Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle

217

Biochimica et Biophysica Acta, 560 (1979) 2 1 7 - 2 4 1 © Elsevier/North-Holland Biomedical Press

BBA 87063

PLATELET-DERIVED GROWTH FACTOR AND THE REGULATION OF THE M A M M A L I A N F I B R O B L A S T C E L L CYCLE

CHARLES D. SCHER a, ROBERT C. SHEPARD b, HARRY N. ANTONIADES c and CHARLES D. STILES b

a Department of Pediatrics, Sidney barber Cancer Institute and Children's Hospital Medical Center, ttarvard Medical School, Boston, MA 02115; b Laboratory o f Tumor Biology, Group W, Sidney Farber Cancer Institute and Department o f Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, and c Center for Blood Research and the Department o f Nutrition, Harvard University School o f Public Health, Boston, MA 02115 (U.S.A.) (Received January 30th, 1979)

Contents I.

Introduction

II.

The platelet-derived growth factor (PDGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structure and function of the platelet . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Recognition of PDGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Purification and chemical characterization of PDGF . . . . . . . . . . . . . . . . . . . . D. Growth factors related to PDGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

218 218 220 221 222

III.

Coordinate control of cell growth by PDGF and somatomedins . . . . . . . . . . . . . . . A. Biology of the 3T3 cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Platelet-poor plasma is required for the optimal growth response to PDGF . . . . . . . C. Somatomedins contained in plasma function coordinately with PDGF to control cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Complementation assay for other growth factors with PDGF-Iike activity . . . . . . .

223 223 224

IV.

............................................

The mammalian fibroblast cell cycle: PDGF initiates an ordered sequence of prereplicative events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General features o f the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The commitment event . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Restriction point control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Transition probability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Synergism of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Ordered sequence of replicative events . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Restriction point is distinct from the commitment (transition) event . . . . . . . . . . H. Exponential growth: PDGF can act during S phase . . . . . . . . . . . . . . . . . . . .

218

226 229 230 230 230 231 232 232 233 234 234

V.

PDGF and neoplastic transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Transformed cell lines: reduced PDGF requirement . . . . . . . . . . . . . . . . . . . . B. Abortive transformation by SV40 reduces the requirement for both PDGF and plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

234 234

VI.

Target cells for PDGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

236

VII.

PDGF and atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237

235

218 viii. Summary: coordinate control of fibroblast growth by PDGF and somatomedins . . . . .

237

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

238 239

I. Introduction Diploid animal fibroblasts and density-inhibited fibroblast cell lines require culture medium supplemented with serum for growth in vitro [1 -4]. The serum requirement for growth is usually reduced or eliminated when these cells are transformed by animal tumor viruses or by oncogenic agents, such as radiation [5-7]. Serum contains many components which function in different ways to support cell growth. Low molecular weight components, including amino acids [8,9], lipids [ 10], and metals [ 11,12], are required in trace amounts for cellular growth and may be provided by the serum supplement to cell culture medium. The transmembrane transport of low molecular weight nutrients may be facilitated by carrier proteins, such as transferrin (which carries iron) or hormones, such as insulin (which regulate glucose and amino acid transport [13]). Finally, serum contains factors which are not required in any nutritional sense for cell growth, but rather play a regulatory role in cell proliferation. In recent years, it has become evident that these growth regulatory factors, contained in serum, constitute a newly recognized class of hormones [13]. The most recent addition to this family of growth regulatory hormones is a low molecular weight polypeptide derived from platelets. The platelet-derived growth factor (PDGF) stimulates the growth of connective tissue cells in vitro [14-191. In whole animals, PDGF may play a role in wound repair and in maintenance of the vascular system [14,15]. The initial studies on PDGF focused on the in vivo role of PDGF and its possible involvement in atherosclerotic disease [15,20,21]. More recently, PDGF has been used as an experimental probe in studies on the hormonal control of the mammalian fibroblast cell cycle [22-26]. Conceptual insights into control of the fibroblast cell cycle gained from these studies with PDGF may prove generally applicable to growth regulation of a variety of animal tissues in vivo [25]. The recent purification of PDGF to homogeneity and its biochemical characterization [26,27] provides a natural vantage point for a review of these early studies with PDGF, which were conducted at the level of cell biology; future research activities on PDGF will undoubtedly be oriented in the directions of protein chemistry, membrane biology and molecular biology. The aim of this article is to consolidate and interpret the investigative data on PDGF on what is, at this writing, the fifth anniversary of the discovery of this novel growth regulatory hormone. II. The platelet-derived growth factor (PDGF)

IIA. Structure and Junction of the platelet Circulating blood platelets are anucleate, disc-shaped cells which have a major role in preventing bleeding. Platelets are produced in the bone marrow by the fragmentation of a precursor cell, tire megakaryocyte [28]. These megakaryocytes are giant, multinucleated cells (average claronaosome number, 16N) which make up less than 1% of all nucleated cells in human bone marrow [28]. Megakaryocytes arise from a multipotential stem cell capable of differentiating into erythrocytic, myelocytic (white cell) or megakaryocytic lines [29]. Recently, mouse megakaryocytes have been grown in tissue culture [30].

219 Electron microscopy studies reveal that megakaryocytes contain a Golgi complex, many ribosomes and prominent tubular channels wtfich extend to the exterior of the cell [31 ]. Granular organelles (the a-granules) are thought to be elaborated in the Golgi region [31]; in the 'mature' megakaryocytes, a-granules are found throughout the cytoplasm [31,32], with the exception of the region adjacent to the cell membrane. Normal adult human bone marrow contains about 6 • 106 megakaryocytes per kg of body weight. At cell maturity, the cytoplasm of each megakaryocyte fragments to form 10 a--104 platelets [28]. These platelets enter the bloodstream and circulate for about 10 days [33]. During its circulatory lifetime, the platelet progressively loses cytoplasmic substance. Little protein synthesis is noted in platelets and most studies have shown a virtual absence of polyribosomes [34]. The blood of healthy humans contains about 200 000 platelets/ml. Platelets play a multifaceted role in the maintenance of hemostasis, but three main functions have been recognized: (1) formation of hemostatic plugs in the walls of damaged blood vessels by platelet aggregation [35]; (2) release of a membrane phospholipid (platelet factor 3) [36] which accelerates clot formation by interacting with plasma coagulation factors VIII and V, ultimately allowing the conversion of fibrinogen to fibrin; and (3) maintenance of 'vascular integrity' through a nurturing action on endothelial cells [37]. The basis of maintaining vascular integrity is poorly understood. Platelets contain a number of organelles which store polypeptides and low molecular weight substances. These organdies release their contents only during the clotting process. The platelet a-granules, which are formed in the megakaryocytes, contain four known proteins: PDGF [38--40], platelet factor4 [41], fl-thromboglobulin [38], and platelet fibrinogen [41]. The presence of these proteins in the a-granules has been demonstrated by separating fractions of disrupted platelets on a sucrose gradient [38,40,41]. In addition, a single human patient with few platelet a-granules has reduced levels of PDGF, /3-thromboglobulin and platelet factor 4 [35]. While in the a-granules, PDGF is in a cryptic state and is not available to stimulate the replication of connective tissue cells. Similarly, the other a-granule proteins are also carried cryptically. The function and the protein chemistry of these other molecules has been studied. Platelet fibrinogen becomes converted to fibrin during clotting, platelet factor 4 neutralizes heparin (an anti-clotting factor) [42], while the function of/3-thromboglobulin remains unclear. Sequence analysis reveals that human platelet factor 4 (molecular weight 7767) and fl-thromboglobulin (molecular weight 8851) have extensive sequence homology with 42 of the 81 residues of/3-thromboglobulin identical to the residues in platelet factor 4 [43,44]. Both molecules have four half-cystine residues which occupy the same relative position. At low ionic strength, both molecules exist as non-covalently bound tetramers [45]. The genes for/3-thromboglobulin and platelet factor 4 arose from the duplication of a common ancestral gene [44]. The relationship of these molecules to PDGF is not known, because PDGF has not been sequenced. However, PDGF is a single chain polypeptide with a molecular weight of 13 000 and appears to contain disulfide bonds (see below). Storage organelles, other than the a-granules include the very dense granules which store serotonin, ATP, ADP, and Ca2+;these organelles are able to remove serotonin from plasma and concentrate it [46]. Another organelle, the membrane-bound vesicle, appears to contain lysosomal enzymes including hydrolytic proteases [47,48]. When circulating platelets are treated with ADP, thrombin, or collagen, or come into contact with traumatized tissue in vivo, a series of morphologic and biochemical changes

