THROMEIOSIS RESEARCH 14; 477-487 Printed in Great Britain @Pergamon Press Ltd.1979. OO@-3848/79/0301-0477
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THE EFFECT OF A PLATELET-DERIVEDGROWTH FACTOR ON THE PROLIFERATIONOF RABBIT ARTERIAL SMOOTH MUSCLE CELLS IN TISSUE CULTURE
I. 0. Ihnatowycz,J.-P. Cazenave, J.F. Mustard and S. Moore Department of Pathology,McMaater University Hamilton, Ontario, Canada
(Received 19.9.1978; in revised form 30.11.1978. Accepted by Editor K.M. Brd.nkhous) ’ ABSTRACT Thrombin and collagen induced the release of a growth factor from washed rabbit plateletswhich requires a serum factor derived from platelet poor plasma for maximum proliferationof the smooth muscle cells. The release of the platelet-derivedgrowth factor by thrombin and collagen parallels the release of serdtonin from theee platelets. Therefore it is possible to use rabbit smooth muscle cells and rabbit platelets and %-thymidine to examine the effect of factors governing the release of the platelet-derivedgrowth factor and the factors governing its effect on smooth muscle cell proliferation. These results show that it is possible to examine the effect of collagen,which is the principal stimulus to platelet release in the vessel wall, in an in vitro system to rapidly study its effect on the release of the platelet-derivedgrowth factor.
INTRODUCTION
Serum contains factors which are essential for the proliferationof diploid, nontransformedcells in culture (1). Sfnce Balk's observationsthat serum was more effective In stimulatingchick embryo fibroblaststhan was plasma (Z).,it has been establishedthat platelets are a source of a factor which stimulates the in vitro proliferationof cells (3-7). The importance of platelets in the smooth muscle cell response to lntlmal Injury has been demonstratedby showing that severe thrombocytopeniain rabbits results in a marked inhibitionof both the developmentof proliferativesmooth muscle cell lesions in response to a single intimal injury (g), and the lipid containing raised lesions caused by continuous intimal injury (9). The aim of the experimentsreported in this paper was to determine whether the incorporationof 3Ii-thymidine into DNA could be used as a rapid 477
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quantitativeassay of the effect of the platelet-derivedgrowth factor (PDGF) on arterial smooth muscle cell proliferationin tissue culture. We examined the relationshipbetween the appearance of serotonln and PDGF in the suspending fluid upon treatment of a washed platelet suspensionwith thrombin or collagen. In addition,we examined the effect of serum derived from plateletpoor plasma on the response of smooth muscle cells to PDGF. Some of the results described in this paper have been reported briefly elsewhere (7).
MATERIALS AND METRODS
The following isotopes were used: [methyl-3Hjl-thymidine t31i-Tdr, s.a. 6.7 Ci./mmole, obtained from New Rngland Nuclear, Boston, Mase.);L4C-serotonln (5-hydroxytryptamine-31-14C-creatinine sulfate; 14C-5HT, 8.8. SOmCi/mmole, obtained from Amersham/Searle,Arlington Reights, Illinois). Eagle's Minimum Essential Medium, F-15, penicillin:streptomycin,trypeln, fetal calf serum; and L-glut-e were all obtained from Grand Island Biological Company of Canada, Burlington,Ontario. Hepes was obtained from Sigma Chemical Co., St. Louis, Missouri. Topical bovine thrombinwas obtained from Parke, Davfs and Co., Detroit, Bovine tendon collagen was obtained from Sigma Chemical Co., Hchigan. St. Louis, msaouri. Arterial smooth muscle cells were grown from explants of segments of media from the thoracic aorta of 3-month old New Zealandwhite rabbits. The method used was similar to that of Ross (lo). A rabbit was anaesthetizsdwith sodium pentobarbital (50 mg/kg; obtained from M.T.C. Pharmaceuticals-, Ramilton, Ont.) and the thoracic aorta excised and stripped of the intimal and adventitial layers. The tunica media was cut into lmm3 sections and incubated at 37'C in Eagle's Minimum RssentialMedium, F-15, supplementedwith L-glutamine, 15% fetal calf serum which had been heat inactivatedat 56'C for 30 minutes, a penicillin: streptomycinsolution (lOOU/ml:lOOmg/ml)and 15mM Eepes at pH 7.3. The smooth muscle cells from the primary cultures were subculturedin the followingmanner for a maximum of five times. The surface of the culture The cells were flask was washed with phosphate buffered saline at pR 1.3. treated with 0.25% trypsin for 3 minutes and sparsely subculturedinto 150 cm2, Falcon tissue culture flasks containing 20 ml of the growth medium. At confluence, the smooth muscle cells from the second to the fifth subculturewere used for experiments. Smooth muscle cell proliferationwas assa ed in the followingmanner. Equal numbers of smooth muscle cells (1.5 x 10s ) were plated per 60 nxaFalcon culture dish containingEagle's Minimum Essential Medium, F-15, supplemented0 with L-glutamine, 15% fetal calf serum which had been heat inactivatedat 56 C for 30 minutes, a penicillin: streptomycinsolution (lOOU/ml:lOOmg/ml),15 mM Heges and 3% sodium bicarbonate. The smooth muscle cells were incubated at 37 C in humidified air containing5% CO? for approximately5 days. At confluence the medium was changed to Eagle s MPH, F-15, supplementedwith the agents mentioned above, but lacking fetal calf serum. At 24 hours the medium was again changed to fetal calf serum-freeEagle's MEM, F-15, prepared as above and containing the material to be tested. At this point one of 2 procedures was followed:
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a) Cell counting technique: Three culture dishes were taken from each experimentaland control group, the medium was removed and the cells were washed twice with phosphatebuffered saline. One millilitre of 0.25% trypsin was added to the culturesand incubated for 3 minutes at 37'~. The cell SUSpension was gently removed by Pasteur pipet and the number of cells counted on a hemacytometer. The remaining dishes were incubated at 37'C in humidified air in 5% CO2 for 12 days. Cell counts were carried out on days 3,6,9, and 12. 3 b) Incorporationof 3H-thymidineinto DNA: H-thymidinewas added to all the culture dishes to a final concentrationof 0.5 pCi/ml. At 48 hours the cultures were washed with phosphate buffered saline (pH 7.3) and solubilizedin 3 ml of 0.5 N NaOH. Five millilitresof 1.0 N HC104 were added to each sample, thoroughlymixed and cooled to 4'C for 30 minutes. After centrifugationat 10,000 g for 20 minutes the supernatantfluid was removed, 1 ml of 1.0 N HC104 was added to each sample and incubated at 70°C for 30 minutes. Each sample was cooled to room temperatureand centrifugedat 10,000 g for 20 minutes. One aliquot from each sample of the second supernatantfluid was taken to count the 3H-thymidineby liquid scintillation. A second aliquot was taken to determine the amount of DNA by a spectrophotometrictechnique (11). The specific radioactivityof the DNA was used as an index of cell proliferation. Platelet-richand latelet-poorplasma was prepared by the method of Ardlie, Packham and Mustard (12P . The platelet-poorplasma was further centrifugedat 12,000 g for 2 minutes to remove any remaining platelets. ‘lhrombin, 5 U/ml, and CaC12, 2mM, were added to the platelet-richand platelet-poorplasma. Following an incubationat 37OC for approximatelytwo hours and centrifugation at 12,000 g for 2 minutes, the supernatantsera were removed, heat inactivatedat 56'C for 30 minutes and dialysed overnight against Tyrode's solution at 4OC. The sera were referred to as serum derived from plateletrich plasma and serum derived from platelet-poorplasma respectively. Suspensionsof twice-washedrabbit platelets were prepared according to Ardlie et al (12), labelledwith 14C-serotonin(13) and finally resus ended at 37'C in Tyrode's solution containing0.35% albumin an Apyrase was prepared by the method of Molnar and Lorand 4dpy'"s~~1~;telet count was adjusted to the requirementsof each individualexperiment. Platelet aggregationstudies and measurementof the release of 14C_ serotonin 5 minutes after the addition of the aggregatingagents were carried out as described previously (16). An aliquot of each sample was assayed for its effect on smooth muscle cell proliferation. lhrombocytopeniain rabbits was produced by irradiationas described by Beimers et al (17) and the injectionof antiplateletserum as prepared by Moore et al (9). Rabbits that were made thrombocytopenicby this method had platelet counts of less than 1,000 per mm3. Blood was collected into 50 ml syringes, allowed to clot and centrifugedat 1000 g for 15 minutes. The supernatantserum was removed, heat inactivatedat 56'C for 30 minutes and referred to as serum from thrombocytopenicrabbits.
BBSULTS
To establish the validity of the assay for the rabbit platelet-derived growth factor, 5% platelet rich or poor serum and platelet supernate or a
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combinationof the platelet poor serum and platelet supernate,containing the material released from the washed platelets,were added to quiescent confluent rabbit smooth muscle cells (3.25 x 105 cells/culturedish). The cells proliferatedonly in response to the platelet rich serum or the combinationof platelet poor serum and the material released from the washed platelets (Fig. 1).
30NUMBER OF CELLS PER loCULTURE DISH (xl+)
0 3 6 9 NUMBER OF DAYS AFTER ADDITIONS
FIG. 1
Smooth muscle cell proliferationin response to serum derived from platelet-richplasma (0), serum derived from platelet-poorplasma (A), the supernatantfluid from a thrombin stimulatedwashed platelet suspension (A), or the combinationof serum derived from platelet poor plasma and the supernatantfluid from a thrombln stimulated washed platelet suspension (0). Mean 2 stsndard error (n-3)
The IncorporatIonof 3H-thymidineinto DNA was measured at 6,9,12,15,18, 21 and 24 hours following the addition of PDGF to confluentquiescent smooth muscle cell cultures (Fig. 2). Synthesisbegins at approximatelysix hours, reaches a maximal point at 15 to 16 hours, and decreases thereafter. Since the maximum incorporationof 3D-thylmidine into DNA occurred at 15 to 18 hours, the experimentswere set up to examine the incoroporationof 3%thymidine into DNA 24 hours after stimulatingwith PDGF and pulsing with 3H-thymidine. Next we examined the effect of culturing the smooth muscle cells in medium containing serum derived from platelet poor plasma and serum derived from plateTwenty-four let rich plasma on 3H-thymidineIncorporationinto DNA (Fig. 3). hours after stimulationthe maximum incorporationof 3H-thymidlneinto DNA was found in the smooth muscle cell cultures in serum derived from platelet rich plasma. Cells cultured in serum derived from platelet poor plasma or in the presence of the material released from washed platelets by thrombin did not The combinationof the lead to much incorporationof 3R-thymidineinto DNA. serum derived from platelet poor plasma and the supernatantfluid from the thrombin-stimulatedplatelets produced nearly as much 3H-thymidine
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TIME (hours) FIG. 2 Time-course of DNA eyntheeis in rabbit smooth muscle cells. 5% rabbit serum prepared from whole blood ( 0 ) or serum-free 3E-thymidine was added to medium (0) were added at time 0. separate culture dishes at 4, 7, 10, 13, 16, 19 and 22 hours The points represent the aaouut of radioactivity respectively. in each culture dish expressed as CPM after a two hour pulse with 31i-thymidine. Mean f standard error (n=3).
S.A.
kYMhgDNA1
FIG. 3 Incorporation of 3Ekthymidine into DNA in response to serum derived from platelet rich or platelet poor plasma, the supernate from thrombin-stimulated platelets , the combination of serum from platelet poor plasma plus the supernate from thrombin stimulated platelets, serum from a thrombocytopenic rabbit and the combination of serum from a thrombocptopeuic rabbit plus the supernate from thrombin stimulated platelets. Each bar represents the specific-radioactiv expressed as CpM/ng DNA after a 24 hour pulse with Mean f standard error (n=4).
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incorpqrationinto DNA as smooth muscle cells cultured in serum prepared from platelet poor plasma (Fig. 3). Since the extent of 3H-thymidineincorporationover 24 hours occurred maximally in those cultureswhich showed the greatest vroliferationin the cell counting assessment,it is possible to use the 24 hour-3N-thymidineapproach to study the effect of mitogenic substanceson smooth muscle cells.
