labeling experiments.Thereupon cells continue to cycle in just the presence ofinsulin. By a study of the effect of actinomycin D and5,6-dichlorobenzimidazole riboside added with insulin tocells that have accumulated in G, phase, we have presentedevidence that RNA synthesis is required for progress to theS phase and that the sensitive time interval is up to 7 hr afterthe insulin addition.3T3 cells, accumulated in G1 phase, as a result of reaching

confluency or from growth in complex medium containinglimiting calf-serum concentrations, initially require platelet-derived growth factor to reach a state of competence. There-upon they are able to synthesize DNA as a result ofepidermalgrowth factor and IGF-I stimulation. Insulin at high concen-trations can apparently substitute for IGF-I by occupying theIGF-I receptor (7). The finding that normal human diploidfibroblasts and many other cell lines as well as CHO-K1respond to an insulin mitogenic signal by insulin binding to itsown receptor indicates that the model proposed for 3T3 is nota universal one for growth control.The fact that CHO-K1 cells accumulate in G1 phase when

starved for insulin might be in variance with the concept thattransformed cells fail to accumulate in G1 because they lackthe restriction point (7). The CHO-K1 cell line does possessmany of the attributes of transformed cells (31). However,when these cells are starved for insulin for 48-72 hr, in ourexperiments, they are changed morphologically into whatappears to be "normal diploid fibroblasts" (16). The fact that(i) insulin is the only hormone required for growth of CHO-K1 cells, (ii) CHO-K1 cells are functionally hemizygous formany traits (31), and (iii) insulin-independent mutants areeasily isolated (16) makes CHO-K1 line an ideal modelsystem for studying transduction of the insulin-insulin re-ceptor mitogenic signal.

We thank M. Jones and P. De Meyts for a careful review of the

manuscript. This work was supported, in part, by National ScienceFoundation Grant DMB-8616536 and the University of California.

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