220 is rapidly initiated [35]. The regular discoid shape of the platelet is immediately transformed into a spherical form with long, slender cytoplasmic protrusions. This shape change is followed rapidly by platelet-to-platelet aggregation and the characteristic platelet release reaction which discharges the contents of the ~-granules, very dense granules, lysosomal enzymes and membrane fractions (containing platelet factor 3) into the microenvironment. The platelets lose about 10% of their total protein during this secretory process. The release reaction is, in part, autocatalytic because the release of ADP causes additional platelets to aggregate and release. The storage organelles are not secreted front the platelet with identical kinetics. Less thrombin is required to initiate the release of the a-granule contents than is required for secretion of serotonin or lysosomal enzymes [49]. Platelets represent a unique delivery system for PDGF. They have an affinity for injured sites, aggregating there and releasing their contents. Thus, the platelet specifically delivers PDGF to injured areas where it may stinmlate connective tissue cell replication and wound repair.

liB. Recognition of PDGF A critical observation which ultimately led to the discovery of PDGF was made by Samuel Balk in 1971 [14,50,51]. For a series of studies on the growth regulation of chick embryo fibroblasts in culture, Balk designed a tissue culture medium to closely approximate the interstitial fluid around cells in vivo. This medium differed from commercially prepared medium in one major way: platelet-poor plasma, which had been heated to 56°C to denature fibrinogen, was used to supplement the culture medium, rather than platelet-rich serum. Balk reasoned that serum is less physiologic than platelet-poor plasma because serum is only found in the vicinity of traumatized tissue. He observed that norreal chicken embryo fibroblasts proliferated more readily in culture medium supplemented with serum than in plasma-supplemented culture medium. Rous sarcoma virustransformed cells grew equally well in both. Balk [14,50] speculated, correctly, that a 'wound hormone' was released into serum or activated during the process of clot formation. These observations were greatly extended in 1974 by Ross and his associates [15] and also by Kohler and Lipton [16]. Working independently, these investigators confirmed that both platelet-poor plasma and serum derived from clotted, platelet-poor plasma were deficient in growth-promoting activity for monkey arterial smooth muscle cells and for mouse Swiss 3T3 cells in culture; a single isolate of SV40-transformed 3T3 cells grew well in plasma-supplemented medium, hnportantly, both of these laboratories demonstrated that an extract of platelets restored tile growth-promoting activity of plasma to a level comparable to that obtained with clotted, platelet-rich serum. Westermarke and Wasteson [17,52,53] have reported that hmnan platelets contain a factor that stimulates the growth of human brain glial cells in culture. Castor et al. [54,55] have studied a similar factor that promotes the growth of human synovial cells in vitro. Concurrent with these investigations, Antoniades and Scher were engaged in the purification, from clotted, human, platelet-rich serum, of a polypeptide growth factor which stinmlated tile division of Balb/c-3T3 cells. This serum-derived growth factor was found to be a heat-stable (IO0°C), cationic polypeptide (molecular weight 13 000) which was distinct from somatomedin [56,57]. It was largely destroyed by trypsin or chymotrypsin and completely inactivated by reduction with 2-mercaptoethanol. It appeared to be a major component of the growth-promoting activity in serum because treatment of serum with a cation-exchange resin ahnost completely removed the growth-stimulating activity

221 [57]. A radioimmunoassay for this serum growth factor demonstrated that it was relatively deficient in platelet-poor, plasma-derived serum and was present in platelets [18]. Furthermore, the growth-stimulating activity of serum and platelet preparations was dependent upon the concentration of the growth factor as determined by radioimmunoassay [18]. A heat-stable growth factor with similar antigenic determinants was also found in extracts of human pituitary glands [58].

IIC. Purification and chemical characterization of PDGF. The work of several independent laboratories [17,26,27,54,55,59] has demonstrated that PDGF is cationic (isoelectric point 9.8-10.0) and has a relatively low molecular weight on Biogel P100 columns. In addition, PDGF retains activity after isoelectric focusing or electrophoresis on SDS-polyacrylamide gels [17,26,27]. It is inactivated by reduction with 2-mercaptoethanol or by trypsin digestion [26,27]. PDGF has recently been purified to homogeneity [26,27]. Clinically outdated human platelets were concentrated by low speed centrifugation, washed and disrupted by freezing and thawing. Because PDGF is stable at 100°C, the platelet lysates were heated to 100°C for 10 rain. PDGF activity was found both in a flocculent precipitate, which was removed by low-speed centrifugation, and in the supernatant. PDGF was recovered from the precipitate by washing with l M NaC1. About 40% of the growth-stimulating activity was recovered after heating. After dialysis against a low ionic strength buffer, the heat: treated platelet extract was applied to a cation-exchange resin of CM-Sephadex at pH 7.4; approx. 95% of the growth-stimulating activity was adsorbed to the resin. After gradient or stepwise elution with NaC1, the PDGF was concentrated. The eluate had about a 400fold higher specific activity than the initial platelet lysates with DNA synthesis occurring at a concentration of 600 ng/ml. In agreement with the results of others [54,55], these eluates showed a single band of 13 000 daltons on an SDS-polyacrylamide gel;however, this material was not pure. Further purification was achieved by gel filtration chromatography under dissociating conditions. Chromatography was carried out on Biogel P150 in 1 M acetic acid;the activity eluted with a peak between 10 000 and 20 000 daltons. Preparative isoelectric focusing between pH 9 and 11 resolved a sharp activity peak at pH 9.8-10.2. The final purification step involved SDS-polyacrylamide gel electrophoresis under non-reducing conditions; the PDGF activity coincided with a single band of Coomassie brilliant blue staining material with an apparent molecular weight of 35 000. About 5 - 1 0 bands of inactive material were noted at 10 0 0 0 - 2 0 000 daltons. The eluted PDGF was active on Balb/c3T3 cells after removal of the SDS with an anion exchange resin [26,27]. The purification scheme is summarized in Table 1. Application of the purified PDGF to another SDS-polyacrylamide gel revealed a single band at 35 000 daltons. Reduction with 2-mercaptoethanol inactivated 95% of the growth-stimulating activity and the 35 000-dalton band disappeared; SDS-polyacrylamide gel electrophoresis revealed a single new band at 13 000 daltons [27]. For true molecular weight estimation, SDS-gel electrophoresis must be carried out under reducing conditions to assure complete binding of the SDS to all amino acid residues [60]. However, the 'aberrant' position of PDGF on unreduced SDS gels allowed the final purification of PDGF, because molecular weight sieving could be used twice for purification. The procedures described above and detailed elsewhere [27] allowed the purification of PDGF to a specific activity 20 • 106 times greater than that in serum and 150 000times greater than that in platelet homogenates. 18/ag pure PDGF were obtained from

222 TABLE l PURIFICATION O1" PDGF FROM CLINICALLY-OUTI)ATED IIUMAN PLATELETS The PDGF activity from 500 units of clinically outdated human platelcts (about 25 000 ml) was pufffled to homogeneity. The recoveries and specific activities of this particular PDGF preparation during the various stages of purification are smnmarized below• For comparative purposes only, the table includes the data which could bc indicated for whole hmnan serum containing PDGF activity equal to that of 500 units platelets. Purification step

Volume (ml)

Clotted serum Platelet lysates Heat (100°C) CM-Sephade x Biogel P-150 Electrofocusing S1)S gcl

1.5 2.0 1.0 1.5 4.0 9.0 4.0

10 s 103 104 101 101 101 101

Protein (rag)

PDGF (units)

