Growth factors and cancer

ICANCER RESEARCH 46, 1015-1029, March 1986] Perspectives in Cancer Research Growth Factors and Cancer1 Anton Scott Goustin, Edward B. Leof, Gary D. Shipley, and Harold L. Moses2 Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 [E. B. L., H. L. M.]; Department of Cell Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 [A. S. G.J; and Department of Cell Biology and Anatomy, The Oregon Health Sciences Center, Portland, Oregon 97201 [G. D. S.¡. Overview GFs3 may be defined as polypeptides that stimulate cell prolif eration through binding to specific high-affinity cell membrane receptors. These GFs differ from the well-known polypeptide hormones such as insulin and adrenocorticotropic hormone not only in the response elicited but also in the mode of delivery from the secreting to the responding cell. GFs do not usually act in an endocrine manner; they presumably diffuse short-range through intercellular spaces and act locally. Plasma contains few growth factors; several of those present in serum are presumed to be derived from platelets and are released during the clotting process (1-4). The presence of growth factors in platelets is thought to facilitate delivery of growth factors to sites of injury where they may play a major role in wound healing. Besides being found in platelets, GFs are present in a variety of tissues, both adult and embryonic, and are thought to be released by many, if not all, cells in culture (5). Membrane receptors for growth factors are also highly ubiquitous with most cells having receptors for more than one growth factor (6-8). Growth factors have differing cell type specificities; some factors such as those of the hematopoietic system (e.g., interleukin 2 or CSF-1 ) stimulate only one or a few cell types while others such as somatomedin C and EGF stimulate a wide variety of cell types, both epithelial and mesenchymal (see below). It has been demonstrated that multiple growth factors are required for max imum stimulation of specific cell types (9, 10). The requirement of nontransformed cells for more than one growth factor for proliferation is also supported by studies on the growth of cells in defined serum-free media. Unless the cells are neoplastically transformed, more than one growth factor supplement is nec essary for growth (11-13). Exposure of a cell to one growth factor can lower the threshold for mitogenicity of a second growth factor (14). Moreover, growth factors operate at different points of the cell cycle (9, 10). For instance, transient treatment of fibroblasts with PDGF will induce a stable state ("competence") whereby cells are made responsive to other circulating plasmaderived factors (15). The multiplicity of growth factors in various tissues, the varying cell type specificity of GFs, and the require ment for multiple GFs for stimulation of specific cell types pre sumably provide the fine tuning of relative proliferation rates Received 8/19/85; revised 11/20/85; accepted 12/2/85. 1This investigation was supported by USPHS Grants CA 16816, CA 27217, CA 09441, and CA 39911 awarded by the National Cancer Institute, Department of Health and Human Services. 1 To whom requests for reprints should be addressed. 3 The abbreviations used are: GF, growth factor; ALV, avian leukosis virus; CSF, colony-stimulating factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor or somatomedin; IL, interleukin; NGF, nerve growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; p21, 21-kDa protein; cDNA, complementary DNA; NRK, normal rat kidney; Con A, concanavalin A. CANCER necessary for coordinated growth of cells to form tissues during development and to maintain tissues in the adult state. Much of the impetus for study of GFs has come through their presumed involvement in cancer. Evidence for this involvement dates to early work showing a decreased serum requirement for growth of neoplastically transformed cells (16-18). With the advent of serum-free culture techniques and the availability of purified growth factors, the altered serum requirement in trans formed cells could be translated into a diminished or absent requirement for specific growth factors (11,19). Loss of require ment for specific growth factors is a common finding in many types of cancer cells (19, 20) and could be mediated by (a) the activation of autologous GF synthesis ("autocrine" activation), (b) synthesis of an altered GF receptor, or (c) activation of a postreceptor pathway that bypasses the GF receptor requirement. Some of the more convincing evidence linking growth factors and cancer has come from recent work linking oncogenes and growth factors. One proto-oncogene, c-s/s, codes for the B chain of PDGF (21, 22). Another (c-enbB) codes for the EGF receptor (23). Similarly, the product of the c-fms oncogene appears very similar to the CSF-1 receptor (24). Moreover, there is evidence to suggest that several other oncogene products are similar to growth factor receptors in that both have transmembrane and tyrosine kinase domains (25). Recent data indicate that the p21 ras oncogene protein is involved in transduction of the growth factor signal and may be an obligatory intermediate in this pathway (26). Growth factors have been shown to increase transcription of certain proto-oncogenes (myc and fos) (27-30), the products of which may in turn regulate the transcription of other genes necessary for stimulation of cell proliferation. These data suggest that many, if not all, of the oncogene products may be involved in the growth factor-receptor-response pathway and indicate points at which alterations may occur leading to the development of neoplastic transformation. Many growth-active polypeptides that fit the definition of growth factors have been described, and this review will concen trate on several well-defined examples. The cellular response to growth factor binding and possible mechanisms of growth factor involvement in the neoplastic process including the oncogene relationship will be addressed. Specific Growth Factors EGF. EGF was first described by Cohen (31) as a peptide which would stimulate precocious eyelid opening and tooth eruption in newborn mice and was purified on this basis; its ability to stimulate the growth of cultured cells was recognized later (32, 33). First purified from male mouse submaxillary glands (31) and later from human urine as urogastrone (34, 35), mature RESEARCH VOL. 46 MARCH 1986 1015 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS EGF is a 6-kDa single polypeptide chain of 53 amino acids displaying 3 internal disulfide bonds (36). EGF is synthesized from a precursor which may be as large as 128 kDa (37). Radiolabeled 46- and 130-kDa species have been detected in mouse male submaxillary gland and mouse kidney, respectively (38). The 4.8-kilobase EGF mRNA from male mouse submaxillary gland has been cloned and sequenced (37,39). The cDNA clones define an open reading frame sufficient to code for 1168 (37) or 1217 amino acids (39). In both cases, native EGF is encoded in residues 977-1029 of the deduced amino acid sequence. Origi nally thought to have a limited range of tissue expression, recent in situ hybridization analyses of sections of whole newborn mice (38) indicate that RNA complementary to cloned EGF probes may be present in a large variety of tissues, including a surpris ingly high expression in the distal tubules of the kidney. The protein translated from this mRNA in kidney remains as a highmolecular-weight protein; little or no 6-kDa EGF is detectable in this tissue (38). Radioimmune (40) and radioreceptor (41) assays have been developed for measuring EGF concentration in extracts; the latter assay detects TGF«as equivalent to native EGF. Not only do EGF and TGFa (see below) both recognize the same cellular receptor, they are apparently equally effective on a mole-formole basis in most systems. It may be the case that EGF is the adult form of the embryonic growth factor TGFa. EGF is mitogenie for a variety of cultured mesenchymal and epithelial cells; its mitogenic activity is strongly potentiated by insulin (42, 43). EGF also acts in synergism with PDGF on BALB/C-3T3 cells (44). Aspects of differentiation are also induced following EGF treatment in certain cell culture models and in vivo (45, 46). No tumors are yet known which synthesize EGF. Consistent with the oncodevelopmental concept which proposes that tu mors may ectopically reactivate embryonic genes, all tumors and tumor cells which synthesize an EGF-like species, in fact, syn thesize a similar molecule called TGFa, to be described later. An EGF-like molecule may also play a role in the benign hyperplasia induced by vaccinia virus which encodes a 140-residue protein processed to 47 residues showing significant homology to both EGF and TGFa, including conservation of the three internal disulfide bonds (47). The cellular receptor for EGF is the best understood GF receptor and has served as a paradigm for other GF receptors. Although present on a large variety of cells, the EGF receptor was first purified from A431 cells (48), a cell line derived from a human squamous carcinoma which has an increased number of EGF receptors (7). The receptor is an integral 170-kDa mem brane protein exhibiting an extracellular binding domain that serves to bind the ligand (EGF or TGFa), a transmembrane region, and an intracellular domain facing the cytoplasm exhibit ing the tyrosine kinase function and presumably binding sites for ATP phosphorylation substrates (48). In response to EGF, the receptor is capable of