%OF
I)0
CONTROL I 0= 0
r
2.5
50
PERCENT IN GROWTH MMUM OF SUPERNATE FROM THROMBH-TREATED PLATELETS (IO6 PL/mm3 1 FIG. 4
Effect of different concentrationsof the supernate from thrombinstimulatedplatelets on smooth muscle cell DNA synthesis. 'Ihe platelet supernatewas added with 5% serum derived from platelet poor plasma (A) and without serum (n). The response to 5% platelet supernate plus serum was assigned a value of 100% and all other values calculatedas a percentage of this value. Hean+standard error (n=4).
The addition of different concentrationsof the supernatantfrom thrombin stimulatedplatelets to the smooth muscle cell cultures did not lead to stimulationof 3H-thymidineincorporationin those cultures to which serum In those derived from platelet poor plasma had not been added (Fig. 4). cultures containingserum derived from platelet poor plasma, the increase in 3H-thymidineincorporationinto the smooth muscle cell DNA was proportional to the amount of supernatantadded to the culture. When washed platelets were stimulatedwith different concentrationsof thrombin, the release of serotoninwas proportionalto the concentrationof thrombinused. The effect of the supernatantfrom the different suspensions of washed platelets treated with different thrombin concentrationson smooth muscle cell proliferationwas proportionalto the extent of thrombln induced release (Fig. 5). The addition of thrombin in combinationwith serum from platelet poor plasma did not significantlystimulate DNA synthesis.
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10-o I.0 THROMBIN CONCENTRATION (unltr/ml)
FIG. 5 Thrombin-inducedeerotonin release and rel ase of FDGF. 4) were treated Washed platelets (2 X lo6 platelets per um~ with several concentrationsof thrombin and the supernatant fluid was mixed with Eagle's MXM (5% v/v) containing 5% serum derived from platelet poor plasma. The amount of %Ithymidine incorporationinto DWA when the smooth muscle cells were exposed to the thrombin-treatedsupernate (5 U/ml) was assigned a value of 1GCl~X and the values obtained when lower concentrationsof thrombinwere used were expreased as a percentageof this value (0). I 14C-serotoninreleaae (0) is expressed as a percentage of the total amount of 14C-serotonin released by the platelets upon stimulationwith thrombin (5 U/ml). Mean *standard error (n=6).
Collagen also induced the release from platelets of the material which stimulated incorporationof %-thymidine into the DNA of cultured smooth The extent of gII-thymidine incorporationcaused by muscle cells (Fig. 6). the supernatantfrom collagen stimulatedplatelets paralleled the extent of serotonin release induced by collagen. In the presence of a constant smount of factor from thrombin stimulated platelets, the addition of increasing concentrationsof platelet poor serum was associatedwith increased incorporationof 3H-thymidineinto the smooth muscle cells (Fig. 7).
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100
OF50
% CONTROL
I/
03 0
111
I*5
UNDIWTED
COLLAGEN FIG. 6 Collagen-inducedserotouin release and release of PDGF. Several concentrationsof collagen were added to washed platelet suspensions (2 X 106 platelets per II&) and the supernatemixed with Eagle's MEH (5% v/v) containing 5% serum derived from platelet poor plasma. The amount of kthynidine incorporationinto DNA when the smooth muscle cells were exposed to the supernste from the undiluted collagen was assigned a value of 100% and the values obtained when dilutions of collagen were sed were expressed as a percentage'of 1t this value (e). Percent C-serotoninrelease is expressed as in Fig. 5 (0). Mean f standard error (n-4).
0
I2
3
4
5
PERCENT (V/V) IN GRGWTH MEblUMGFPtATELETPOOR PLASMA FIG. 7 Several concentrationsof serum prepared from platelet poor plasma were added to the smooth muscle cell cultures containing the supernatant fluid from thrombin stimulatedplatelets. The response to a serum concentrationof 5% was assigned a value of 100% and all other values expressed as a percentage of this value. Mean f standard error (n=5).
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DISCUSSION These results show that in addition to other species rabbit platelets 'alsocarry amitogenic factor(s) that stimulatesDNA synthesis in quiescent confluent rabbit arterial smooth muscle cells within 24 hours after its addition. This is in agreement with Rutherfordand Ross who have shown that quiescentmonkey smooth muscle cells synthesizeDNA 16 to 21 hours after the addition of PDGF (18). Furthermore,this stimulationof DNA synthesisway be estimated by measuring the incorporationof 3H-thymidineinto DNA over a 24 hour period and used as an index of cell proliferation. The use of this bioassay assumes that the substancesadded to the smooth muscle cells do not block uptake of thymidine or that they inhibit proliferationbut do not block In comparisonto the cell counting incorporationof 3B-thymidineinto DNA. technique or autoradiographythis method makes it possible to rapidly estimate the effect of growth factors on smooth muscle cell proliferation. The results are in agreement with those of Busch et al who observed that in human platelets, the appearance of PDGF seems to parallel to a large extent the release of serotonin induced by thrombin or collagen (19). PDGF is probably stored in a granules and appears to be released by lower concentrations of thrombln than those required to release the dense bodies or the acid hpdrolase granules (20). Thrombln has been reported to be atltogenicfor chick embryo flbroblasts (21). However, thrombinwas not mitogenic for rabbit arterial smooth muscle cells when added alone or in combinationwith serum derived from platelet poor plasma. The mechanism by which the growth factor stimulatescell proliferation islJuknown. Nishikawa and his colleaguespostulated that in addition to growth factors, serum also contains a "survival factor" of pituitary origin without which an ovarian cell line will not proliferate (22). Others have observed that 3T3 cell proliferationdepends upon the presence of both serum and the platelet-derivedmitogen (23-25). The present studies show that DNA synthesis and proliferation of arterial smooth muscle cells also occur only when both the platelet-derivedmitogen and platelet poor serum are present, and support the view that there is a factor in plasma which acts in cmbiuation with the platelet mitogen to stimulate DNA synthesis and cell proliferation. In agreementwith Busch et al (19) these results show that collagen, which is believed to be the principal factor In sub-endothellumwhich Induces the platelet release reaction from platelets adhering to the damaged vessel wall, like thrombin,also induces the release of the platelet mitogcn. The advantages of studying the effect of collagen on the platelet release of the mitogen are couaiderable,since it Is the platelets interactingwith the &ollagen surface of the vessel wall that will most likely deliver the mitogen in the highest concentrationto affect the smooth muscle cells just belw the basement membrane.