1.1 7.8 3.8 6.0 1.0 5.0 1.8

2.6 2.6 1.0 7.2 7.2 2.0 3.7

- 107 104 103 101 101 10 -1 10-2

% Recovery

106 106 106 lOs lOs l0 s

100 40 28 28 7.8

104

1.5

Specific activity (units/rag) 2.4 3.25 2.66 1.2 7.2 4.0 5.0

•

10-1 101 102 104 104

l0 s 106

approx. 500 units o f clinically outdated human platelets; this quantity of human platelets represents the circulating platelet content of approx. 50 adult humans. Concentrations of pure PDGF as low as 10 -1 o M (1 ng/ml) stimulate replicative DNA synthesis in quiescent cultures of Balb/c-3T3 cells. Other polypeptide hormones such as insulin characteristiccally exert their biologic effect at 10 -1° M. PDGF fulfills tire biological criteria o f a polypeptide hormone. It is an informational molecule, carried in the blood, which acts at nanomolar concentrations on specific target cells. Unlike other polypeptide hormones, however, PDGF is not found in plasma, but is carried in the a-granules of platelets [ 3 8 - 4 0 ] where it is not available to stimulate the replication of cells. Platelets specifically adhere to injured areas and release PDGF [15,49]. Packaging a growth-stimulating hormone within the platelets provides a mechanism for the selective, hormone-modulated growth stimulation o f connective tissue cells, which are ubiquitously distributed throughout the b o d y . liD. Growth factors related to PDGF Platelets may contain more than one growth factor. An anionic stimulator of DNA synthesis, constituting about 5 - 1 0 % o f the total growth-stimulating activity for Balb/c3T3 and glial cells has been described [17,27]. This growth-stimulating activity has not been purified to homogeneity. Sirbasku [61] has demonstrated that platelets contain a factor that stimulates the growth of rat epithelial mammary cells. This growth factor is distinct from PDGF because it is destroyed by heating at 70°C. Gel filtration indicates an apparent molecular weight o f 30 0 0 0 - 5 0 000. The activity is partially destroyed by trypsin. A platelet protein that helps sustain the growth of SV40-transformed Swiss 3T3 cells has also been described [62]. This protein is destroyed by sulfhydryl reduction; gel filtration indicates an apparent molecular weight o f about 70 000. Growth factors have been isolated from other tissues, including the mouse submaxillary gland [63] and the bovine pituitary [64]. Some o f these factors resemble PDGF biochemically. Sato, Armelin, Holley, Gospodarowicz and others have shared in the important discovery that the pituitary gland contains growth factors for fibroblast cells.

223 Gospodarowicz [65] has isolated a polypeptide growth factor from bovine pituitary glands, termed pituitary FGF. Like PDGF, this polypeptide has a molecular weight of 13 400 on reduced SDS-gels and an isoelectric point of 9.6. Two half-cystine residues are present [65]. Unlike PDGF, however, bovine pituitary FGF is acid-labile and inactivated at 70°C. It maximally stimulates cellular DNA synthesis at a concentration of 1 - 1 0 ng/ ml [65,66]. Similar growth factors have been isolated from bovine brain after mild acid extraction. Two forms of bovine FGF are active in stimulating DNA synthesis. Both are degradation products of brain basic myelin [66,67]. However, basic myelin itself has no growthstimulatory activity [67]. FGF-1 has a molecular weight of 13 000 and FGF-2 of 11 700. Both have the same N-ternainal amino acid, phenylalanine. Furthermore, tryptic digests and mapping show that FGF-1 has 16 peptides and FGF-2 has 13, all identical with those of FGF-1. Both FGF-1 and FGF-2 have isoelectric points of 9.6. No half-cystine residues are present in either form. Like pituitary FGF, both are acid-labile and inactivated at 70°C. FGF-1 is maximally active at 100 ng/ml and FGF-2 at 50 ng/ml [66]. Serum also contains growth factors that resemble PDGF; Nishikawa et al. [68] described a basic polypeptide growth factor (termed CGF) in fetal calf serum which stimulates the growth of normal ovarian cells in culture. This basic ovarian growth factor may be identical to PDGF, which is also cationic and is liberated into serum during the clotting process. I!I. Coordinate control of cell growth by PDGF and somatomedin

IliA. Biology of the 3T3 cell PDGF stimulates the replication of a variety of mesenchyme-derived cells in vitro (see Section IV). However, the most detailed studies of the cellular response to PDGF have been conducted with mouse 3T3 cells in vitro [16,19,22-26,59,110-113]. There is good justification for using 3T3 cells to study the biologic action of PDGF. Clonal isolates of 3T3 cells are used in laboratories throughout the world. A variety of well-characterized RNA and DNA tumor viruses can transform 3T3 cells and alter their growth requirements for PDGF (see Section V). Finally, 3T3 cells have been extensively used as model systems to study control of the mammalian cell cycle [22-26,59,69]. As will be noted in this section and in section IV, PDGF seems to function by controlling an early event in the Go or G1 phase of the fibroblast cell cycle. Because so many of the studies on PDGF function have been conducted with 3T3 cells in culture, a brief synopsis of 3T3 cell history and growth characteristics is in order prior to a detailed discussion of PDGF function. Tile original 3T3 cell line was established in 1963 by Todaro and Green [70]. These investigators disaggregated mouse embryo cells and placed them into tissue culture; the cells were passaged on a rigid subcultivation schedule being transferred every three days at a density of 3 • l0 s cells per 60 mm culture dish. After 10-20 serial passages in vitro, the mouse embryo cells became subtetraploid and acquired cellular immortality. The original Swiss 3T3 cell line established in this fashion was derived from random-bred Swiss mouse embryos [70]. Subsequently, the same subcultivation protocol was used to establish 3T3 cell lines from inbred Balb/c mice (Balb/c-3T3) [71] and from random-bred NIH Swiss mice (NIH/3T3) [72]. Like normal fibroblasts, all of the 3T3 cell lines exhibit anchorage dependence [73] and density inhibition of cell growth [74]. Furthermore, the 3T3 cell lines do not form tumors when inoculated into immunosuppressed animal hosts such as nude mice [19,75,76]. The growth and final saturation density of the 3T3 lines in

224 vitro is controlled by the concentration of serum added to the culture medium [77,78]. Exposure to tumor viruses or to other oncogenic agents in vitro drastically alters the growth behavior of 3T3 cells. They lose density-dependent inhibition of growth [79], often lose anchorage-dependence [73] and often have a decreased serum requirement [80]. Furthermore, many of these transformed cell lines are tumorigenic in animal hosts [19,75,76]. When 3T3 cells are plated sparsely in medium containing an optimal concentration of either human or bovine serum, they replicate in an exponential fashion with a generation time of about 18 h [81]. The growth cycle of exponentially dividing 3T3 and other cells classically is divided into four discrete phases: G1, the period between mitosis and DNA synthesis (duration 6 h); S, the period of DNA synthesis (duration 8 h); G2, the period between DNA synthesis and mitosis (duration 3 h) and M, mitosis (duration 1 h) [81]. When cultures of 3T3 cells growing in 10% calf serum supplemented medium reach the confluent monolayer stage, replicative DNA synthesis and cell division are arrested [81 ]. These density growth-arrested 3T3 cells contain a G1 content of DNA, indicating that growth-arrest occurs at a discrete region of the cell cycle prior to the S phase. However, these cells do not appear to be arrested in the Gl phase of the cell cycle because the stimulation of DNA synthesis, by either the addition of serum, or by seeding the cells in fresh medium at a low density, occurs after a constant lag of at least 12 h in duration. This quiescent state has been termed Go and it has been proposed that it lies outside the proliferative stage of the cell cycle [82,83]. Augenlicht and Baserga [84] have demonstrated that growth arrested human embryo fibroblasts may progressively go into a 'deeper' Go.

IIIB. Platelet-poor plasma is required Jbr the optimal growth response to PDGF Serum, which contains both PDGF and plasma, supports the growth of 3T3 cells. Pledger et al. [22] and, later, Vogel et al. [59] demonstrated that the components of platelet-poor plasma are required for an optimal growth response to PDGF. Pledger et al. [22] added partially purified PDGF to confluent Go-arrested Babl/c-3Te cells in the presence of various quantities of platelet-poor plasma. Continuous incubation of cells with partially purified PDGF alone or with plasma alone could induce a significant fraction of the cells to synthesize DNA but only when high concentrations of either agent were used. In contrast, a low concentration of PDGF and of plasma stimulated replicative DNA synthesis when cells were incubated with these agents in combination (Fig. 1). Thus, PDGF and plasma function synergistically to promote S phase entry in quiescent density-arrested Balb/c-3T3 cells. The induction of replicative DNA synthesis in Balb/c3T3 cells exposed to PDGF and plasma was always preceded by a minimum lag time of 12 h characteristic of Go-arrested cells. A kinetic analysis demonstrated that PDGF was not required continuously in the culture medium for the induction of DNA synthesis. The length of time during which PDGF is required in the culture medium for subsequent DNA synthesis to occur is a function of PDGF concentration [22]. At high concentrations, PDGF preparations are required only briefly in the culture medium [21,22,25] ; whereas at lower concentrations, PDGF is required for a prolonged time [22] (Fig. 2). Similarly, the length of time during which PDGF is required in the culture medium for an optimum mitogenic response is a function of temperature; at 37°C, PDGF functions rapidly, whereas treatment with PDGF at 25°C or 4°C is less effective [22] (Fig. 2). The concentration of plasma controls, in part, the rate of entry of PDGF-treated cells into the S phase. Confluent Balb/c-3T3 cells were pulsed with partially purified PDGF