autophosphorylation on tyrosine residues (49). A second form of the receptor missing the transmembrane domain is found in a secreted form in the A431 cell line (50), although the significance of this molecule is not clear. The oncogene v-erbB codes for a product homologous to a portion of the EGF receptor in which the EGF-binding domain has been deleted. Evidence exists suggesting that this truncation of the EGF receptor may lead to constitutive activation without require CANCER RESEARCH AND CANCER ment for ligand binding (see below). Platelet-derived Growth Factor. PDGF is a major mitogen in serum; moreover, it elicits a chemotactic response in fibroblasta and smooth muscle cells (51). PDGF is a potent mitogen, suffi cient in some cells to induce both DNA synthesis and cell division even in the absence of other growth factors (52). It is thought that most transformed mesenchymal cells produce PDGF or a PDGF-like molecule (53, 54). PDGF purified from outdated human platelets is a mixture of polypeptides with molecular weights in the 30-32-kDa range. The platelet-derived dimer is composed of a 14-18 kDa A chain disulfide bonded to a 16-kDa B chain (21, 55); the size hetero geneity probably reflects differential degradation of the A chain ends as well as differential addition of carbohydrate side chains. The B chain (or PDGF-2) is encoded in the c-s/'s proto-oncogene (21, 22, 54, 56, 57); its cellular transcript appears as a 4.2kilobase mRNA in denaturing gels. Parts of the human c-s/s locus have been cloned from genomic libraries (58, 59). There are 7 exons to the human c-s/s locus of chromosome 22, encompassing at least 23 kilobases of DNA; no promoter has yet been found (59). Although PDGF from platelets is apparently a heterodimer, transformed cells may actually secrete a B-B homodimer. Se quencing of a c-s/s cDNA clone reveals an open reading frame sufficient to encode 241 amino acids or 27 kDa of protein (57, 59). A dimer of pro-B chains could thus include 54 kDa of peptide; carbohydrate addition would presumably add to the size of this pro-B chain dimer. PDGF may be synthesized as a high-molec ular-weight precursor (54, 59-61) which is presumably disulfide bonded and processed to the 32-kDa secreted form observed in cultures of osteosarcoma cells (60-62) and glioma cells (63). A radioreceptor assay for PDGF has been developed (64-66) which affords a specific and sensitive quantitation of PDGF in extracts and conditioned media. Scatchard analyses of 125Ilabeled PDGF binding allows analysis of receptor number per cell (400,000 receptors/cell for human fibroblasts) as well as the dissociation constant (Ka) for the factor-receptor complex (101000 pw). The concentration of PDGF exerting half-maximal stimulation of DNA synthesis varies widely, between 11 and 310 pw (0.4-10 ng/ml). This large variation may reflect the interaction of other growth factors with the cell which may lower the cell's threshold of response to PDGF. PDGF was originally purified from blood platelets where it is stored as a component of the a granules (67). PDGF synthesis has been demonstrated in large vessel endothelial cells (68) and aortic smooth muscle cells of newborn but not adult rats (69). The 4.2-kilobase c-s/s transcripts are present in the cytotrophoblastic shell of human placenta, and placenta! expiants synthe size a PDGF-like molecule (70). Cell lines cultured from early placentas also express cell surface receptors for PDGF and respond to exogenous PDGF with an activation of the c-myc gene and DNA synthesis (70). Since the cells of the cytotrophoblastic shell are the most invasive and proliferative normal cells known, the expression of PDGF receptors in this tissue may help account for their "pseudomalignant" phenotype (71). Receptors for PDGF are found on a variety of mesenchymal cells (65, 66) as well as human placental cytotrophoblasts (70). Other than the trophoblastic cells, receptors for PDGF are not found on most epithelial cells (66). Stimulation of cells with PDGF induces an autophosphorylation of a 185-kDa protein (72) which VOL. 46 MARCH 1986 1016 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS turns out to be the PDGF receptor (73). An antibody to phosphotyrosine has been used in the purification of the receptor from BALB/C-3T3 cells; purified receptors inserted into liposome re constitute the GF binding characteristics of the native receptor (74). Transforming Growth Factor Type a. TGFs can be defined operationally by their ability to stimulate the anchorage-inde pendent growth in soft agar of cells which are otherwise anchor age dependent (75). This definition has led to the isolation and characterization of two very divergent molecular entities: TGF/3, a 25-kDa disulfide-linked homodimer (described below); and TGFa, a 5.6-kDa species consisting of a single chain of 50 amino acids (76). TGF«was first described as sarcoma growth factor, now known to be composed of both TGF/3 and a 5.6-kDa species (TGFa) that are secreted into the medium conditioned by the growth of murine sarcoma virus-transformed cell lines and that compete with 125l-labeled EGF for binding to a common cell surface receptor (77, 78). As it turns out, purified TGFa alone in serum-containing medium only weakly stimulates soft agar col ony formation (79). The apparent colony-stimulating ability of sarcoma growth factor was presumably due to the interaction of TGFa and TGF/3 on NRK indicator cells (see TGF0 below). The sequence of native rat cell-derived TGFa shows a signifi cant homology to both human and mouse EGF (76, 80). Like EGF, TGFa is presumably synthesized from a precursor; the open reading frame of the cloned human TGFa gene is sufficient to encode a protein of 160 amino acids of which residues 4089 encode native TGFa (81). Transcripts of 4.8 kilobases have been detected in the cell line 1072 F57, derived from a human renal cell carcinoma (81). Besides being found in a variety of virally transformed cells, TGFa has also been demonstrated in a variety of nonneoplastic tissues, including human placenta (82) and mouse and rat embryos (83, 84). However, TGFa has thus far not been demonstrated in nonneoplastic adult tissues and may represent the embryonic form of EGF that is inappropriately expressed in certain neoplastic cells. Transforming Growth Factor Type ß.TGF/3 is very different from TGFa in molecular composition, biological response elicited, and membrane receptor binding. TGF/3 is one of the most interesting growth-regulatory polypeptides because it has been demonstrated to both stimulate and inhibit cell proliferation with the response obtained depending largely on cell type (85-87). TGF0 was first described as a factor stimulating the growth in soft agar of AKR-2B (88) and NRK cells (89) that did not compete with 125l-labeled EGF for receptor binding. Although TGF/3 was active in the soft agar assay on AKR-2B (clone 84A) cells alone, the soft agar response of NRK (clone 49F) cells to TGF/3 required the presence of EGF or TGFa (89). It was not clear until later that the TGF activity in the NRK and AKR-2B assays was due to the same molecule, now called TGF/3 (1, 90, 91). NRK cells seem to be unusual in their requirement for EGF in the soft agar assay, and thus the EGF requirement originally included in the definition of TGFß(89) has since been removed (92). TGF/3 has been purified to homogeneity from four sources including bovine kidney (93), human placenta (94), human plate lets (95), and feline sarcoma virus-transformed rat cells (96). These sources reveal a 25-kDa disulfide-linked apparently homodimeric molecule. Derynck ef a/. (97) have cloned the gene for TGF/3 from a human genomic library and from cDNA libraries derived from human term placenta and the human fibrosarcoma CANCER RESEARCH AND CANCER line HT1080. Amino acid sequencing of reduced platelet-derived TGF/3 confirms the conclusion that the 2 chains of TGF/3 are identical; in conjunction with the sequencing of overlapping cDNA clones, these studies define the native molecule as a homodimer of 2 disulfide-linked chains of 112 amino acids each (97). These studies furthermore suggest a precursor encoded in the 391residue open reading frame defined by the overlapping clones where native TGF/3 is encoded by residues 280-391. The gene for TGF/3 is transcribed into a 2.5-kilobase mRNA present in a wide variety of normal and transformed cells; its abundance in human peripheral blood lymphocytes is increased severalfold by mitogen stimulation (97). In addition, TGF/3 protein itself has been detected in normal liver, lung, kidney, submaxillary gland, brain, and heart tissue as well as embryos and placenta (1, 89, 94, 98, 99). A number of cells in culture both produce TGF/3 and have specific TGF/3 membrane receptors, yet they do not constitutively exhibit the phenotype induced by adding TGF/3. A partial explanation for these observations has been provided by recent work demonstrating that the TGF/3 released by cells in culture was in an inactive form; activation occurred irreversibly with acid treatment (100, 101). Some evidence has been pre sented that the inactive TGF/3 precursor might have a higher molecular weight than the active molecule (92), perhaps through association with a binding protein in a manner analogous to that of somatomedin C in plasma (see below). Considering the ubiq uity of TGF/3 (and its receptor), activation of a precursor molecule could represent an important regulatory step in TGF/3 action. TGF/3 is mitogenic for a variety of fibroblastic cell types in monolayer culture (52, 86, 87, 96). In