We wish to thank Mrs. D. Bloudwska for excellent technicalassistauce.
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This worlcwas supported by grants from the Medical Research Council of Canada (ET-2168and MT 1309) and from the Ontario Reart Foundation. Dr. J.-P. Cazenave is a Senior Research Fellow of the Ontario Heart Foundation.
1.
RQLLKY, B.A., KIERNAW, J.A. "Contact inhibzkion"of cell division in 3T3 cells. Proc. Natl. Acad. Sci. USA. 60, 300, 1968.
2.
BALK, S.D. Calcium as a regulator of the proliferationof normal, but not of transformed,chicken fibroblastsin a plasma-containing medium. Proc. Natl. Acad. Sci. USA. 68, 271, 1971.
3.
RQSS, R.J., GLQMSET, J., KARIYA, B. and RARKRR, L. A plateletdependent serum factor that stimulates the proliferationof arterial smooth muscle cells In vitro. Proc. Natl. Acad. Sci. USA. 71, 1207, 1974.
4.
KORLRR, N. and LIPTON, A. Platelets as a source of fibroblastgrowthpromoting activity. E&p. Cell Res. 87, 297, 1974.
5.
WBSTBRMARK, B. and WASTESON, A. A platelet factor stimulatinghuman normal glial cells. Exp. Cell Ree. 98, 170, 1976.
6.
AETONIADES,H.N. and SCRER, C.D. Radioimmrmoassayof a humaa serum growth factor for Balb/c-3T3 cells: Derivation from platelets. Proc. Natl. Acad. Sci. USA. 74, 1973, 1977.
7:
IRNATOWYCZ, X.0., MOORE, S. and MUSTARD, J.F. Platelet growth factor stimulationof DNA synthesis in smooth muscle cells in vitro. Fed. Proc. 36, 430, 1977.
8.
FRIEDMAN, R.J., SD, M.B., WENZ, B., MQORE, S., GAULIDE, J., GENT, R ., TIELL, M.L. and SPAKT, T.H. The effect of thrombocytopenlaon expermental arterioscleroticlesion formation la rabbits. I. Smooth muscle cell proliferationand re-endothelialization.J. Clin. Invest. 60, 1191, 1977.
9.
MOORE, S., FRIEDHAN, R.F., SINGAL, DS., GAULDIE, J., BLAJCBMAN,M.A., and ROBERTS, R.S. Inhibition of injury-inducedthrombo-atherosclerotic lesions by anti-plateletserum in rabbits. Thromb. Raemoetas. 35, 70, 1976.
10. ROSS, R. The smooth muscle cell. II. Growth of smooth muscle in culture aad formation of elastic flbres. J. Cell Blol. 50, 172, 1971. 11. LKYVA, A., aad KBLLBY, W.N. Measurement of DNA in cultured human cells. Analytical Biochem. 62, 173, 1974. and MUSTARD, J.F. Adenosine diphosphate12. ARDLIE, N.G., PACKRAI4,'M.A. induced platelet aggregation in suspensionsof washed rabbit platelets. Br. J. Raematol. 19, 7, 1970.
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13. CAZENAVE, J.-P., REIMERS, H.-J., KINLOUGH-RATHBONE,R.L., PACKHAM, M.A., and MUSTARD, J.F. Effects of sodium periodate on platelet functions. Lab. Invest. 34, 471, 1976. 14. MUSTARD, J.F., PERRY, D.W., ARDLIE, N.G. and PACKHAM, M.A. Preparation of suspensionsof washed platelets from humans. Br. J. Haematol. 22, 193, 1972. 15. MOLNAR, J., and LORAND L. Biophys. 93, 353, 1961.
Studies on apyrases. Archives Biochem.
16. GREENBERG, J., PACKHAM, M.A., CAZENAVE,J.-P., REAMERS, H.-J., and MUSTAtZD,J.F. Effect on platelet function of removal of platelet sialic acid by neuraminidase. Lab. Invest. 32, 476, 1975. 17. REIMERS, H.-J., KINLOUGH-BATHBONE,R.L., CAEENAVE, J.-P., SENYI, A.F., HIRSH, J., PACKHAM, M.A. and MUSTARD, J.F. In vitro and in vivo functions of thrombin-treatedplatelets. Thromb. Haemostas. 35, 151, 1976. 18. RUTHERFORD,R.B., and ROSS, R. Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate. J. Cell Biol. 69, 196, 1976. 19. BUSCH, C., WASTESON, A., and WESTFRMARK,B. Release of a cell growth promoting factor from human platelets. Thromb. Res. 8, 493, 1976. 20. WITTE, L.D., KAPLAN, K.L., NOSSEL, H.L., LAGES, B.A., WEISS, H.J., and GOODMAN, D.S. Studies of the release from human platelets of the growth factor for cultured human arterial smooth muscle cells. Circ. Res. 42, 402, 1978. 21. CHEN, L.B., and BUCHANAN, J.M. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc. Natl. Acad. Sci. USA. 72, 131, 1975. 22. NISHIKAWA, R., ARMELIN, H.A., and SATO, G. Control of ovarian cell growth in culture by serum and pituitary factors. Proc. Natl. Acad. Sci. USA 72. 483, 1975. 23. PLEDGER, W.J., STILES, C.D., ANTONIADES,H.N., and SCHER, C.D. Induction of DNA synthesis in Balb/c-3T3cells by serum components: re-evaluationof the commitmentprocess. Proc. Natl. Acad. Sci. USA 74, 4481, 1977. 24. VOGEL, A., RAINES, E., KARIYA, B., RIVEST, M.J. and ROSS;R. Coordinate control of 3T3 cell proliferationby PDGF and plasma components. Proc. Natl. Acad. Sci. USA. 75, 2810, 1978. 25. PLEDGER, W.J., STILES, C.D., ANTONIADES,H.N. and SCHER, C.D. An ordered sequence of events is required before BALB/c-3T3cells become committed to DNA synthesis. Proc. Natl. Acad. Sci. USA. 75, 2839, 1978.