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Fig. 1. Effect of concentration of platelet-poor plasma on platelet extract-induced DNA synthesis. Platelet extract (e, 100 ug: o 10 ~g; a, 5 ug; or X, 0 ug) was added to cultures in 0.2 ml medimn containing [3tlldThd and various concentrations of platelet-poor plasnla, Cultures were fixed 36 h later and processed for autoradiography. Fig. 2. Temperature dependence of platelet extract-induced commitment to DNA synthesis. Cultures were incubated with the platelet extract in 0.2 ml medium at 37°C (I, 100#g;A, 50 ug), 25°C (~, 100 /~g; n,, 50/~g), or 4°C (a, 100 ~g, ~, 50/~g). At the indicated times, the platelet extract was removed and the cultures were washed with 28 mM 2-mercaptoethanol followed by medium. The cultures were placed in 0.2 ml medium containing [3H]dThd and 5% platelet-poor plasma. Cultures were fixed and processed for autoradiography at 36 h.

and transferred to medium containing various concentrations o f platelet-poor plasma. The data were plotted graphically according to the fashion of Smith and Martin [85] with the log of the percent unlabeled nuclei plotted versus hours. The rate and number of cells that entered the S phase was a function of the plasma concentration (Fig. 3). In 5% plasma, cells entered the S phase more rapidly than in 0.25% plasma. In these experiments, all cells received the same treatment with PDGF; hence the number of cells that synthesized DNA was determined by the plasma concentration. Because PDGF and plasma are both needed for the optimal stimulation of DNA synthesis, it seemed likely that they control different events in the cell cycle. To demonstrate that PDGF and, plasma regulate different events, quiescent cultures of confluent Balb/c3T3 cells were pulsed with partially purified PDGF and transferred to medium containing no plasma; at intervals (0, 5, I0 and 13 h later) the cells were transferred to medium conraining an optimal (5%) ~ , suboptimal (0.25%) concentration of plasma. The cells rapidly began to enter the S phase 12 h after the addition of 5% plasma, whether plasma was added at the time that PDGF was removed or as long as 13 h later. The addition o f 0.25% plasma did not increase the rate of cell entry into the S phase. Thus, a platelet-poor plasma concentration of 0.25% did not allow the entry of competent cells into the S phase of the cell cycle, whereas a concentration of 5% did (Fig. 4). In summary, the experiment depicted in Fig. 4 demonstrates that PDGF and plasma control different events in the cell cycle. PDGF induces cells to become ' c o m p e t e n t ' to synthesize DNA. Plasma allows competent cells, but not incompetent cells (that had not been treated with PDGF), to 'progress' through Go and G~ and enter the S phase. It appears likely that PDGF primes cells to respond to other growth factors in plasma, which in turn regulate DNA synthesis. The PDGF-induced competent state is stable for at

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Fig. 3. Effect of concentration of platelet-poor plasma on the rate of cell entry into the S phase. Cultures were treated with 50 ug platelet extract at 3 7 ° C i n 0.2 ml medium supplemented with [3H]dThd and platelet-poor plasma ( . , 0.25%, o, 2.5%, ~, 5%). Cultures were fixed at the times indicated and processed for autoradiography.

Fig. 4. The stability of the platclet extract-induced committed state. (A) Cultures were treated with 50 ~g platelet extract at 37°C in 0.2 ml medium containing 5% platelet-poor plasma and [3H]dThd. At the indicated times, cultures were fixed and processed for autoradiography. (B-E) Cultures were treated with 50 ug platelet extract in 0.2 ml medium for 5 h (3,) at 37°C washed, and returned to 0.2 ml medium containing [3H]dThd but lacking platelet-poor plasma. At the times indicated (l) the medium was supplemented with platelet-poor plasma (o, 5%; o, 0.25%). The cultures were fixed and processed for autoradiography at time intervals.

least 13 h after PDGF is removed; cells enter the S phase 12 h after plasma addition [22]. The stability of the competent state, together with the requirement for plasma growth factors, allowed Stiles et al. [25] to define and detect other growth factors with competence activity (See Section IIID). IIIC. Somatomedins, contained in plasma, function coordinately with PDGF to control cell growth Temin [86] demonstrated that G0-arrested chick embryo fibroblasts can be stimulated to replicate by the addition of insulin at hyperphysiological concentrations. Insulin bears an amino acid sequence relationship to a family of polypeptide hormones collectively termed 'somatomedins' [87]. The concentration of somatomedins in blood is controlled by the level of pituitary growth hormone. Human patients with acromegaly contain elevated levels of somatomedin, whereas hypopituitary dwarfs contain very little somatomedin. For this reason, somatomedins have been postulated to play a central role in developmental growth of animals by mediating the effects of pituitary growth hormone on skeletal and extraskeletal tissue [87]. In accord with the postulated growth-mediating action of somatomedins, these factors stimulate the formation of chondroitin sulfate by cartilage explants in vitro [87] ; however, in contrast to prediction, somatomedins have shown only weak growth activity in vitro. Somatomedins, like insulin, stimulate DNA

227 synthesis in chick e m b r y o fibroblasts in vitro [ 8 8 - 9 1 ] ; however, DNA synthesis is not stimulated to the extent observed when serum is added to the culture medium, and relatively high concentrations of somatomedins are required. The addition o f highly purified somatomedins to G0-arrested Balb/c-3T3 cells does not stimulate DNA synthesis [56,57], demonstrating that somatomedins alone do not control S phase entry [25]. The role of somatomedins and of insulin in regulating the growth of fibroblast cells in culture was recently clarified by Stiles et al. [25]. Plasma from hypophysectomized rats was 20-fold less efficient in allowing PDGF-treated competent cells to enter the S phase than plasma from normal rats (Fig. 5). Addition o f physiological concentrations o f pure somatomedin C (10 -9 M) to PDGF-treated cells in hypophysectomized plasma, allowed the cells to rapidly enter the S phase (Fig. 6). Pure somatomedin C alone did not promote the DNA synthesis o f PDGF-treated cells, indicating the presence o f other materials in hypophysectomized rat plasma which were essential for cellular growth. These experiments demonstrated that somatomedin C is a critical component o f plasma which is required for PDGF-treated, competent cells to progress through the Go/G1 phase o f the cell cycle and synthesize DNA. The use of hypophysectomized rat plasma allowed the development of an assay for progression factors under pituitary control [25]. Confluent growth-arrested Balb/c-3T3

60

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5o

20: I

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Ol

I0

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% PLATELET-POOR PLASMA

o'h2

18

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30

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HOURS AFTER 40DITION OF PDGF

Fig. 5. Plasma prepared from hypophysectomized rats is deficient in progression activity. Densityarrested Balb/c-3T3 cells were treated with 57 ~g partially-purified PDGF for 3 h. The culture medium was removed. Cell monolayers were washed once with saline containing 28 mM 2-mercaptoethanol and once with saline only. Fresh medium supplemented with 5/aCi/ml [3H]dThd was added to the cultures. Plasma from hypophysectomized (e) or normal (o) rats was added to the indicated concentration. After 24-h incubation at 37°C, the monolayers were fixed and processed for autoradiography. Fig. 6. The addition of pure somatomedin C to hypophysectomized rat plasma restores progression factor activity. Density-arrested Balb/c-3T3 cells were treated with PDGF, washed and placed in [3H]dThd medium as in Fig. 5. The cells were fixed at intervals and processed for autoradiography. The data are plotted after the fashion of Smith and Martin [103]. (A) The culture medium was supplemented with 3% plasma from control (o) or hypophysectomized (o) rats. (B) Culture medium was supplemented with 3% plasma from hypophysectomized animals (o), 3% plasma from hypophysectomized animals plus 3 ng/ml pure somatomedin C (e) or 3 ng/ml pure somatomedin C only (zx).