AKR-2B cells, this mito genic activity is apparently conveyed through an indirect action involving PDGF.4 TGF/3 will induce DNA synthesis in AKR-2B cells after a prolonged prereplicative phase of 24 h instead of the 12-14 h seen with PDGF or EGF (52). In this instance, the mitogenic action of TGF/3 is proposed to be indirect, acting to induce c-s/s expression (increasing rapidly at 4 h, although already apparent at 20 min after TGF/3 addition) and the appear ance of a PDGF-like activity within the medium (detectable first at 8 h); it is thought this induced PDGF is the direct mitogen, accounting for the delay in DNA synthesis of 12 h.4 This inter esting twist in the growth factor story not only suggests a mode of action for TGF/3 involving PDGF but also provides support again for a model in which several growth factors might act in concert to increase the proliferative capacity of a cell. If the mitogenic activity of TGF/3 is mediated through PDGF, then it would not be expected that epithelial cells which do not have receptors for PDGF would be stimulated by TGF/3. Intriguingly, the action of TGF/3 can be inhibitory to cell growth in certain circumstances. Evidence has been presented (85) indicating that TGF/3 is the same molecular entity as the growth inhibitor de scribed by Holley ef a/. (102, 103) in the medium conditioned by the growth of BSC-1 monkey kidney cells. The growth-inhibitory action of TGF/3 has since been demonstrated for a variety of neoplastically transformed epithelial cells (86, 87). In certain circumstances, transformation of epithelial cells may involve a loss of the inhibitory response to TGF/3. Whereas the growth of normal human prokeratinocytes is inhibited by TGF/3 in a serum4E. B. Leof, J. A. Proper, A. S. Goustin, G. D. Shipley, P. E. DiCorleto, and H. L. Moses. Induction of c-s/s mRNA and platelet derived growth-like activity by transforming growth factor, type-/i: a proposed model for indirect mitogenesis involving autocrine activity. Proc. Nati. Acad. Sci. USA, in press, 1986. VOL. 46 MARCH 1986 1017 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS free medium (87), it has been shown that a squamous carcinoma cell line grown in the same medium is not inhibited by TGF0.5 This is consistent with a model in which the repression of a growth-inhibitory response in transformation might have the same consequences as the induction of a growth-stimulatory response (52, 87, 104). Radioreceptor assays for TGFßhave recently been developed (105-107), allowing for the quantitation of dissociation constant (25-140 PM) and receptor number per cell (10,000-40,000). The AND CANCER 3 internal disulfide bonds. It has also been prepared in milligram quantities by solid-phase synthesis (124). The human genes for IGF-I and IGF-II have been cloned (125); a recombinant soma tomedin C identical to human somatomedin C except for the substitution of methionine with isoleucine at position 59 is avail able commercially. Interestingly, the human IGF-II gene is, in fact, closely linked to the human insulin gene, on human chromosome 11, band p15 (126). The genes for both IGFs indicate that the native 7 kDa proteins may be processed from 15-21-kDa pre cursors; the 70-residue native IGF-I has a 130-residue precursor, TGFßreceptor was detected on a wide variety of cell types, both whereas the 67-residue native IGF-II has a 180-residue precursor epithelial and mesenchymal (105). It is apparently quite different (125). It has been speculated that IGF-I may be an adult soma from either the EOF or PDGF receptors; affinity labeling of the tomedin, whereas IGF-II would be its embryonic counterpart receptor in mouse cells identifies a 565-kDa complex, which (127). IGF-I and IGF-II each appear to have their own receptor dissociates in the presence of disulfide reagents to two 280to which they preferentially bind, although cross-reaction is seen 290-kDa subunits (108). The receptor is apparently a glycoproat high GF concentrations (128). Chemical cross-linking of radiotein (108) and shows slightly larger species in human cells (615 labeled IGF to cells has allowed the definition of quite distinct and 330 kDa, respectively, for unreduced and reduced receptor). Other Transforming Growth Factors. In addition to TGF« molecular entities (128). The cellular receptors for IGF-I (type I and TGF0, other TGFs have been described that appear to be receptors) show homology to the insulin receptor, a heterotetrameric 450-kDa complex consisting of two transmembrane ß distinct from TGF« and ß.An acid-labile factor, TGF72, that stimulates the growth in soft agar of BALB/C-3T3 cells has been subunits (98 kDa each), each disulfide bonded to one «subunit (130 kDa each) (129). The «subunits provide the insulin- (or described (109). This factor has been purified and an amino acid IGF)-binding domains (130), whereas the ßsubunits possess composition has been determined (110). Another factor is the epithelial tissue-derived factor which stimulates the growth in ATPase and tyrosine kinase activities (131). At least for the insulin receptor now cloned, both subunits are encoded in a soft agar of the carcinoma cell line, SW 13(111). Interestingly, single polyprotein cleaved posttranslationally to yield the heterthis factor is released into media conditioned by the growth of otetrameric receptor. A cDNA encoding the insulin receptor these same cells, suggesting the possibility that this TGF may polyprotein (1370 amino acids) has now been cloned (132). The be involved in autocrine growth regulation of this carcinoma cell a region shows a surprising homology to the extracellular domain line. Insulin-like Growth Factors (IGF-I and IGF-II). First described of the human EGF receptor. Not so surprising, however, is the as a "sulfation factor" by Salmon and Daughaday (112), somahomology of the ßdomain to members of the src family of tyrosine kinases; homology is highest, however, with the ros tomedin C is the best known member of a family of insulin-like oncogene (132). These homologies strongly suggest that one or peptides, ancestrally related to proinsulin (113); members include IGF-I and IGF-II. IGF-I corresponds to human somatomedin C more of these oncogenes may encode growth factor receptors. The type II receptor (preferential for IGF-II) is simpler, exhibiting and IGF-II corresponds to human somatomedin A and rat multi only a 250-kDa component which may be single chain (133). plication-stimulating activity. In the literature, however, somato medin C still generally goes by the original name. Produced in Type II IGF receptors may not undergo ligand-induced down regulation (134). response to circulating growth hormone, somatomedin C is one Interleukin 2. Upon treatment of human peripheral blood Tof the important growth factors found in serum and plasma (114) cells with the lectin Con A, soluble factors are released that active in stimulating the proliferation of a large number of cultured stimulate the proliferation of activated T-cells (135, 136). One cells (115). Supraphysiological concentrations of insulin (>100 factor, first called T-cell growth factor or TCGF and later interleunw) can replace the IGF requirement in defined media through kin 2, was isolated which supported the long-term in vitro culture cross-reaction with ubiquitous IGF receptors (116). Somatomeof clonal populations of normal cytotoxic T-lymphocytes (136). dins apparently circulate in plasma noncovalently bound to a Using mRNA from the overproducer tumor cell line JURKAT, the specific carrier protein (117). Somatomedins have been hypoth gene for human IL-2 has been cloned as a cDNA; the sequence esized to stimulate cell growth in an autocrine fashion (118). indicates a peptide of 153 amino acids (137) that is cleaved to BRL-3A cells secrete large amounts of IGF-II into the medium form the mature 133-residue secreted sialoglycoprotein display (119); however, they do not require the IGF-II for proliferation ing one internal disulfide bond (138). The human gene for IL-2 and thus do not satisfy the autocrine hypothesis (120). Recent spans about 8 kilobases and consists of 4 exons; it shows no evidence argues for a paracrine or autocrine role for somato medin C in the stimulation of fetal mouse growth (121). A significant rearrangements in the JURKAT tumor cell line (139). In addition, a cDNA-encoding mouse IL-2 has been cloned which monoclonal antibody to human somatomedin C has recently exhibits 76% homology at the amino acid level to human IL-2 been shown to strongly inhibit the mitogenic effect of plasma on with an open reading frame sufficient to encode a protein of 169 competent BALB/C-3T3 cells (122). residues (140). Treatment of the JURKAT cells with Con A Somatomedin C (IGF-I) has been purified from human serum induces an IL-2 transcript of 1.5 kilobases (137). Stimulation with and sequenced (123); it is a single chain of 70 amino acids with the lectin phytohemagglutinin results in a 30-fold induction of IL5G. D. Shipley, M. R. Pittelkow, J. J. Wille, Jr., R. E. Scott, and H. L. Moses. 