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THE EFFECT OF A PLATELET-DERIVEDGROWTH FACTOR ON THE PROLIFERATIONOF RABBIT ARTERIAL SMOOTH MUSCLE CELLS IN TISSUE CULTURE
I. 0. Ihnatowycz,J.-P. Cazenave, J.F. Mustard and S. Moore Department of Pathology,McMaater University Hamilton, Ontario, Canada
(Received 19.9.1978; in revised form 30.11.1978. Accepted by Editor K.M. Brd.nkhous) ’ ABSTRACT Thrombin and collagen induced the release of a growth factor from washed rabbit plateletswhich requires a serum factor derived from platelet poor plasma for maximum proliferationof the smooth muscle cells. The release of the platelet-derivedgrowth factor by thrombin and collagen parallels the release of serdtonin from theee platelets. Therefore it is possible to use rabbit smooth muscle cells and rabbit platelets and %-thymidine to examine the effect of factors governing the release of the platelet-derivedgrowth factor and the factors governing its effect on smooth muscle cell proliferation. These results show that it is possible to examine the effect of collagen,which is the principal stimulus to platelet release in the vessel wall, in an in vitro system to rapidly study its effect on the release of the platelet-derivedgrowth factor.
INTRODUCTION
Serum contains factors which are essential for the proliferationof diploid, nontransformedcells in culture (1). Sfnce Balk's observationsthat serum was more effective In stimulatingchick embryo fibroblaststhan was plasma (Z).,it has been establishedthat platelets are a source of a factor which stimulates the in vitro proliferationof cells (3-7). The importance of platelets in the smooth muscle cell response to lntlmal Injury has been demonstratedby showing that severe thrombocytopeniain rabbits results in a marked inhibitionof both the developmentof proliferativesmooth muscle cell lesions in response to a single intimal injury (g), and the lipid containing raised lesions caused by continuous intimal injury (9). The aim of the experimentsreported in this paper was to determine whether the incorporationof 3Ii-thymidine into DNA could be used as a rapid 477
47%
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quantitativeassay of the effect of the platelet-derivedgrowth factor (PDGF) on arterial smooth muscle cell proliferationin tissue culture. We examined the relationshipbetween the appearance of serotonln and PDGF in the suspending fluid upon treatment of a washed platelet suspensionwith thrombin or collagen. In addition,we examined the effect of serum derived from plateletpoor plasma on the response of smooth muscle cells to PDGF. Some of the results described in this paper have been reported briefly elsewhere (7).
MATERIALS AND METRODS
The following isotopes were used: [methyl-3Hjl-thymidine t31i-Tdr, s.a. 6.7 Ci./mmole, obtained from New Rngland Nuclear, Boston, Mase.);L4C-serotonln (5-hydroxytryptamine-31-14C-creatinine sulfate; 14C-5HT, 8.8. SOmCi/mmole, obtained from Amersham/Searle,Arlington Reights, Illinois). Eagle's Minimum Essential Medium, F-15, penicillin:streptomycin,trypeln, fetal calf serum; and L-glut-e were all obtained from Grand Island Biological Company of Canada, Burlington,Ontario. Hepes was obtained from Sigma Chemical Co., St. Louis, Missouri. Topical bovine thrombinwas obtained from Parke, Davfs and Co., Detroit, Bovine tendon collagen was obtained from Sigma Chemical Co., Hchigan. St. Louis, msaouri. Arterial smooth muscle cells were grown from explants of segments of media from the thoracic aorta of 3-month old New Zealandwhite rabbits. The method used was similar to that of Ross (lo). A rabbit was anaesthetizsdwith sodium pentobarbital (50 mg/kg; obtained from M.T.C. Pharmaceuticals-, Ramilton, Ont.) and the thoracic aorta excised and stripped of the intimal and adventitial layers. The tunica media was cut into lmm3 sections and incubated at 37'C in Eagle's Minimum RssentialMedium, F-15, supplementedwith L-glutamine, 15% fetal calf serum which had been heat inactivatedat 56'C for 30 minutes, a penicillin: streptomycinsolution (lOOU/ml:lOOmg/ml)and 15mM Eepes at pH 7.3. The smooth muscle cells from the primary cultures were subculturedin the followingmanner for a maximum of five times. The surface of the culture The cells were flask was washed with phosphate buffered saline at pR 1.3. treated with 0.25% trypsin for 3 minutes and sparsely subculturedinto 150 cm2, Falcon tissue culture flasks containing 20 ml of the growth medium. At confluence, the smooth muscle cells from the second to the fifth subculturewere used for experiments. Smooth muscle cell proliferationwas assa ed in the followingmanner. Equal numbers of smooth muscle cells (1.5 x 10s ) were plated per 60 nxaFalcon culture dish containingEagle's Minimum Essential Medium, F-15, supplemented0 with L-glutamine, 15% fetal calf serum which had been heat inactivatedat 56 C for 30 minutes, a penicillin: streptomycinsolution (lOOU/ml:lOOmg/ml),15 mM Heges and 3% sodium bicarbonate. The smooth muscle cells were incubated at 37 C in humidified air containing5% CO? for approximately5 days. At confluence the medium was changed to Eagle s MPH, F-15, supplementedwith the agents mentioned above, but lacking fetal calf serum. At 24 hours the medium was again changed to fetal calf serum-freeEagle's MEM, F-15, prepared as above and containing the material to be tested. At this point one of 2 procedures was followed:
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a) Cell counting technique: Three culture dishes were taken from each experimentaland control group, the medium was removed and the cells were washed twice with phosphatebuffered saline. One millilitre of 0.25% trypsin was added to the culturesand incubated for 3 minutes at 37'~. The cell SUSpension was gently removed by Pasteur pipet and the number of cells counted on a hemacytometer. The remaining dishes were incubated at 37'C in humidified air in 5% CO2 for 12 days. Cell counts were carried out on days 3,6,9, and 12. 3 b) Incorporationof 3H-thymidineinto DNA: H-thymidinewas added to all the culture dishes to a final concentrationof 0.5 pCi/ml. At 48 hours the cultures were washed with phosphate buffered saline (pH 7.3) and solubilizedin 3 ml of 0.5 N NaOH. Five millilitresof 1.0 N HC104 were added to each sample, thoroughlymixed and cooled to 4'C for 30 minutes. After centrifugationat 10,000 g for 20 minutes the supernatantfluid was removed, 1 ml of 1.0 N HC104 was added to each sample and incubated at 70°C for 30 minutes. Each sample was cooled to room temperatureand centrifugedat 10,000 g for 20 minutes. One aliquot from each sample of the second supernatantfluid was taken to count the 3H-thymidineby liquid scintillation. A second aliquot was taken to determine the amount of DNA by a spectrophotometrictechnique (11). The specific radioactivityof the DNA was used as an index of cell proliferation. Platelet-richand latelet-poorplasma was prepared by the method of Ardlie, Packham and Mustard (12P . The platelet-poorplasma was further centrifugedat 12,000 g for 2 minutes to remove any remaining platelets. ‘lhrombin, 5 U/ml, and CaC12, 2mM, were added to the platelet-richand platelet-poorplasma. Following an incubationat 37OC for approximatelytwo hours and centrifugation at 12,000 g for 2 minutes, the supernatantsera were removed, heat inactivatedat 56'C for 30 minutes and dialysed overnight against Tyrode's solution at 4OC. The sera were referred to as serum derived from plateletrich plasma and serum derived from platelet-poorplasma respectively. Suspensionsof twice-washedrabbit platelets were prepared according to Ardlie et al (12), labelledwith 14C-serotonin(13) and finally resus ended at 37'C in Tyrode's solution containing0.35% albumin an Apyrase was prepared by the method of Molnar and Lorand 4dpy'"s~~1~;telet count was adjusted to the requirementsof each individualexperiment. Platelet aggregationstudies and measurementof the release of 14C_ serotonin 5 minutes after the addition of the aggregatingagents were carried out as described previously (16). An aliquot of each sample was assayed for its effect on smooth muscle cell proliferation. lhrombocytopeniain rabbits was produced by irradiationas described by Beimers et al (17) and the injectionof antiplateletserum as prepared by Moore et al (9). Rabbits that were made thrombocytopenicby this method had platelet counts of less than 1,000 per mm3. Blood was collected into 50 ml syringes, allowed to clot and centrifugedat 1000 g for 15 minutes. The supernatantserum was removed, heat inactivatedat 56'C for 30 minutes and referred to as serum from thrombocytopenicrabbits.
BBSULTS
To establish the validity of the assay for the rabbit platelet-derived growth factor, 5% platelet rich or poor serum and platelet supernate or a
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combinationof the platelet poor serum and platelet supernate,containing the material released from the washed platelets,were added to quiescent confluent rabbit smooth muscle cells (3.25 x 105 cells/culturedish). The cells proliferatedonly in response to the platelet rich serum or the combinationof platelet poor serum and the material released from the washed platelets (Fig. 1).
30NUMBER OF CELLS PER loCULTURE DISH (xl+)
0 3 6 9 NUMBER OF DAYS AFTER ADDITIONS
FIG. 1
Smooth muscle cell proliferationin response to serum derived from platelet-richplasma (0), serum derived from platelet-poorplasma (A), the supernatantfluid from a thrombin stimulatedwashed platelet suspension (A), or the combinationof serum derived from platelet poor plasma and the supernatantfluid from a thrombln stimulated washed platelet suspension (0). Mean 2 stsndard error (n-3)
The IncorporatIonof 3H-thymidineinto DNA was measured at 6,9,12,15,18, 21 and 24 hours following the addition of PDGF to confluentquiescent smooth muscle cell cultures (Fig. 2). Synthesisbegins at approximatelysix hours, reaches a maximal point at 15 to 16 hours, and decreases thereafter. Since the maximum incorporationof 3D-thylmidine into DNA occurred at 15 to 18 hours, the experimentswere set up to examine the incoroporationof 3%thymidine into DNA 24 hours after stimulatingwith PDGF and pulsing with 3H-thymidine. Next we examined the effect of culturing the smooth muscle cells in medium containing serum derived from platelet poor plasma and serum derived from plateTwenty-four let rich plasma on 3H-thymidineIncorporationinto DNA (Fig. 3). hours after stimulationthe maximum incorporationof 3H-thymidlneinto DNA was found in the smooth muscle cell cultures in serum derived from platelet rich plasma. Cells cultured in serum derived from platelet poor plasma or in the presence of the material released from washed platelets by thrombin did not The combinationof the lead to much incorporationof 3R-thymidineinto DNA. serum derived from platelet poor plasma and the supernatantfluid from the thrombin-stimulatedplatelets produced nearly as much 3H-thymidine
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CPM PER CULTURE DISH (xtcJ-9
TIME (hours) FIG. 2 Time-course of DNA eyntheeis in rabbit smooth muscle cells. 5% rabbit serum prepared from whole blood ( 0 ) or serum-free 3E-thymidine was added to medium (0) were added at time 0. separate culture dishes at 4, 7, 10, 13, 16, 19 and 22 hours The points represent the aaouut of radioactivity respectively. in each culture dish expressed as CPM after a two hour pulse with 31i-thymidine. Mean f standard error (n=3).
S.A.
kYMhgDNA1
FIG. 3 Incorporation of 3Ekthymidine into DNA in response to serum derived from platelet rich or platelet poor plasma, the supernate from thrombin-stimulated platelets , the combination of serum from platelet poor plasma plus the supernate from thrombin stimulated platelets, serum from a thrombocytopenic rabbit and the combination of serum from a thrombocptopeuic rabbit plus the supernate from thrombin stimulated platelets. Each bar represents the specific-radioactiv expressed as CpM/ng DNA after a 24 hour pulse with Mean f standard error (n=4).
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incorpqrationinto DNA as smooth muscle cells cultured in serum prepared from platelet poor plasma (Fig. 3). Since the extent of 3H-thymidineincorporationover 24 hours occurred maximally in those cultureswhich showed the greatest vroliferationin the cell counting assessment,it is possible to use the 24 hour-3N-thymidineapproach to study the effect of mitogenic substanceson smooth muscle cells.