228 cells were e x p o s e d b r i e f l y to p a r t i a l l y p u r i f i e d P D G F ; these c o m p e t e n t cells were t h e n t r a n s f e r r e d to c u l t u r e m e d i u m c o n t a i n i n g p l a s m a f r o m h y p o p h y s e c t o m i z e d rats. The samples to be assayed for progression activity were a d d e d t o this assay m i x t u r e . All a u t h e n t i c s o m a t o m e d i n s h a d progression activity in this assay. G r o w t h h o r m o n e , ' s o m a t o m e d i n ' B ( w h i c h is n o t a true s o m a t o m e d i n ) , a n d t h y r o i d h o r m o n e did n o t s t i m u l a t e D N A synthesis. H y d r o c o r t i s o n e did, b u t o n l y at h y p e r p h y s i o l o g i c a l c o n c e n t r a t i o n s ( 1 0 -s M). Insulin also s t i n m l a t e d D N A s y n t h e s i s at h y p e r p h y s i o l o g i c a l c o n c e n t r a t i o n s ( 1 0 -T M), as w o u l d be e x p e c t e d , since insulin b i n d s w e a k l y to the cellular s o m a t o m e d i n r e c e p t o r [ 2 5 ] . Nerve g r o w t h f a c t o r , w h i c h shows partial a m i n o acid s e q u e n c e h o m o l o g y w i t h p r o i n s u l i n [ 9 4 ] , h a d n o activity. These e x p e r i m e n t s , w h i c h are s u m m a r i z e d in Table I1, d e m o n s t r a t e t h a t s o m a t o m e d i n s regulate, in p a r t , the e n t r y o f P D G F - t r e a t e d B a l b / c - 3 T 3 cells i n t o tire S phase. The w e a k progression activity o f insulin p r o b a b l y a c c o u n t s for the g r o w t h p r o m o t i n g activity o f this h o r m o n e (at h y p e r p h y s i o l o g i c a l c o n c e n t r a t i o n s ) in a variety o f cell c u l t u r e s y s l e m s [ 8 6 , 9 2 , 9 3 ] .

TABLE II QUANTITATION OF PROGRESSION OR COMPETENCE ACTIVITY Progression activity was titrated (see Ref. 25): a unit of progression acticity equals the quantity which must be added to 3% hypophysectomized rat plasma to promote DNA synthesis to the degree obtained with 354 normal rat plasma, l:or competence testing, cells were exposed for 3 h to serial dilutions of growth factor; control cultures were exposed to 592 normal human plasma. The cell monolayers were then washed and incubated 36 h with Dulbecco's modified Eagle's medium containing 5c,:; normal human plasma and [3lt]dThd (5 gCi/ml). The cells were fixed and processed for autoradiography. Under these conditions, less than 5% of cells from control cultures synthesized DNA; a unit of competence activity equals the quantity of material which must be added for 3 h to stimulate subsequent DNA synthesis in 6 0 - 9 5 % of the cells. Each tissue culture well contained 200 ~sl medium; hence, final concentrations of all growth factor can be derived by multiplying the quantities indicated X 5 m1-1. Treatment

Progression activity

Competence activity

Somatomedin A * Somatomedin C 1LA * MSA BRLC medium * Insulin Hydrocortisone

Potent: 20 ng/unit Potent: 2 ng/unit Potent: 100 ng/unit Potent: 2 ng/unit Potent: 6 ~l/unit Weak: 160 ng/unit Weak: 724 ng/unit

Weak: 300 ng/unit None: at 200 ng Not tested None: at 200 ng Weak: 50 ~l/unit None: at 120 ng None: at 724 ng

Growth Hormone * Somatomedin B T-3 NGF EGF

None: at 200 ng None: at 5 ng None: at 132 ng None: at 200 ng Partial: see text

None: at 200 ng None: at 5 ng Not tested None: at 200 ng None: at 200 ng

FGF PDGF * Ca3(PO4) 2 Wounding Human serum Human plasma

None: at 200 ng None: at 50 ~g None: at 558/~g None Potent: 2 ~l/unit Potent: 2 ~l/unit

Potent: 2 ng/unit Potent: 5 ng/unit Potent: 558 ug/unit Potent Potent: 10 ~l/unit None: at 200 ~1

SV40

Potent: 6 pfu/cell

Potent: 6 pfu/eell

* These growth factors were only partially purified.

229

IIID. Complementation assay/'or other growth factors with PDGF-like activity FGF [65,66] and precipitates of Ca3(PO4)2 [95,96] stimulate DNA synthesis in Balb/ c-3T3 cells. However, these agents did not allow PDGF-treated competent cells to enter the S phase in the presence of hypophysectomized rat plasma [25]. Thus, these potent stimulators of DNA synthesis did not have progression activity. A simple complementation test was developed to assay growth factors for the 'competence' activity which characterizes PDGF [25]. This test exploited the finding that competence factors such as PDGF function synergistically with the progression factors found in normal platelet-poor plasma to induce DNA synthesis in confluent Balb/c-3T3 cells; additionally, cells require only transient, early exposure to competence factors for DNA synthesis, whereas the progression factors, contained in normal plasma, are required continuously [22,23]. Stiles et al. [25] demonstrated that FGF and precipitates of Caa(PO4)2 function by inducing competence. Transient treatment (3 h) with either pure FGF or Ca3(PO4)= was sufficient to induce subsequent DNA synthesis. The induction of DNA synthesis by either of these agents was promoted synergistically by the progression factors found in normal platelet-poor plasma, but not by partially purified PDGF (Fig. 7) [25]. 'Wounding' of confluent 3T3 cell monolayers, by scraping a clear area through the cell sheet, induces replicative DNA synthesis in those cells bordering the wound [25]. Wounding induces competence since plasma, but not PDGF, promoted DNA synthesis in the Balb/c-3T3 cells adjacent to the wound. The cells in the intact monolayer did not respond. These results are summarized in Table II. Other growth factors were assayed for competence activity. It was fotmd that the somatomedins, insulin, hydrocortisone, thyroid hormone and growth hormone did not have competence activity. Furthermore, those agents which had competence activity did 8

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20 25

5

7'5

10

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50

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150

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PLATELET EXTRACT (]~]/ml)

Fig. 7. A c o m p l e m e n t a t i o n test for competence factors. (A) Density-arrested Balb/c-3T3 cells were exposed for 3 h to 10 ng FGF (=), 57 ~g PDGF (e), 18 m M Ca3(PO4)2 (~) or were w o u n d e d by streaking a cleared area through the cell m o n o l a y e r (o). Control cultures (A) were exposed for 3 h to 5% normal h u m a n plasma. After 3 h, all cultures were washed as described for Fig. 1. Fresh m e d i u m containing 5 #Ci/ml [3H]dThd and normal h u m a n plasma at the indicated concentration was added. After 36 h, the cultures were fixed and processed for autoradiography. (B) All cultures were incubated for 3 h with partially purified PDGF at the concentration indicated on the abscissa. After 3 h, the PDGF was removed. The cells were washed and [ 3 H l d T h d was added as in Fig. 1. Some o f the PDGFtreated cell cultures were then ' w o u n d e d ' (e). Other cultures were s u p p l e m e n t e d with 10 ng F G F (m) or 18 mM calcium p h o s p h a t e (~). Control cultures (o) were incubated in m e d i u m only. After 36 h, the cell cultures were fixed and processed for autoradiography.

230 not have progression activity and vice versa (Table II). Thus, distinct agents control different events in the cell cycle. Serum induces DNA synthesis in Balb/c-3T3 cells because it contains all classes of growth regulatory factors [25,26]. IV. The mammalian fibroblast cell cycle: PDGF initiates an ordered sequence of prereplicative events

IVA. General features o f the cell cycle The replicative cycle of fibroblast cells such as 3T3 is under stringent regulatory control. The cells proliferate in an exponential fashion until they deplete the medium of an essential nutrient or growth factor. They then become growth-arrested in the Go phase of the cell cycle. Cells transformed by retroviruses or DNA viruses may continue to profifcrate under these conditions. Thus, normal cells are distinguished from transformed cells by their ability to undergo growth-arrest [97]. The molecular signals that regulate cell proliferation and growth arrest are not understood. Studies of the cell cycle to date have been largely descriptive in nature. Nonetheless, these studies have provided an important and necessary framework for future investigations at a molecular level. Several models of cell cycle regulation have been proposed. Each of these models has been a reflection of the tools employed to study growth. Initial investigators employed whole serum to stimulate arrested populations to proliferate. More recently, several groups have used defined growth factors. PDGF has proved to be an exceptionally useful tool for cell cycle analysis. IVB. The commitment event Because 3T3 cells become growth-arrested 12 h prior to DNA synthesis, it has been proposed that tile critical growth regulatory event(s) occur in the Go or G1 phase of the cell cycle. In addition, there is a marked variation in the length of both Go/G1 in arrested populations and G~ in exponentially growing populations of cells [85]. Tire other stages of the cell cycle (S, G2, and mitosis) are relatively invariant in duration [85,98]. It appears that each cell in a population, whether growing or arrested, makes a critical decision, at some point before the S phase, to synthesize DNA. Each cell in the population takes a different length of time to reach this decisive point, giving rise to a marked variability in the duration of the Go/G~ phase of the cell cycle. This critical event has been termed the commitment point, implying an irreversible cellular decision to enter the S phase and complete the growth cycle. Other terms for this event, transition point [85] or restriction point [97], are also widely used and discussed below. Todaro and Green [74] were the first investigators to study the commitment of Goarrested 3T3 cells to DNA synthesis. They grew populations of Swiss 3T3 cells in 10% serum until they reached confluence and became growth-arrested. It now appears that the density-arrested cells stopped replicating because they had depleted serum of PDGF; addition of fresh PDGF to the serum-depleted medium stimulates DNA synthesis and cell replication [18], whereas the addition of whole plasma [18] or somatomedins [56,57] does not. Todaro and Green [741 studied the 'conrnlitment' event by treating cells with fresh whole serum for various lengths of time and then returning them to serum-depleted medium. They found that only a short period of time ( 4 - 6 h) was required to 'commit' the majority of cells to DNA synthesis. In contrast to the results of Todaro and Green, Brooks [98,99] found that a long period of time was required to commit cells to DNA synthesis. Swiss 3T3 cells were shifted