2 transcription in normal human lymphocytes (141). Interestingly, Reversible inhibition of normal human prokeratinocyte proliferation by type rf the immunosuppressive drug cyclosporin A deactivates the IL-2 transforming growth factor/growth inhibitor in serum-free medium. Cancer Res., gene in phytohemagglutinin-induced JURKAT cells (142), sug46: in press, 1986 CANCER RESEARCH VOL. 46 MARCH 1986 1018 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS AND CANCER gesting a role for the activation of the IL-2 gene during T-cell single-chain proteins in the 14-18-kDa size. Other members of activation. Cell surface receptors for IL-2 have been purified from both normal and transformed lymphocytes (143); although the recep tor molecules are slightly different in size (55 and 60 kDa, respectively), the significance of this difference is unclear. This receptor is apparently quite different from those described for other growth factors; the sequencing of cloned cDNAs (144, 145) indicates an open reading frame of only 272 amino acids (33 kDa), containing a cytoplasmic region of only 13 residues, insufficient to encode a tyrosine kinase activity (144). The cyto plasmic domain does, however, contain one serine and one threonine which can be phosphorylated. The discrepancy be tween the observed size of purified receptor (55 kDa) and this the FGF family include the factors described as endothelial cell growth factor, chondrosarcoma growth factor, and heparin-binding growth factors. Proteins that are apparently acidic and basic in the neural extracts show similar features (153-155). Several factors have now been purified to apparent homogeneity; an acidic form of FGF from bovine brain (156) and a cationic form from bovine pituitary (157) have been isolated by multistep procedures and an NH2-terminal sequence was reported for the cationic form (158). Both factors have a molecular weight of 16,000. Several factors that have properties similar to those of FGF have been recently purified by heparin affinity chromatography. An 18-kDa endothelial growth factor from chondrosarcomas was open reading frame capable only of coding for 33 kDa of protein is problematic; 22 kDa of added carbohydrate would be surpris ing. However, it is possible that neither cDNA clone represents a functional IL-2 receptor; the functional receptor cDNA encoding the first to be purified by this technique (159). Subsequently, it has been shown that the cationic 16-kDa pituitary FGF and cationic brain FGF can be purified by this technique and are identical (160). An 18-kDa form of the heparin-binding growth factor has also been observed in preparations from bovine pituitary (161) and hypothalamus (162). Others have reported that multiple forms of FGF activity can be isolated by heparin leukemia virus 1 transformed) cells contain an additional mRNA in which a 216-base region has been spliced out; this mRNA affinity, including both the cationic and anionic FGFs from brain could encode a 200-residue protein identical to the normal IL-2 (163). An amino acid composition for both forms has been reported (162) and the acidic form of the molecule isolated by receptor except for a deletion of 72 amino acids at or near the presumed IL-2 binding domain (144). The significance of this this technique has the same molecular weight and amino acid alternative receptor species is not clear, although this might be composition as the molecule isolated by the multistep procedure similar to the truncated EGF receptor coded for by the erfaß (156). The complete sequence of bovine pituitary basic FGF is oncogene that is missing the ligand-binding site. In addition, the now available (164); the sequence describes a molecule of 146 so-called anti-Tac antibody recognizes both a canonical, func amino acids (16.4 kDa). This sequence agrees with the partial sequence obtained for bovine basic FGF obtained from other tional form and an alternative form of the receptor which displays 100-fold lower affinity for IL-2 (146). Both the 272-codon cDNA tissues, including brain, adrenal gland, retina, corpus luteum, and the 200-codon cDNA have been transfected into COS cells; and kidney (164). Since this sequence differs substantially with the larger cDNA transfectants both bind anti-Tac and radiolathat reported for the bovine acidic form (165), there are probably beled IL-2, although with a 1000-fold lower affinity than expected at least two genes encoding FGFs corresponding to the acidic (145). The 200-codon transfectants seem to produce neither and basic FGFs. However, there is antigenic and sequence functional receptor nor Tac-reactive material. Intriguingly, treat relatedness between these two gene products (164). Further ment of human lymphocytes with IL-2 induces a down regulation more, there is a slight amount of homology between acidic FGF of canonical IL-2 receptors but at the same time induces an and ¡nterleukin1 (165). The factor described as endothelial cell increase in the amount of the alternative receptor on the cell growth factor is related to the FGF family. Purified endothelial cell GF has been radioiodinated for use in a receptor assay, surface (146). In conclusion, the current status of the cloning and purification of IL-2 receptors has thus failed to provide a allowing the estimation of dissociation constant (200-800 pw) and receptor number per cell (20,000-40,000) (166). clear understanding of either receptor structure or its genetic A radioreceptor assay for FGF might allow for a survey of the regulation. Functional receptors for IL-2 are not found on resting T-cells distribution of FGF content and FGF production by various (147); the action of Con A or antigen in T-cell proliferation thus tissues; no such survey has yet been done. The significance of involves the induction not only of IL-2 production by the T-helper an endothelial cell growth factor concentrated in brain or pituitary cells but also of IL-2 receptors on T-killer cells, a two-step is yet unclear. The possible scenario of FGF as an endocrine process (148). The control of IL-2 receptor presentation in the growth factor would stand in contrast to patterns of other GFs immune response is in this way a key control of normal T-cell as locally produced and locally acting paracrine or autocrine growth factors. The production of a FGF by chondrosarcoma is proliferation. Fibroblast Growth Factors (Heparin-binding Growth Fac more in keeping with the general scheme, if one imagines a tors). Extracts of bovine neural tissue contain growth factors paracrine role for this growth factor in the stimulation of tumor mitogenic for cultured fibroblasts and vascular endothelial cells angiogenesis, as has been suggested (167). (149). Reported by Gospodarowicz ef al. (150) in bovine pituitary Nerve Growth Factors. Although NGF has been around as a defined substance for a number of years, its role as a factor for and then in bovine brain (151), the molecular characterization of these factors has been elusive until recently. It was claimed at the maintenance and differentiation of sensory and sympathetic neurons argues against its inclusion in a strict list of growth one time that FGF was derived from brain myelin protein frag factors. Nevertheless, NGF fits into the general scheme of growth ments (152); it has now been shown that this claim was mistaken factors in many ways. Indeed, recent evidence indicates that (153). There are several factors present in these neural extracts which have been given the name FGF; they are all apparently NGF may play a mitogenic role in cultured rat adrenal chromaffin a significant cytoplasmic domain may remain to be cloned. More puzzling yet is the observation that HuT-102B2 (human T-cell CANCER RESEARCH VOL. 46 MARCH 1986 1019 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS cells (168). First detected as a factor released by transplanted tumors (169), NGF was first purified from snake venom (170) and then mouse submaxillary gland (171). NGF isolated from submaxillary glands is found in a 7S complex, containing three protein subspecies labeled a, ß,and y (172). NGF activity resides in the ßchain, a 26-kDa dimer of two identical NGF chains (118 amino acids per chain) which has been sequenced (173). Se quencing of mouse and human cDNA clones suggests that NGF is synthesized as a much larger precursor (174); proNGF is apparently a dimer of 307 residues per chain, with native NGF encoded in residues 188-305 of the precursor. Receptors for NGF are present on a variety of normal sym pathetic and sensory neurons as well as normal and neoplastic chromaffin cells. The rat pheochromocytoma cell line, PC12, has been used extensively in studies concerning NGF. PC12 cells respond to NGF treatment by an inhibition of proliferation and a stimulation of differentiation (175). The mechanisms controlling this response are presently unknown. The PC12 receptor has been defined in ligand-cross-linking studies as a single chain protein of 130 kDa, although a smaller receptor of 100 kDa, possibly a degradation product, is present (176). The receptor in A875 melanoma cells has been partially purified by affinity chromatography; again a 98-kDa species is present, although larger species of 138 and 190 kDa are present (177). It is not known how these multiple NGF receptor species correspond to the two receptor species defined by their apparent dissociation constants of 2 PM and 2 nw (178, 179). Recently, six cDNA clones repre senting mRNAs induced in PC12 cells by NGF have been iso lated; one of the clones, VGF8a, encodes a 90-kDa protein the mRNA of which is induced more than 50-fold by NGF (180). Colony-stimulating Factors (CSF-1, CSF-2, Multi-CSF). The soft agar colony assay developed by Metcalf and Johnson (181) has led to the identification of a number of factors, called CSFs, that regulate the growth and differentiation of hematopoietic precursor cells. In common with other tissue GFs, CSFs are synthesized at a large number of sites in the body and are active in the low pw level. These factors include CSF-1 [formerly called macrophage CSF (182)], CSF-2 [granulocyte-macrophage