%OF
I)0
CONTROL I 0= 0
r
2.5
50
PERCENT IN GROWTH MMUM OF SUPERNATE FROM THROMBH-TREATED PLATELETS (IO6 PL/mm3 1 FIG. 4
Effect of different concentrationsof the supernate from thrombinstimulatedplatelets on smooth muscle cell DNA synthesis. 'Ihe platelet supernatewas added with 5% serum derived from platelet poor plasma (A) and without serum (n). The response to 5% platelet supernate plus serum was assigned a value of 100% and all other values calculatedas a percentage of this value. Hean+standard error (n=4).
The addition of different concentrationsof the supernatantfrom thrombin stimulatedplatelets to the smooth muscle cell cultures did not lead to stimulationof 3H-thymidineincorporationin those cultures to which serum In those derived from platelet poor plasma had not been added (Fig. 4). cultures containingserum derived from platelet poor plasma, the increase in 3H-thymidineincorporationinto the smooth muscle cell DNA was proportional to the amount of supernatantadded to the culture. When washed platelets were stimulatedwith different concentrationsof thrombin, the release of serotoninwas proportionalto the concentrationof thrombinused. The effect of the supernatantfrom the different suspensions of washed platelets treated with different thrombin concentrationson smooth muscle cell proliferationwas proportionalto the extent of thrombln induced release (Fig. 5). The addition of thrombin in combinationwith serum from platelet poor plasma did not significantlystimulate DNA synthesis.
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10-o I.0 THROMBIN CONCENTRATION (unltr/ml)
FIG. 5 Thrombin-inducedeerotonin release and rel ase of FDGF. 4) were treated Washed platelets (2 X lo6 platelets per um~ with several concentrationsof thrombin and the supernatant fluid was mixed with Eagle's MXM (5% v/v) containing 5% serum derived from platelet poor plasma. The amount of %Ithymidine incorporationinto DWA when the smooth muscle cells were exposed to the thrombin-treatedsupernate (5 U/ml) was assigned a value of 1GCl~X and the values obtained when lower concentrationsof thrombinwere used were expreased as a percentageof this value (0). I 14C-serotoninreleaae (0) is expressed as a percentage of the total amount of 14C-serotonin released by the platelets upon stimulationwith thrombin (5 U/ml). Mean *standard error (n=6).
Collagen also induced the release from platelets of the material which stimulated incorporationof %-thymidine into the DNA of cultured smooth The extent of gII-thymidine incorporationcaused by muscle cells (Fig. 6). the supernatantfrom collagen stimulatedplatelets paralleled the extent of serotonin release induced by collagen. In the presence of a constant smount of factor from thrombin stimulated platelets, the addition of increasing concentrationsof platelet poor serum was associatedwith increased incorporationof 3H-thymidineinto the smooth muscle cells (Fig. 7).
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100
OF50
% CONTROL
I/
03 0
111
I*5
UNDIWTED
COLLAGEN FIG. 6 Collagen-inducedserotouin release and release of PDGF. Several concentrationsof collagen were added to washed platelet suspensions (2 X 106 platelets per II&) and the supernatemixed with Eagle's MEH (5% v/v) containing 5% serum derived from platelet poor plasma. The amount of kthynidine incorporationinto DNA when the smooth muscle cells were exposed to the supernste from the undiluted collagen was assigned a value of 100% and the values obtained when dilutions of collagen were sed were expressed as a percentage'of 1t this value (e). Percent C-serotoninrelease is expressed as in Fig. 5 (0). Mean f standard error (n-4).
0
I2
3
4
5
PERCENT (V/V) IN GRGWTH MEblUMGFPtATELETPOOR PLASMA FIG. 7 Several concentrationsof serum prepared from platelet poor plasma were added to the smooth muscle cell cultures containing the supernatant fluid from thrombin stimulatedplatelets. The response to a serum concentrationof 5% was assigned a value of 100% and all other values expressed as a percentage of this value. Mean f standard error (n=5).
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DISCUSSION These results show that in addition to other species rabbit platelets 'alsocarry amitogenic factor(s) that stimulatesDNA synthesis in quiescent confluent rabbit arterial smooth muscle cells within 24 hours after its addition. This is in agreement with Rutherfordand Ross who have shown that quiescentmonkey smooth muscle cells synthesizeDNA 16 to 21 hours after the addition of PDGF (18). Furthermore,this stimulationof DNA synthesisway be estimated by measuring the incorporationof 3H-thymidineinto DNA over a 24 hour period and used as an index of cell proliferation. The use of this bioassay assumes that the substancesadded to the smooth muscle cells do not block uptake of thymidine or that they inhibit proliferationbut do not block In comparisonto the cell counting incorporationof 3B-thymidineinto DNA. technique or autoradiographythis method makes it possible to rapidly estimate the effect of growth factors on smooth muscle cell proliferation. The results are in agreement with those of Busch et al who observed that in human platelets, the appearance of PDGF seems to parallel to a large extent the release of serotonin induced by thrombin or collagen (19). PDGF is probably stored in a granules and appears to be released by lower concentrations of thrombln than those required to release the dense bodies or the acid hpdrolase granules (20). Thrombln has been reported to be atltogenicfor chick embryo flbroblasts (21). However, thrombinwas not mitogenic for rabbit arterial smooth muscle cells when added alone or in combinationwith serum derived from platelet poor plasma. The mechanism by which the growth factor stimulatescell proliferation islJuknown. Nishikawa and his colleaguespostulated that in addition to growth factors, serum also contains a "survival factor" of pituitary origin without which an ovarian cell line will not proliferate (22). Others have observed that 3T3 cell proliferationdepends upon the presence of both serum and the platelet-derivedmitogen (23-25). The present studies show that DNA synthesis and proliferation of arterial smooth muscle cells also occur only when both the platelet-derivedmitogen and platelet poor serum are present, and support the view that there is a factor in plasma which acts in cmbiuation with the platelet mitogen to stimulate DNA synthesis and cell proliferation. In agreementwith Busch et al (19) these results show that collagen, which is believed to be the principal factor In sub-endothellumwhich Induces the platelet release reaction from platelets adhering to the damaged vessel wall, like thrombin,also induces the release of the platelet mitogcn. The advantages of studying the effect of collagen on the platelet release of the mitogen are couaiderable,since it Is the platelets interactingwith the &ollagen surface of the vessel wall that will most likely deliver the mitogen in the highest concentrationto affect the smooth muscle cells just belw the basement membrane.