231 down to a very low (0:25%) serum concentration to achieve a Go growth arrest. The cells were then pulsed with whole serum for various lengths of time and returned to 0.25% serum. At a concentration of 0.25% all serum growth factors, including both PDGF and somatomedin, are limiting. Brooks [99] found that some cells had entered the S phase before the majority became committed to DNA synthesis. The demonstration that whole serum contains several classes of growth factors that regulate different cell cycle events [22,25,26] reconciles the seemingly disparate results of these two groups of investigators. Todaro and Green were predominantly studying the effect of PDGF, whereas Brooks, who returned the cells to 0.25% serum, was studying the time required for cells to respond to all classes of growth factors. Thus, it appears that the irreversible commitment event occurs late in G1, while a separate PDGF-induced event occurs earlier (see below).

IVC. Restriction point control Pardee [97] proposed that either serum or nutrient deprivation of Syrian hamster cells (BHK and Nil 8) causes them to become arrested at the same point in the cell cycle, the restriction point. The lag period until DNA synthesis was the same after the addition of isoleucine to isoleucine-deprived cells or serum to serum-deprived cells. Furthermore, isoleucine-deprived cells did not synthesize DNA if isoleucine was returned but serum was withdrawn. Polyoma virus-transformed cells were not subject to this restriction point control and continued to cycle tinder adverse conditions [97]. Eventually, these transformed cells stopped replicating at various random points in the growth cycle and died. Recent evidence suggests the existence of more than one restriction point. After the addition of complete medium containing both serum and amino acids to BHK, CHL, or Swiss 3T3 cells, the lag period until DNA synthesis depends on whether the cells were initially starved for serum or amino acids [100-102]. IVD. Transition probability Smith and Martin [85] recognized the importance of studying the inherent variability of the Go/G~ phase of the cell cycle. They found that both growth-arrested and exponentially-growing populations of cells entered the S phase and the mitotic phase with firstorder kinetics [85,103]. This data, which had been repeatedly confirmed in both mammalian cells and yeast [104,105], supported the concept that the commitment to DNA synthesis is a single random event characterized by a first-order rate constant, the transi, tion probability. According to this model, the 'initiation of cell replication processes is random in the sense that radioactive decay is random'. Because cells enter S phase with first-order kinetics, it was proposed that the rate limiting commitment event divides the cells into two fundamentally different parts: (1) a deterministic 'B' state of relatively constant duration, including a portion of late G1 phase and all of the S, G2, and M phases; and (2) a non-deterministic 'A' state which includes most of G~ and in growth-arrested cells, Go. According to the transition probability model, sometime after mitosis a cell enters the A state. While in the A state, the cell's activity is not directed toward replication, and the cell does not progress towards division [85]. In support of the transition probability theory, Shields [106] found that the daughter cells of clonal isolates of Swiss 3T3 cells or mouse embryo fibroblasts have highly variable generation times. These generation times could be plotted as a first-order function. The daughters of cloned SV40-transformed Balb/c-3T3 cells also had first-order generation times implying that the replication of both transformed and non-transformed populations

232 of cells is governed by the same critical commitment event. Brooks [99] found that quiescent populations of Swiss 3T3 cells enter the S phase with first-order kinetics 12-14 h after stimulation with serum. Because the serum concentration determined the rate at which cells entered the S phase, he proposed that serum commits cells to enter the S phase by increasing the transition probability. The 12 14 h lag period required for growth-arrested cells to begin S phase entry was interpreted to be the minimal interval needed to increase the transition probability. The actual commitment (transition) event occurred near the G~-S phase boundary because the addition of cycloheximide to a population of cells entering the S phase inhibited further cellular entry into the S phase within 2 h [107]. The basis of the transition probability theory is the finding that cells enter the S phase with first-order kinetics, which can be plotted senti-logarithmically. However, such cell replication data can be plotted to fit other statistical curves, implying that a deterministic model of the cell cycle is equally valid [108]. Furthermore, studies supporting first-order kinetics rely heavily on data obtained after 30 50% of the cells have synthesized DNA, and often do not take into account the period when the first cells enter the S phase [108]. During this period, the slope is often not first-order but is increasing rapidly until a constant slope is reached.

IVE. Synergism of growth factors De Asua et al. [92,93] used pure growth factors to dissect the lag phase of Swiss 3T3 cells into distinct temporal regions. In an important series of investigations, De Asua [92,93] demonstrated that the pure hormones, prostaglandin Fza and insulin, act synergistically to induce DNA synthesis in quiescent cells. Swiss 3T3 cells were grown to confluence and left in serum-depleted medium; the addition of prostaglandin 122oeto such cultures induced DNA synthesis after a lag period of 15 h. Addition of insulin to these cultures did not alter the lag period, but did increase the rate that prostaglandin F2c~-treated cells entered the S phase. Insulin had this effect when added as late as 8 h after prostaglandin F2~. Furthermore, hydrocortisone inhibited growth stimulation by the continuous treatmeut of cells with prostaglandin F2c~ only if added within 3 h. The authors postulated that there are two hormonal regulatory signals in Go/G1 which control the entry of cells into the S phase. In the presence of serum-depleted medium, a first signal is regulated by prostaglandin F2c~ and is inhibited by hydrocortisone; insulin regulates the second signal which controls the rate that cells enter the S phase. Other investigators have also shown that growth factors act synergistically to induce an optimal proliferative response. Nishikawa et al. [68] found that the optimal response of ovarian cells with carboxymethyl celhdose adsorbed growth factor, isolated from fetal calf serum, occurred only alter the addition of a more anionic serum fraction. Furthermore, pituitary FGF induces DNA synthesis optimally in 3T3 cells in tire presence of a corticosteroid and insulin [ 109].

I VF. Ordered sequence of replicative events Pledger et al. [22] showed that serum, which is widely used to support the growth of cells in tissue culture, contains at least two hormonal components which regulate different cell cycle events. PDGF causes Go-arrested Balb/c-3T3 cells to become competent to enter the cell cycle. Cells remain competent for at least 13 h after the removal of PDGF. Addition of platelet-poor plasma allows PDGF-treated competent cells to enter the S phase after a lag period of 13 h (Fig. 4). One of the critical regulatory components in