CSF (183)], and granulocyte CSF (184). Another factor called interleukin 3, IL-3, is active in stimulating colonies of mixed cell type (185) and has been dubbed multi-CSF. This factor goes by various names in the literature, reflecting its stimulation of growth and differentiation of a variety of cell types: P-cell-stimulating factor (186); mast cell-stimulatory factor (187); hematopoietin 2 (188); burst-promoting activity (189); and hematopoietic cell growth factor (190). Unlike most GFs which are purified on the basis of a cell growth bioassay, IL-3 was described and purified on the basis of its ability to induce an enzyme (20a-hydroxysteroid dehydrogenase) in mouse spleen T-lymphocytes (191 ). The activity of IL-3 (multi-CSF) includes the promotion of growth and differentiation of granulocytes, macrophages, and multipotential stem cells as well as colony formation from early erythroid, eosinophilic, megakaryocytic, and mast cell progenitors (192). Multi-CSF has been purified and partially sequenced (185); cDNA clones corresponding to both human and mouse multi-CSF have been isolated (187, 193). CSF-1 has been purified to homogeneity from mouse L-cells AND CANCER two possibly identical chains linked by disulfide bonds (194). The variation in size is due in large part to variable carbohydrate side chain modification; the polypeptide chain itself may account for only 15 kDa of the size of the reduced chain. The human gene encoding CSF-1 has now been cloned (197); sequence of the cDNA clone indicates a pre-proCSF-1 of 252 residues with a 32residue leader peptide. The proCSF-1 peptide (224 residues) may be further processed to a 20-kDa form by proteolytic processing after residue 188. Incubation of bovine marrow ad hesive cells with either CSF-1 or multi-CSF will induce up regu lation of the number of CSF-1 receptors (198). It has recently been reported that the product of the c-fms proto-oncogene is the receptor for CSF-1 (24). The c-fms protein is a 170-kDa transmembrane glycoprotein which displays tyrosine kinase ac tivity (199, 200). As in the case of the v-enbB gene and the EGF receptor, the v-frns gene may encode a truncated version of the CSF-1 receptor (199, 201). Unlike the EGF receptor case, how ever, the v-fms protein does not appear to be significantly truncated. Since the c-fms gene is located on human chromo some 5 (202), it is interesting to note that a deletion in the long arm of this chromosome in bone marrow cells is associated with a syndrome in which patients are predisposed to myeloid leu kemia (203) or polycythemia vera (204); patients displaying this 5 q- marker are hemizygous for a deletion of chromosome 5 which does include the c-fms locus (205). The macrophage-granulocyte factor, CSF-2, is a glycoprotein that has been purified from endotoxin-treated mouse lung (re viewed in Ref. 206). The factor has now been cloned from three species, mouse, gibbon ape, and humans. Both the human and gibbon ape CSF-2 cDNA clones encode a protein of 144 amino acid residues (207) which is thought to be cleaved to form a mature protein of 127 residues (14 kDa). Sequencing of the mouse cDNA clone (208) reveals substantial sequence homology at the amino acid level to the corresponding residues in human CSF-2; there is 54% amino acid homology between mouse and human CSF-2 (207, 209). CSF-2 has also been called neutrophil migration-inhibitory factor (210). Much less is known about other colony-stimulating factors, although significant progress has been made in purification. The murine factor called granulocyte-CSF (211) has been purified to homogeneity and runs as a 24.5-kDa band on a sodium dodecyl sulfate-polyacrylamide gel (212). This factor is apparently distinct from the differentiation factor [D factor (213)] now purified to homogeneity as a 62-kDa band (214). This latter protein may be identical to MGI-2 (215) and differentiation-inducing factor (216). The D factor induces differentiation of the human promyelocytic leukemia cell line HL-60 (216); chemical treatment of HL-60 cells leads to an induction of the c-fms proto-oncogene (205) and thus presumably receptors for CSF-1. Although the evidence is yet fragmentary, the induction of CSF-1 receptors by another CSF (factor D) would certainly be in keeping with a model of hema topoietic cell differentiation involving the regulation of hematopoiesis mediated through a complex cascade of intercellular protein factor signals. None of these factors have yet been cloned. Autocrine and Paracrine Stimulation in Cancer (182) and human urine (194), and radioreceptor and radioimmune assays have been developed (195, 196). Native CSF-1 from mouse L-cells is a 65-80-kDa sialoglycoprotein composed of CANCER RESEARCH Autologous production of a growth factor by a cell bearing receptors for that same factor could result in a growth advantage. VOL 46 MARCH 1986 1020 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS AND CANCER (i.e., neoplastic and stromal cells) cross-feed each other with factors could explain, in some cases, why it has not been possible to grow presumptive malignant cells in culture (e.g., certain carcinoma cells). In line with a paracrine regulatory model, one might propose that the transformed epithelial cells are de pendent on factors produced by the nonimmortalized, nontrans formed stromal cells found in the tumor which might be unable to survive in long-term cultures. A second explanation would involve the growth-inhibitory feature of TGF/3 on epithelial cell The implications of such autostimulation for the growth of trans formed cells are readily apparent (217-219). Hypotheses invok ing autostimulatory models have also been proposed for smooth muscle cells, human osteosarcoma cells, chemically transformed mouse fibroblasts, and T-cell leukemia involving IGF-1, PDGF, TGF0, and IL-2, respectively. Testing of the autocrine model in these systems has led to a mixed result. In three of these cases, it has been possible to use an anti-GF antibody which inhibits binding of the GF to its receptor. The smooth muscle cells have been shown to produce an IGF-1-like molecule and monoclonal antibodies to IGF-1 inhibit proliferation in a defined culture system (220). In the osteosarcoma case, it has been possible to dem onstrate production of PDGF and functional PDGF receptors in the cloned cell line U-2 OS, as well as significant inhibition of growth (87). Because TGF/8 is a component of serum (1), the routine culture of tumor expiants in serum-containing media might inhibit the outgrowth of TGFß-inhibitedepithelial cells. This explanation is consistent with the observation that most epithelial cell lines tested exhibit some degree of inhibition by TGF/3 (8587,103). growth in the presence of a polyclonal PDGF antibody (221). Another circumstance in which specific cells have been shown to both produce and respond to the same factor is with TGF0 in Growth Factors, Oncogenes, and the Cellular Response chemically transformed fibroblasts (87, 88, 91), although TGF/î antibody inhibition experiments have not yet been performed Dissection of the cellular events intervening between growth due to the lack of high-affinity antibodies to TGF/3. Interestingly, factor binding to cell surface receptors and the initiation of DMA the change in the chemically transformed cells relative to their synthesis is one of the major tasks of cell biology and cancer nontransformed parents is a greatly increased sensitivity to the biology. The machinery that transduces the growth factor signal TGFßproduced by the cells (and present in serum) and not to the cell nucleus includes the growth factor receptors, their increased production of TGF^ (222). substrates, a number of key enzymes (including kinases and In the T-lymphocyte system, evidence indicates that antigenupases), cytoskeletal proteins, transcriptional factors, DNA-bindinitiated IL-2-dependent T-cell growth occurs normally through ing proteins, and lastly a complex of enzymes which channel both autocrine and paracrine mechanisms. T-helper cells both deoxy- and ribonucleotide precursors into the growing forks of produce and respond to IL-2, whereas the majority of cytolytic DMA replication (226). Possible scenarios for GF induction of and suppressor T-cells do not produce IL-2 but proliferate in DMA synthesis and alterations in neoplastic transformation (see response to IL-2 derived from helper T-cells (paracrine stimula Fig. 1) might proceed as follows. tion) (222, 223). The gibbon ape leukemia cell line MLA-144 1. GF binds to its cognate cell surface receptor. In response provides an excellent model for autocrine growth regulation. AGF? MLA-144 cells both produce and respond to IL-2 (224); further more, an anti-IL-2 antibody strongly inhibits the growth of this cell line.6 It has not been possible to extend the autocrine stimulatory observations, however, to human T-cell leukemias. Freshly isolated leukemic cells and cell lines established from children with T-cell acute lymphoblastic leukemia do not produce or respond to IL-2. On the other hand, cells and cell lines from patients with adult T-cell leukemia which is associated with human T-leukemia virus 1 express IL-2 receptors but do not produce IL-2 (225). It is not known whether this constitutive display of IL-2 receptors on virally infected cells could operate in the same fashion as v-eroB in virus-induced erythroblastic leu / Autocrine Stimulation / kemias (see below). The autocrine model may well be adequate to explain growth in soft agar and relative growth factor independence of chemi cally