We wish to thank Mrs. D. Bloudwska for excellent technicalassistauce.
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This worlcwas supported by grants from the Medical Research Council of Canada (ET-2168and MT 1309) and from the Ontario Reart Foundation. Dr. J.-P. Cazenave is a Senior Research Fellow of the Ontario Heart Foundation.
1.
RQLLKY, B.A., KIERNAW, J.A. "Contact inhibzkion"of cell division in 3T3 cells. Proc. Natl. Acad. Sci. USA. 60, 300, 1968.
2.
BALK, S.D. Calcium as a regulator of the proliferationof normal, but not of transformed,chicken fibroblastsin a plasma-containing medium. Proc. Natl. Acad. Sci. USA. 68, 271, 1971.
3.
RQSS, R.J., GLQMSET, J., KARIYA, B. and RARKRR, L. A plateletdependent serum factor that stimulates the proliferationof arterial smooth muscle cells In vitro. Proc. Natl. Acad. Sci. USA. 71, 1207, 1974.
4.
KORLRR, N. and LIPTON, A. Platelets as a source of fibroblastgrowthpromoting activity. E&p. Cell Res. 87, 297, 1974.
5.
WBSTBRMARK, B. and WASTESON, A. A platelet factor stimulatinghuman normal glial cells. Exp. Cell Ree. 98, 170, 1976.
6.
AETONIADES,H.N. and SCRER, C.D. Radioimmrmoassayof a humaa serum growth factor for Balb/c-3T3 cells: Derivation from platelets. Proc. Natl. Acad. Sci. USA. 74, 1973, 1977.
7:
IRNATOWYCZ, X.0., MOORE, S. and MUSTARD, J.F. Platelet growth factor stimulationof DNA synthesis in smooth muscle cells in vitro. Fed. Proc. 36, 430, 1977.
8.
FRIEDMAN, R.J., SD, M.B., WENZ, B., MQORE, S., GAULIDE, J., GENT, R ., TIELL, M.L. and SPAKT, T.H. The effect of thrombocytopenlaon expermental arterioscleroticlesion formation la rabbits. I. Smooth muscle cell proliferationand re-endothelialization.J. Clin. Invest. 60, 1191, 1977.
9.
MOORE, S., FRIEDHAN, R.F., SINGAL, DS., GAULDIE, J., BLAJCBMAN,M.A., and ROBERTS, R.S. Inhibition of injury-inducedthrombo-atherosclerotic lesions by anti-plateletserum in rabbits. Thromb. Raemoetas. 35, 70, 1976.
10. ROSS, R. The smooth muscle cell. II. Growth of smooth muscle in culture aad formation of elastic flbres. J. Cell Blol. 50, 172, 1971. 11. LKYVA, A., aad KBLLBY, W.N. Measurement of DNA in cultured human cells. Analytical Biochem. 62, 173, 1974. and MUSTARD, J.F. Adenosine diphosphate12. ARDLIE, N.G., PACKRAI4,'M.A. induced platelet aggregation in suspensionsof washed rabbit platelets. Br. J. Raematol. 19, 7, 1970.
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13. CAZENAVE, J.-P., REIMERS, H.-J., KINLOUGH-RATHBONE,R.L., PACKHAM, M.A., and MUSTARD, J.F. Effects of sodium periodate on platelet functions. Lab. Invest. 34, 471, 1976. 14. MUSTARD, J.F., PERRY, D.W., ARDLIE, N.G. and PACKHAM, M.A. Preparation of suspensionsof washed platelets from humans. Br. J. Haematol. 22, 193, 1972. 15. MOLNAR, J., and LORAND L. Biophys. 93, 353, 1961.
Studies on apyrases. Archives Biochem.
16. GREENBERG, J., PACKHAM, M.A., CAZENAVE,J.-P., REAMERS, H.-J., and MUSTAtZD,J.F. Effect on platelet function of removal of platelet sialic acid by neuraminidase. Lab. Invest. 32, 476, 1975. 17. REIMERS, H.-J., KINLOUGH-BATHBONE,R.L., CAEENAVE, J.-P., SENYI, A.F., HIRSH, J., PACKHAM, M.A. and MUSTARD, J.F. In vitro and in vivo functions of thrombin-treatedplatelets. Thromb. Haemostas. 35, 151, 1976. 18. RUTHERFORD,R.B., and ROSS, R. Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate. J. Cell Biol. 69, 196, 1976. 19. BUSCH, C., WASTESON, A., and WESTFRMARK,B. Release of a cell growth promoting factor from human platelets. Thromb. Res. 8, 493, 1976. 20. WITTE, L.D., KAPLAN, K.L., NOSSEL, H.L., LAGES, B.A., WEISS, H.J., and GOODMAN, D.S. Studies of the release from human platelets of the growth factor for cultured human arterial smooth muscle cells. Circ. Res. 42, 402, 1978. 21. CHEN, L.B., and BUCHANAN, J.M. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc. Natl. Acad. Sci. USA. 72, 131, 1975. 22. NISHIKAWA, R., ARMELIN, H.A., and SATO, G. Control of ovarian cell growth in culture by serum and pituitary factors. Proc. Natl. Acad. Sci. USA 72. 483, 1975. 23. PLEDGER, W.J., STILES, C.D., ANTONIADES,H.N., and SCHER, C.D. Induction of DNA synthesis in Balb/c-3T3cells by serum components: re-evaluationof the commitmentprocess. Proc. Natl. Acad. Sci. USA 74, 4481, 1977. 24. VOGEL, A., RAINES, E., KARIYA, B., RIVEST, M.J. and ROSS;R. Coordinate control of 3T3 cell proliferationby PDGF and plasma components. Proc. Natl. Acad. Sci. USA. 75, 2810, 1978. 25. PLEDGER, W.J., STILES, C.D., ANTONIADES,H.N. and SCHER, C.D. An ordered sequence of events is required before BALB/c-3T3cells become committed to DNA synthesis. Proc. Natl. Acad. Sci. USA. 75, 2839, 1978.