233 plasma is the somatomedin family of polypeptide hormones. PDGF appears to induce the first event in the cell cycle [23]. Treatment of Goarrested incompetent cells with plasma, before the addition of PDGF, did not shorten the lag phase until DNA synthesis. Such a shortening might be expected if plasma controlled the first replicative events. Distinct growth-arrest points have been noted in the plasma mediated progression sequence [23]. PDGF-treated competent cells were treated with an optimal concentration of plasma (5%) for various lengths of time and were then transferred to medium lacking plasma; plasma was then added back and the cells entered the S phase. The lag period until DNA synthesis in these experiments was determined by the length of the initial exposure to plasma: (1) PDGF-treated competent cells that were incubated with 5% plasma for 5 h during the initial exposure, remained competent, but did not progress through Go/G~; they began DNA synthesis 12 h after the readdition of plasma. (2) Cells treated with an optimal concentration of plasma for I0 h, before plasma was withdrawn, became arrested at a point (termed V) midway through Go/G1; i.e. 6 h before the S phase. (3) Cells treated with plasma for 15 h during the first addition, became arrested at another growth arrest point (termed W), immediately before the S phase. Thus, there appear to be at least four growth arrest points in the Go/G1 phase of the Balb/c-3T3 cell cycle: 1. Go; incompetent, arrested 12 h before S. 2. PDGF-treated; competent, arrested 12 b before S. 3. PDGF- and plasma-treated; V, arrested 6 h before S. 4. PDGF- and plasma-treated; W, arrested at the G1/S phase boundary. Cells arrested at the W point, at the Ga/S phase boundary, were studied extensively [23] because they had not been irrevocably committed to DNA synthesis. Addition of plasma to these W point arrested cells induced immediate entry into the S phase with first-order kinetics. The rate of entry into the S phase was directly dependent upon the plasma concentration. This plasma-dependent entry into the S phase was blocked by cycloheximide, demonstrating, as Brooks [107] had previously done, that commitment required protein synthesis. Thus, the actual commitment to DNA synthesis occurs as cells leave the W point, at the GI/S phase boundary, and enter the S phase. The identification of four distinct growth-arrest points in the Go/G~ phase of the cell cycle, before the commitment to DNA synthesis, demonstrates that there are deterministic growth regulatory events which occur in the Go/G~ phase of the growth cycle. Serum growth factors, PDGF and plasma, regulate the ordered cell cycle events which precede commitment and plasma also regulates the commitment event itself. Because these serum factors control several distinct events in Go/G~, cells can spend an indeterminate amount of time in this phase of the cell cycle. The transition probability describes only one step (commitment) in an ordered series of replicative events which occur during Go/G1. Clearly, the transition probability model cannot account for all replicative events which occur during the Go/G~ phase of the cell cycle. Commitment cannot be solely determined by a single random event. An ordered sequence of deterministic replicative events must occur before cells can become committed [23]. IVG. Restriction point is distinct from the commitment (transition) event Deprivation of several essential amino acids prevents serum-stimulated cells from entering the S phase. PDGF induces amino acid-deprived cells to become competent [24]. Addition of dialyzed 5% plasma to these amino acid-deprived cells causes the majority of

234 tile cells to progress to a point, V, 6 h prior to the S phase, where they become growth arrested. These cells will synthesize DNA, with a 6 h lag period, when the missing amino acids are added to the medium. Thus, deprivation of either amino acids [24] or plasma [23] can cause growth arrest at a point V, 6 h before S phase. Because this V point is sensitive to either plasma or amino acid deprivation, it appears to be a true restriction point as defined by Pardee [97]. This restriction point (V) is clearly distinct from the commitment (transition) point which occurs as cells leave the W point and enter the S phase. V may represent the first recognizable point in G1 because in exponentially growing populations of Balb/c-3T3 cells, G1 is approx. 6- 7 h in duration.

IVH. Exponential growth." PDGF can act during S phase Vogel et al. [59] demonstrated that 3T3 cells treated with an optimal concentration of both PDGF and plasma grew exponentially. Like all exponentially growing populations, these cells had a short interval between M and the next S phase, and did not appear to enter Go. Scher et al. (in preparation) reasoned that cells may not enter Go if they are treated with PDGF during the preceeding S phase. Confluent Go-arrested Balb/c-3T3 cells were treated with PDGF and plasma in the presence of methotrexate, an inhibitor of dihydrofolate reductase, an enzyme needed for de now) purine and thymidine synthesis. Because the cells could not synthesize DNA, they became growth-arrested in an early part of the S phase. Removal of the methotrexate and addition of an optimal concentration of plasma allowed these cells to complete the cell cycle, but they did not enter the S phase of the subsequent cell cycle. Addition of PDGF and plasma to these cells after M allowed them to enter the S phase of the second cycle, with a lag of 12 h, demonstrating that cells that lack PDGF are incompetent and become arrested at Go. Methotrexate-blocked cells were released from methotrexate and treated with a short pulse of partially purified PDGF while traversing the S phase; the cells were then returned to 5% plasma and allowed to undergo mitosis. The daughter cells, which remained in plasma, entered a second S phase, with a lag period of 6 - 7 h from the time of mitosis. Thus, the daughter cells 'remembered" that their parents had been treated with PDGF during the S phase of the preceeding cell cycle. Because the parent had been treated with PDGF, the daughters did not enter Go, but synthesized DNA after a 6 7 h lag period from the preceeding M. Thus, treatment of the parent cells with PDGF during S phase prevented the daughter cells t'rom becoming arrested at Go (Scher, C.D., Stone, M. and Stiles, C.D., unpublished data). V. PDGF and neoplastic transformation VA. Transformed cell lines." reduced PDGF requirement Balk [14,50,51] found that chick embryo fibroblasts transformed by Rous sarcoma virus do not appear to require PDGF for long-term growth because they proliferate equally well in either serum or plasma supplemented medium. Similarly, a single line of SV40-transformed Swiss 3T3 cells grow well in plasma-supplemented mediuln [ 16]. Nontransformed chick embryo fibroblasts [14] or Swiss 3T3 cells [16,59] require the PDGF present in serum for growth under these conditions. Recently, Scher et al. [19] tested a large number of clonal lines of both 3T3 cells and human fibroblasts transformed by SV40 or retroviruses for growth in plasma. Both SV40transformed 3T3 cells and SV40-transformed human embryo fibroblasts grew to similar cell densities in serunr- or plasma-supplemented medium. In contrast, the non-trans-

235 formed parent cell lines grew to far higher cell densities in the presence of serum. Thus, SV40-transformed cells can proliferate for prolonged periods of time in medium lacking or having only a low level of PDGF. Vogel et al. [110] isolated clones of SV40-transformed Swiss 3T3 cells that appear to be density-inhibited. These flat revertants, as well as flat SV40-transformed Balb/c-3T3 cells, do not display all of the phenotypic characteristics of transformed cells. Unlike typical transformants, they do not attain high cell densities in vitro; the final cell density is determined by an equilibrium between cell division and cell death [111]. These flat, SV40-transformed clonal cell lines grew equally well in either serum- or plasma-supplemented medium [ 19] demonstrating a decreased requirement for PDGF. Most SV40-transformed 3T3 cells can grow in medium supplemented with a low concentration of serum. Vogel and Pollack [112] isolated several SV40-transformed clones that required a high serum concentration for growth. These cell lines grew well in either serum- or plasma-supplemented medium [19]. In spite of their need for a high concentration of serum, they have lost the requirement for PDGF. Thus, the loss of the requirement for PDGF appears to be a characteristic of all SV40-transformed 3T3 cell lines. The Kirsten murine sarcoma virus and the Abelson routine leukemia virus can morphologically transform NIH 3T3 cells in vitro. Clonal cell lines transformed by these retroviruses grew to the same final density in medium supplemented with either plasma or serum [19]. In contrast, NIH 3T3 cells infected by a non-transforming retrovirus, the Moloney leukemia virus, grew to a far higher density in tile serum supplemented medium. The Moloney virus has a sequence of genetic information in common with the Abelson virus [113], but this common sequence does not allow morphological transformation or growth in plasma. These studies demonstrate that cells transformed by murine retroviruses have a reduced requirement for PDGF. Furthermore, the reduction of tile need for PDGF is a transformation-specific process. Human and mouse cell lines were tested for tumorigenic potential by inoculation into athymic nude mice. Without exception, every cell line which formed tumors in nude mice was capable of growth in plasma-supplemented medium [19]. Every cell line which showed poor growth in plasma-supplemented medium failed to grow as tumors when inoculated into nude mice. However, some cell lines which were capable of growth in plasma-supplemented medium failed to form tumors. Thus, a reduction in the growth requirement for PDGF may be a necessary though not a sufficient step for tumorigenicity in vivo [19].