transformed fibroblasts, several instances of simian sar coma virus transformation, the serum factor independence of osteosarcoma cells, and perhaps even the pseudomalignant behavior of normal placental trophoblast (70). A second pathway involving a paracrine model might be at least as likely an expla nation of how growth factor production might operate in the development of cancer. GFs produced by cancer cells could stimulate proliferation of stromal cells (e.g., fibroblasts and vas cular cells), a necessary occurrence for the development of large tumors. Alternatively, stromal cells may produce GFs that stim ulate cancer cells. Such a situation in which tumor components IGF- Fig. 1. Involvement of proto-oncogene cell products in the growth factor-recep tor-response pathway. Specific high-affinity receptors (ft) for GFs are indicated as rectangles in the plane of the cell membrane, each with its own specific site for GF binding; subunit structure is indicated. The c-myc and c-s/s proto-oncogenes are indicated as double helices within the cell nucleus; their mRNA transcripts are indicated by a single wavy line. The phosphatidylinositol pathway (PI —» PIP2 —• DAG + IP3) is indicated as taking place in the plane of the membrane. The protein product of the c-fos oncogene is indicated in a nuclear compartment. Although this fictional cell is indicated to bear receptors for seven different GFs, the various GF receptors show a degree of cell type specificity (see Table 1). No attempt is made to indicate the process of receptor intemalization and/or down regulation. See text for further explanation, pi 70, 170-kDa protein (other proteins are similarly desig nated); pp36, 36-kDa phosphoprotem. • K. A. Smith, personal communication. CANCER RESEARCH VOL. 46 MARCH 1986 1021 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS to GF binding, the receptor may undergo an allosteric change, a redistribution in the membrane, or an association with other membrane proteins. The EGF receptor, for example, is a 170- AND CANCER treatment (240); TGF/3 will induce cytoskeletal changes similar to those seen for PDGF.7 Likewise, EGF will induce transient ruffling behavior and a long-term reorganization of A431 monolayer morphology (241). Recent evidence indicates that the p21 product of the ras gene may be involved in growth factor signal transduction. strate(s) for phosphorylation (48). In the presence of EGF, the Besides induction of morphological transformation (242, 243), receptor density on the cell surface decreases ("down-regula microinjection of p21 induces DMA synthesis (244). Even more tion") as the GF-GF receptor complex is internalized into "recepmeaningful are the studies with microinjection of monoclonal tosomes" (227). antibodies to p21 (26). The antibodies block serum or EGF/ 2. The activated GF receptor activates a number of ¡ntracellular insulin stimulation of DMA synthesis indicating that the ras p21 substrates. Although the EGF receptor can phosphorylate itself is an obligatory intermediate in the transduction of the growth (49), it may also lead to the phosphorylation of a 35-kDa protein factor stimulus. Further, it suggests another step at which a in a Ca2+-dependent fashion (228), a 36-kDa protein (229-231), lesion may occur as a step in neoplastic transformation. If a possibly a 42-kDa protein (232), or even phosphatidylinositol postreceptor mechanism is constitutively activated, the cell may (233). Other possible targets for phosphorylation include vinculin continuously receive a proliferative stimulus without the need for (234) and the glycolytic enzymes enolase and phosphoglycerate a growth factor or its receptor. Such may be the mechanism of mutase (235). transformation by activated ras. Activation of the receptor can sometimes occur in the absence 4. GF stimulation of quiescent cells brings about transcriptional of growth factor. Sequence homology between the eròB gene activation of a number of genes in the middle time frame (20 min-4 h). One of the most striking consequences of GF stimu product and the cellular receptor for EGF (23) suggested that lation is the induction of c-oncogene transcription. Treatment of the chief feature distinguishing the two is the absence of the EGF-binding domain in the retroviral version, suggesting a mode fibroblasts with PDGF brings about a 40-fold elevation of c-myc of oncogene activation in which the erbB protein might function mRNA levels within 2 h (27) and a similar increase in c-fos mRNA to relay a mitogenic signal even in the absence of ligand (EGF) levels within 45 min (28-30). Recent evidence using Chinese binding. Recent evidence confirms this model in ALV-induced hamster lung fibroblasts, however, indicates that the increased chicken erythroleukemias; every case analyzed in one study accumulation of c-myc transcripts may be posttranscriptional apparently involved the integration of an intact ALV genome into (245). FGF and EGF share this ability to induce c-fos gene the c-enbB locus in a fashion that would lead to the overexprestranscription (29). PDGF induces the c-myc gene in cultured sion of a truncated EGF receptor under the control of the placenta! trophoblast cells as well (70). Other genes induced by introduced ALV promoter (236). In this way, the cell expressing growth factors include ß-and 7-actin by EGF (246) and three a truncated GF receptor might be constitutively activated to a mRNAs of unknown function, KC, JE, and JC [related to c-fos "turned-on" state regardless of the presence of the growth factor. (247)], after PDGF treatment (248). In addition, TGF/3 induces a Moreover, evidence has led to an identification of the protein peak of actin mRNA the magnitude of which is TGF/? dose product of the c-fms oncogene as the cell surface receptor for dependent (249) which correlates with the degree of morpholog the hematopoietic stem cell growth factor CSF-1 (24); the v-frns ical transformation apparent at 24 h after TGF treatment (43). As has been mentioned, TGF/8 also induces the c-s;'s proto oncogene may encode an altered form of the receptor. EGF receptor gene (c-erbB) homology to other members of oncogene in mouse fibroblasts, the translation product of which the src gene family has led to the speculation that one or more (a PDGF-related mitogen) is suggested to serve as the mitogen of these c-oncogenes may encode GF receptors (25). Some of mediating the action of TGF/îon AKR-2B cells.4 In this middle the src-related proto-oncogenes may encode enzymes involved time frame, GF treatment brings about a 2- to 4-fold rise in the in the increased ¡ntracellularformation of inositol triphosphate rate of protein synthesis, accompanied by the phosphorylation and diacylglycerol (233). Although both the 35- and 36-kDa of ribosomal protein S6 (250). proteins are phosphorylated on tyrosines as well as serines and 5. Several GF-induced proteins are localized to the nucleus of threonines, the significance of the tyrosine phosphorylation has stimulated cells and may be involved in the pleiotropic activation not provided the key to growth control as had first been hoped. of growth-regulated genes. The products of the c-myc and c-fos 3. The increased concentrations of inositol triphosphates and genes (251, 252) are presumably DNA-binding proteins (253) diacylglycerol is followed by a transient increase in cytosolic-free found chiefly in the cell nucleus (29,254,255). The c-fos-encoded calcium, an activation of protein kinase C and adenyl cyclase, protein increases rapidly in concentration after PDGF stimulation and a reorganization of the cytoskeleton. These middle early and is found localized to the cell nucleus 1 h after stimulation events occur within several min after GF stimulation; it is not (29). Similarly, an unidentified 29-kDa protein is rapidly induced known whether their temporal proximity reflects any causal by PDGF in BALB/C-3T3 cells and becomes localized in the relationship. However, recent evidence points to an interplay nucleus (256). It appears that the level of c-myc gene expression between diacylglycerol and the interaction of a-actinin with the correlates well with the level of proliferative activity in placental cell membrane (237) and between phosphatidylinositol 4,5-bistrophoblast (257) and the state of lymphocyte proliferation (258). phosphate and actin polymerization (238). PDGF induces a rapid These results would suggest that the c-myc product may reflect reorganization of the cytoskeleton of human fibroblasts within 2 the cell's commitment to proliferation, perhaps through an acti min marked by the formation of circular membrane ruffles within vation of other growth-related genes. 15 min (239). In addition to its effects on cytoskeletal actin, 7W. J. Pledger, personal communication. PDGF also induces a redistribution of vinculin within minutes of kDa glycoprotein located at the cell surface, possessing an extracellular EGF binding domain, a transmembrane region, and a cytoplasmic face bearing domains which bind ATP and sub- CANCER RESEARCH VOL. 46 MARCH 1986 1022 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS Constitutive activation of growth factor-regulated genes such as c-myc in some circumstances results in an apparently contin uous stimulus to proliferate. Such may be the case with certain B-cell tumors, such as mouse plasmacytoma and Burkitt's lymphoma, where derived cell lines all show characteristic chromo somal translocations involving the c-myc locus (259, 260). In these tumors, the chromosomal rearrangements presumably transcriptionally activate the c-myc locus, resulting in a high constitutive level of c-myc mRNA; it is this transcriptional acti vation of c-myc, it is argued, that drives their uncontrolled proliferation (261). The constitutive high-level synthesis of myc mRNA is not sufficient to transform fibroblastic cells, however. Transfection of primary rat fibroblasts with an activated myc gene is not sufficient to cause formation of transformed foci (262). Full transformation seems to require a second cooperating oncogene from the ras family (262). Armelin ef al. (263) have helped to clarify this puzzling observation by transfecting 3T3 cells with a c-myc gene construct that allowed high-level contin uous presence of c-myc mRNA in the presence of glucocorticoids. Instead of transforming the target cell, the c-myc gene activation led to an independence of the cells from PDGF stim ulation (263). The situation becomes more clear in light of the competence- AND CANCER second GF signal which might come from stimulation by insulinlike growth factors or EGF [progression factors for BALB/C-3T3 cells (44)]. In light of the c-myc gene induction by PDGF (27), one might therefore speculate that expression of c-myc protein could be part of the state of competence (264). However, recent evidence suggests that c-myc gene induction is necessary, but not sufficient for induction of competence in normal human Blymphocytes (265). One model might thus divide not only growth factors into competence and progression groups but their cellular targets as well. In this way, certain oncogene cell products may be involved in competence (e.g., myc, myb, E1a, fos, sis), others in progression (e.g., ras, Blym, raf/mil), and still others in both (polyoma middle T). If myc expression is a competence phenom enon (and not a growth phenomenon per se) reflecting exposure to GFs (70,266), then it is not surprising to learn that cells grown in the presence of serum would show myc transcripts and myc protein regardless of their particular cell cycle phase (267). Summary and Conclusions Growth factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in progression model of Pledger ef al. (9, 10) in which growth factors can be divided into two groups. Competence factors, such as PDGF, induce a state of "competence" to respond to a culture and probably also for cells in vivo. Nontransformed cells show an absolute requirement for growth factors for proliferation in culture and generally more than one growth factor is required. Under usual culture conditions, growth factors are more rapidly Table 1 factor translation sourceSubmaxillary cellWide size6 product1168 factorEGFTGF«PDGFTGF/3IGF-IIGF-IIIL-2FGF(3-NGFCSF-1CSF-2 Growth aa"160 or 1217 gland.Brunner's epithelialand variety of aa)5.6 kOa (53 gland.possibly mesenchymalcellsSame kDatyrosine gene; 170 kinaseSame 31-4175-8455-57, parietalcellsTransformed aa241 Achain aa (B chain); Bchainunknown; c-sis encoded in proto-oncogene391 aa130 cells.placenta, aa)32kDa(16kDaBchain; kOa (50 em bryosBlood en-dothelial platelets, 14-18-kDaA cells,placentaBlood CHO25kDa(2x chain), + EGFMesenchymal as EGF185 as cells,smooth pla-cental muscle, trophoblastFibroblastic kDa tyrosine ki nase565-615 112aa)7 platelets,kidney, placenta,cultured cellsAdult kerati-nocytes, cells, complex(2 kDa kDa)450 x 280-290 mammaryepithelial cells, carci melanomalinesEpithelial, noma, and aa180 aa)7 kDa (70 andother liver sites,smooth musclecellsFetal mesenchymalEpithelial, achains kDa complex (2 kDa;2 of 130 85kDa)Single ft chains of aa153 placentaT-helper liver, aa)15kDa(133aa);some kDa (67 mesenchymalCytotoxic polypeptidechain kDa55 of 260 kDa (33 kDa pro CHO)Unknown130 tein + 22 kDa cellsBrain, aa(human)Unknown307 aa (mouse); 169 aa252 aa144 (granulocyte-macrophageCSF)Multi-CSF aa144aaMature CHO14-1 (basicFGF 8 kDa 146aa)26 is aa)70 kDa (2 x 118 35kDa); kDa (2 x CHO15-28 60% T-lympho cytesEndothelial pituitary.chondrosarcomaSubmaxillary cells, fibro blastsSympathetic glandMouse and sen neuronsMacrophage sory L-cellsEndotoxin-inducedlung; progenitorsMacrophage granu-locyte and aa)(1-50% kDa (127 CHO)28kDa(134aa)(50% placentaT-lymphocytesTarget progenitorsEosinophil, (IL-3)Primary 105-108123-134123-134137-140,14 kDa (possibly ki nase)c-frns proto-oncogene;170 194-205183, kDa tyrosine ki naseUnknownUnknownRef.23, 192,206-210185, cell.granulocyte, mast macro phage progenitors; T-lymphocytesReceptorc-erbB CHO)Cell 67-70, 73-7485-99, 188,190-192 a aa, amino acid residues; CHO, carbohydrates. CANCER RESEARCH VOL. 46 MARCH 1986 1023 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS depleted than other media components and thus become rate limiting for proliferation. The loss of or decreased requirement for specific growth factors is a common occurrence in neoplast- AND CANCEL References 1. Childs, C. B., Proper, J. A., Tucker, R. F., and Moses, H. L. Serum contains a platelet-derivedtransforming growth factor. Proc. Nati. Acad. Sci. USA, 79: 5312-5316,1982. 2. Holley, R. W., and Kieman, J. A. Control of the initiation of DNA synthesis in 3T3 cells: serum factors. Proc. Nati. Acad. Sci. USA, 71: 2908-2911,1974. 3. Oka, Y., and Orth, D. N. Human plasma epidermal growth factor/0 urogastrone is associated with blood platelets. J. Clin. Invest., 72.-249-259,1983. 4. Vogel, A., Raines, E., Kariya, B., Rivest, M.-J., and Ross, R. Coordinate control of 3T3 cell proliferation by platelet-derivedgrowth factor and plasma components. Proc. Nati. Acad. Sci. USA, 75: 2810-2814,1978. 5. Shields, R. Growth factors for tumours. Nature (Lond.),272: 670-671,1978. 6. Wrann, M., Fox, C. F., and Ross, R. Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derivedgrowth factor. Science(Wash.DC), 270:1363-1365,1980. 7. Fabricant, R. N., DeLarco, J. E., and Todaro, G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc. Nati. Acad. Sci. USA, 74:565-569,1977. 8. Bowen-Pope, D. F., DiCorieto, P. E., and Ross, R. Interactions between the receptors for platelet-derivedgrowth factor and epidermal growth factor. J. Cell Bid., 96: 679-683, 1983. 9. 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. Nati. Acad. Sci. USA, 75: 2839-2843,1978. 10. Leof, E. B., Wharton, W., Van Wyk, J. J., and Pledger, W. J. Epidermal growth factor (EGF)and somatomedin C regulate G, progression in compe tent BALB/C-3T3cells. Exp. Cell Res., 141:107-115,1982. 11. Barnes. D., and Sato. G. Serum-free cell culture: a unifying approach. Cell. 22: 649-655, 1980. 12. Walthall, B. J., and Ham, R. G. Multiplication of human diploid fibroblasts in a synthetic medium supplemented with EGF, insulin, and dexamethasone. Exp. Cell Res., 734: 301-309,1981. 13. Tsao, M. C., Walthall, B. J., and Ham, R. G. Clonal growth of normal human keratinocytes in a defined medium. J. Cell. Physiol., 110: 219-229,1982. 14. Wharton, W., Leof, E., Olashaw, N., O'Keefe, E. J., and Pledger, W. J. Mitogenic response to epidermal growth factor (EGF)modulated by plateletderived growth factor in cultured fibroblasts. Exp. Cell Res., 747: 443-448, 1983. 15. O'Keefe, E. J., and Pledger, W. J. Review: a model of cell cycle control: ically transformed cells and may lead to a growth advantage, a cardinal feature of cancer cells. Recent work with transforming growth factors, the platelet-derived growth factor, and oncogenes has produced some insight into the mechanisms through which alterations in growth factor-receptor-response pathways could lead to a growth advantage. Evidence has been derived for autocrine secretion in which the cell produces its own growth factor. Many transformed mesenchymal cells produce PDGF (the product of the c-s/s proto-oncogene) and certain transformed cells both produce and respond in a growth-stimulatory manner to TGF0. With TGF/3, which is a growth inhibitor for certain epithelial and other cell types, the loss of the normal inhibitory response in transformed cells could have the same result as the activation of a growth-stimulatory response. Two proto-oncogenes, erbB and fms, encode growth factor receptors. In the erbB case, the viral erbB aberrant receptor produced is truncated and appears to be constitutively activated without the need for a growth factor. Recent studies suggest that the p21 product of the ras oncogene may be an obligatory intermediate in transducing the growth factor signal. Activation of ras may, therefore, activate the growth factor pathway without the need for either a growth factor or its receptor. The transcrip tion of myc and fos is induced by growth factor stimulation of quiescent cells. The protein products of both are nuclear asso ciated and conceivably could be involved in regulating other genes important in the control of cell proliferation. Activation or inappropriate expression of either myc or tos could produce the same end result as stimulation of a growth factor pathway leading to a growth advantage. Study of the molecular mechanism(s) of growth factor action has just begun. The excitement and attention focused on cellular oncogenes in recent years is now turning toward growth factors, not only as they concern the control of normal cell growth but also the involvement of growth factor-initiated pathways in the 16. 17. 18. 19. etiology of cancer. One important implication of the molecular dissection of growth control is the identification of specific genes important in growth regulation. The genes encoding growth factors, growth factor receptors, and the post-receptor machinery (i.e., the prod ucts of the sis, erbB, fms, ras, fos, myb, and myc proto-onco 20. 