VB. Abortive transformation by SV40 reduces the requirement for both PDGF and plasma A wide range of SV40 and retrovirus-transformed cell lines have a reduced requirement for PDGF. However, all of these virus-transfornled lines were subjected to selection procedures in culture at some time during the derivation of the clonal cell line. It was therefore important to determine if the ability of transformed cells to grow in plasma-supplemented medium was a direct consequence of transfornling virus gene activity or an indirect consequence of the selection procedures employed to obtain the transformants. SV40 directly induces the transient proliferation (abortive transformation) of growtharrested Balb/c-3T3 cells in depleted agamma calf serum [7]. To show that the SV40transforming gene(s) can directly override the non-transformed cell's requirement for the platelet-derived growth factor, quiescent Balb/c-3T3 cells in 5% plasma supplemented medium were infected with SV40, and the growth of the cells was monitored. 100 infec-

236 tious units of purified SV40 per cell induced several rounds of cell division. The growth induced by SV40 infection was comparable to that achieved by the addition of either partially purified PDGF or serum to the culture medium. These data indicate that SV40 rapidly and directly reduces the growth requirement for PDGF [19]. PDGF interacts synergistically with hormonal growth factors in plasma to induce DNA synthesis in Go-arrested Balb/c-3T3 cells (Fig. 1). Similarly, the somatomedins, or hyperphysiological concentrations of insulin, interact synergistically with PDGF to allow DNA synthesis in the presence of plasma from hypophysectomized animals (Fig. 6, Table II). In contrast to these growth-regulating polypeptide hormones, the induction of DNA synthesis in confluent, Go-arrested Balb/c-3T3 cells by SV40 is strictly a function of the virus nmltiplicity of infection [25,26]. Neither increasing the concentration of plasma in the culture medium nor treating the cell with increasing concentrations of PDGF enhanced cellular DNA synthesis in the SV40-infected cells (Fig. 8, Table II). Thus, SV40, which devotes only a small quantity of genetic information to the transformation process [25,26], overrides the growth requirement for two functionally distinct sets of hormones. VI. Target cells of PDGF PDGF stimulates the growth of cells derived from the mesenchyme in vitro. Diploid embryo fibroblasts from humans [19], mice [19], and chickens [14] all respond to PDGF, as do human brain glial cells [17] and monkey arterial smooth muscle cells [15]. Diploid embryo fibroblasts from chickens or humans [19] only display a growth requirement for PDGF in culture medium containing hypophysiological concentrations of Ca 2+. Balk [14,50,51] has suggested that the growth factors present in serum sensitize their target cells to utilize available calcium. In contrast to the behavior o f normal fibroblasts in culture, normal kidney epithelioid A

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Fig. 8. SV40 induces both competence and progression. (A) SV40 was added to density-arrested Balb/ c-3T3 cells at the multiplicity of infection indicated in parenthesis. After 3 h, the cells were washed as in Fig. 1 and transferred to medium plus 5 ~Ci/ml [3H]dThd and the concentration of normal human plasma indicated on the abscissa. After 36 h the cells were fixed and processed for autoradiography. (B) Density-arrested Balb/c-3T3 cells were exposed for 3 11to the concentration of PDGF indicated on the abscissa. The medium was removed and the cell monolayers were washed as in Fig. 1. Fresh medium containing [3H]dThd and 0.25% normal plasma was added to all cultures together with SV40 at the multiplicity of infection indicated in parenthesis. A supplement of 0.25% normal human plasma sustains cell attachment to the tissue culture dish but is not sufficient to cause progression of PDGFtreated cells into S phase (see Fig. 7). After 36 h, all cultures were fixed and processed for autoradiography.

237 cells and normal rat liver cells grow equally well in serum- or plasma-supplemented medium (Stiles, C.D., unpublished data). Mouse spleen lymphocytes, activated by concanavalin A, proliferate equally well in serum or plasma supplemented culture medium (Fahri, J. and Stiles, C.D., unpublished data). Thus, PDGF selectively stimulates the growth of connective tissue cells only. Epithelioid cells and lymphoid cells may respond to tissuespecific growth factors other than PDGF. FGF also only stimulates cells of mesenchymal origin [114]. VII. PDGF and atherosclerosis A single layer of endothelium lines the internal surface (intima) of arteries. Under the, intima is the media composed of multiple layers of smooth muscle cells. Disruption of the continuity of the endothelial cell layer allows the underlying smooth muscle cells to proliferate. Ross [15,21] has proposed that PDGF initiates the proliferative response of these smooth muscle cells and has suggested that this proliferative response is the sine qua non of atherosclerosis. This subject has been reviewed extensively [115,116] and will only be briefly covered here. Endothelial cell injury causes platelets to adhere to the injured site [117] and release PDGF from the a-granules. Smooth muscle cells respond to PDGF and plasma by proliferating [15,21]. PDGF is needed for the proliferative response because plasma alone does not stimulate cell replication in vitro [15,21]. Of interest is the recent finding that the proliferative response of smooth muscle cells in vivo after endothelial cell disruption can be significantly reduced by ablation of the pituitary gland [118]. PDGF and somatomedins probably regulate the proliferation of vascular smooth muscle cells in vivo. The role of PDGF in promoting endothelial cell proliferation requires further study. Haudenschild [119] demonstrated that neither serum nor plasma stimulates the growth of confluent, density-inhibited, endothelial cells in vitro. However, FGF allows the clonal growth of sparse cultures of vascular endothelial cells [120]. PDGF may fulfill a similar role, and thus may be involved in endothelial cell repair growth after injury of the vascular intima. VIII. Summary: coordinate control of fibroblast growth by PDGF and somatomedins In a relatively short period of time since the perceptive observations of Balk [14] and Ross [ 15], considerable progress has been made towards understanding the role of PDGF in control of cell growth. PDGF has been purified to homogeneity. It is a heat-stable cationic polypeptide with a molecular weight of 13 000. Disulfide groups are essential for its activity. Concentrations of pure PDGF as low as 10 -1 o M initiate a round of replicative DNA synthesis and cell division in Balb/c-3T3 cells. PDGF fulfills the biological criteria of a polypeptide hormone. It is an 'informational' molecule, carried in the blood, which acts at nanomolar concentrations or less on specific target cells. Unlike other polypeptide hormones , however, PDGF is not found in plasma, but is carried in the a-granules of platelets. Thus, the molecule is in a cryptic state in the platelet and is not available to stimulate the replication of cells. Platelets specifically adhere to traumatized tissues and release PDGF; connective tissue cells near the traumatized region (but not epithelioid cells or lymphoid cells) respond by initiating the process of replication. By packaging a growth stimulating hormone within the platelets, nature has provided a mechanism for the selective, hormone-modulated growth stimulation of connective tissue cells.

238 PDGF initiates tire process of cell replication by making cells competent to respcmd to other growth factors contained in platelet-poor plasma. In confluent Go-arrested Balb/c3T3 cells, this competence is the first event in an ordered replicative sequence. PDGF also acts in the S phase to initiate the process of cell replication in post-mitotic daughter cells. Plasma is required to allow PDGF-treated competent cells to progress through Go/G1 and enter tile S phase. A crucial regulatory component in plasma that controls, in part, the progression sequence is the somatomedin family of hormones. Since tile concentration of somatomedin in plasma is a function of pituitary growth hormone (somatotropin), a portion of the fibroblast cell cycle is under pituitary control. Progression is a complex process which requires low molecular weight nutrients and a variety of growth factors contained in platelet-poor plasma; these growth factors include, but are not restricted to, the somatomedins, because plasma from hypophysectomized rats is also required for progression. Tile use of hypophysectomized plasma allowed the development of an assay for progression factors under pituitary control. The merit of this progression assay lies in physiological relevance. Any component of tissue culture medium (for example, amino acids and plasma proteins) can be manipulated at will and most of these manipulations affect progression. In contrast, most chemical constituents of blood in vivo (amino acids and most plasma proteins) are maintained in relative homeostasis. Those agents which regulate growth in animals constitute a minority of tile blood chemical components whose concentration fluctuates to meet the demmld for cell proliferation. PDGF probably plays a regulatory role in connective tissue growth in vivo since it is only released within the microenvironment of traumatized tissue. Sonlatomedins also may have a growth regulatory role in tissue growth in vivo because their concentration in blood fluctuates in proportion to pituitary growth hormone activity. PDGF is a mitogen with an apparently narrow range of target tissues, whereas somatomedins appear to interact with a wide range of tissues in vitro. Somatomedins coordinate the balanced growth response of all body tissues as animals progress from infancy towards adulthood; humans with chronic hypophysiological levels of somatomedins show a balanced retardation of growth. We have proposed that the control of Balb/c-3T3 cell growth in vitro by a tissue-specific competence factor and by somatomedins is a specific example of a common pattern for growth regulation in animal tissues. In vivo, tissue-specific competence factors could play a predominant role in repair growth and in differentiation while somatomedins would control the balanced, maturational growth of body tissues from infancy to adulthood. Malignant transformation could result from the impairment of either or both of these pathways at the cellular level. This model accounts for the diversity of growth patterns observed in neoplastic disease and explains why some tumors (e.g., mammary and prostatic carcinomas) exhibit hormonedependent growth. Studies with SV40 indicate that this tumor virus disrupts both the competence and the progression pathways. It will ultimately be of great interest to determine whether the growth regulation of various differentiated mammalian cells can be understood in the context of competence factors such as PDGF and progression factors such as the somatomedins.

Acknowledgements We thank Drs. A. Pardee, W.J. Pledger, and G. Sato for helpful discussions. Portions of the work summarized within were supported by Grants CA 18662, CA 06515, CA 15388, CA 22427, and CA 22042 from the National Institutes of Health. C.D. Scher is a Scholar of the Leukemia Society of America.

239

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