21. genes as well as the p53 gene) may be a significant subset of these pivotal regulatory genes. The cell specificity of these genes (see Table 1) may imply that it would be possible to treat neoplastic diseases with a more targeted arsenal of therapeutic agents which focus their effects on a narrower range of proliferative cells than today's drugs with more generalized actions. 22. 23. In this way, an agent which might interfere with the TGF/3-s/sPDGF pathway might inhibit mainly mesenchymal cell prolifera tion in a sarcoma, leaving untouched the proliferation of normal cells in the hemopoietic lineage and the intestinal epithelium, so often a side effect of the current generation of chemotherapeutic agents. 24. 25. 26. 27. Acknowledgments 28. 29. The authors wish to thank Patricia Hart for typing the manuscript. CANCER RESEARCH sequential events regulated by growth factors. Mol. Cell. Endocrinol., 37: 167-186,1983. Temin, H. M. Studies on carcinogenesis by avian sarcoma viruses, VI. The differential effect of serum and polyanionson multiplicationof uninfected and converted cells. J. Nati. Cancer Inst., 37: 167-175,1966. Paul, D., Upton, A., and Klinger, I. Serum factor requirements of normal and simian virus-40-transformed 3T3 mouse fibroblasts. Proc. Nati. Acad. Sci. USA, 68: 645-648, 1971. Dulbecco, R. Topoinhibition and serum requirement of transformed and untransformed cells. Nature (Lond.), 227: 802, 1970. Kaplan, P. L., Anderson, M., and Ozanne, B. Transforming growth factor production enables cells to grow in the absence of serum: an autocrine system. Proc. Nati. Acad. Sci. USA, 79: 485-489,1982. Moses, H. L, Proper, J. A., Volkenant, M. E., Wells, D. J., and Getz, M. J. Mechanismof growth arrest of chemicallytransformedcells in culture. Cancer Res., 38. 2807-2812,1978. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C. H., Huang, J. S. and Deuel, T. F. Platelet-derivedgrowth factor is structurally relatedto the putative transform ing protein p28s/s of simiansarcomavirus. Nature(Lond.),304:35-39,1983. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. Simian sarcoma virus one gene, v-sis, is derived from the gene (or genes) encoding a platelet-derivedgrowth factor. Science (Wash. DC), 227: 275-277, 1983. Downward, J., Yarden, Y., Mayes, E., Scarce, G., Totty, N., Stockwell, P., Ullrich,A., Schlessinger,J., and Waterfield, M. D. Close similarityof epidermal growth factor receptor and v-ero-B oncogene protein sequences. Nature (Lond.),307: 521-527, 1984. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T., and Stanley, E. R. The c-frns proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell, 41: 665-676 1985. Hunter, T. The proteins of oncogenes. Sei. Am., 257: 70-79, 1984. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. Requirementfor ras protooncogene function during serum-stimulatedgrowth of NIH 3T3 cells. Nature (Lond.), 373: 241-243, 1985. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. Cell-specificregulation of the c-myc gene by lymphocytemitogens and platelet-derivedgrowth factor. Cell, 35:603-610, 1983. Greenberg,M. E., and Ziff, E. B. Stimulationof 3T3 cells induces transcription of the c-fos proto-oncogene. Nature (Lond.), 377: 433-438,1984. Muller, R., Bravo, R., Burckhardt, J., and Curran, T. Induction of c-fos gene VOL. 46 MARCH 1986 1024 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. FACTORS and protein by growth factors precedes activation of c-myc. Nature (Lond.), 312: 716-720, 1984. Kruijer, W., Cooper, J. A., Hunter, T., and Verma, I. M. Platelet-derivedgrowth factor induces rapid but transient expression of the c-fos gene and protein. Nature (Lond.), 312: 711-716.1984. Cohen, S. Isolation of a mouse submaxillarygland protein acceleratingincisor eruption and eyelid opening in the new-born animal. J. Biol. Chem., 237: 1555-1562,1962. Carpenter, G., and Cohen, S. Human epidermal growth factor and the proliferation of human fibroblasts. J. Cell. Physiol., 88: 227-238, 1975. Hollenberg, M. D., and Cuatrecasas, P. Epidermal growth factor: receptors in human fibroblasts and modulation of action by cholera toxin. Proc Nati. Acad. Sci. USA, 70: 2964-2968, 1973. Gregory, H. Isolation and structure of urogastrone and its relationship to epidermalgrowth factor. Nature (Lond.), 257: 325-327,1975. Cohen, S., and Carpenter, G. Human epidermal growth factor: isolation and chemical and biological properties. Proc. Nati. Acad. Sci. USA, 72: 13171321,1975. Taylor,J. M., Mitchell, W. M., and Cohen,S. Epidermalgrowth factor: physical and chemical properties. J. Biol. Chem., 247: 5928-5934, 1972. Gray, A., Dull, T. J., and Ullrich,A. Nucleotide sequenceof epidermalgrowth factor cDNA predicts a 128,000-molecularweight protein precursor. Nature (Lond.),303: 722-725, 1983. Rail, L. B., Scott, J., and Bell, G. I. Mouse prepro-epidermalgrowth factor synthesis by the kidney and other tissues. Nature (Lond.), 373: 228-231, 1985. Scott, J., Urdea, M., Quiroga, M., Sánchez-Pescador,R., Fong, N., Selby, M., Rutler, W. J., and Bell, G. I. Structure of a mouse submaxillarymessenger RNA encoding epidermal growth factor and seven related proteins. Science (Wash. DC), 22J: 236-240, 1983. Dailey, G. E., Krause, J. W., and Orth, D. N. Homologous radioimmunoassay for humanepidermalgrowth factor (urogastrone).J. Clin. Endocrinol.Metab., 48:929-936, 1978. Carpenter, G., Lembach, K. J., Morrison, M. M., and Cohen, S. Characteri zation of the binding of 1Ml-labeledepidermal growth factor to human fibro blasts. J. Biol. Chem., 259: 4297-4304, 1975. Rose, S. P.. Pruss, R. M., and Herschman, H. R. Initiation of 3T3 fibroblast cell division by epidermalgrowth factor. J. Cell. Physiol.,86: 593-598,1975. Shipley, G. D., Childs, C. B., Volkenant, M. E., and Moses, H. L. Differential effects of epidermalgrowth factor, transforming growth factor, and insulinon DNA and protein synthesis and morphology in serum-free cultures of AKR2B cells. Cancer Res.. 44: 710-716. 1984. Leof, E. B., Van Wyk, J. J.. O'Keefe. E. J., and Pledger, W. J. Epidermal 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. growth factor (EGF)is required only during the traverse of early d in PDGF stimulated density-arrested BALB/C-3T3cells. Exp. Cell Res., 747:202-208. 1983. Oka. Y., Ghishan, F. K., Greene, H. L., and Orth, D. N. Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine. Endocrinology, 772: 940-944, 1983. Weissman, B. E., and Aaronson, S. A. BALB and Kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines. Cell, 32: 599-606,1983. Brown, J. P., Twardzik, D. R., Marquardt, H.. and Todaro, G. J. Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature (Lond.), 373:491-492,1985. Cohen, S., Ushiro, H., Stoscheck, C., and Chinkers, M. A native 170,000 epidermalgrowth factor receptor-kinase complex from shed membrane ves icles. J. Biol. Chem., 257: 1523-1531,1982. Ushiro, H., and Cohen, S. J. Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A431 cell membranes. J. Biol. Chem., 255: 8363-8365,1980. Weber, W., Gill. G. N., and Spiess, J. Production of an epidermal growth factor receptor-related protein. Science (Wash. DC), 224: 294-297, 1984. Seppä,H. E. J., Grotendorst, G. R., Seppä,S. !.. Schiffmann.E., and Martin. G. R. The platelet-derivedgrowth factor is a chemoattractant for fibroblasts. J. Cell. Biol.. 92: 584-588,1982. Shipley, G. D., Tucker, R. F., and Moses, H. L. Type /3-transforminggrowth factor/growth inhibitor stimulates entry of monolayercultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc. Nati. Acad. Sci. USA, 82: 4147-4151, 1985. Bowen-Pope, D. F., Vogel, A., and Ross, R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide variety of agents. Proc. Nati. Acad. Sci. USA, 87: 2396-2400, 1984. Niman, H. L., Houghton, R. A., and Bowen-Pope, D. F. Detection of high molecularweight forms of platelet-derivedgrowth factor by sequence-specific antisera. Science (Wash. DC), 226: 701-703, 1984. Johnsson, A., Heldin, C.-H., Westermark, G., and Wasteson, A. Plateletderived growth factor: identification of constituent polypeptide chains. Biochem. Biophys. Res. Commun., 704: 66-74, 1982. Johnsson, A., Heldin,C.-H., Wasteson, A., Westermark, B., Deuel,T., Huang, J. S., Seeburg, P. H., Gray, A., Ullrich, A., Scrace, G., Stroobant, P., and CANCER RESEARCH AND CANCER 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. Waterfield, M. The c-s/s gene encodes a precursor of the B chain of plateletderived growth factor. EMBO J., 3: 921-928, 1984. Josephs, S. F., Quo, C., Ratner, L., and Wong-Staal, F. Human proto oncogene nucleotidesequences corresponding to the transforming region of simian sarcoma virus. Science (Wash. DC), 223. 487-491,1984. Dalla Pavera,R., Gelmann,E. P., Gallo, R. C., and Wong-Staal, F. A human one gene homologous to the transforming gene (v-s/s) of simian sarcoma virus. Nature (Lond.), 292: 31-35,1981. Gazit, A., Igarashi, H., Chiù,I.-M., Srinivasan, A., Yaniv, A., Tronick, S. R., Robbins, K. C., and Aaronson, S. A. Expression of the normal human sis/ PDGF-2 coding sequence induces cellular transformation. Cell, 39: 89-97, 1984. Graves, D. T., Owen, A. J., Barth, R. K., Temps!, P., Winoto, A., Fors, L., and Hood, L. E. Detection of c-s/'s transcripts and synthesis of PDGF-like proteins by human osteosarcoma cells. Science (Wash. DC), 226: 972-974, 1984. Pantazis,P., Pelicci,P. G., Dalla-Favera,R., and Antoniades, H. N. Synthesis and secretion of proteins resemblingplatelet-derivedgrowth factor by human glioblastoma and fibrosarcoma cells in culture. Proc. Nati. Acad. Sci. USA, 82:2404-2408,1985. Betsholtz, C., Heldin, C.-H., Nistér, M., Ek, B., Wasteson, A., and Wester mark, B. Synthesis of a PDGF-like growth factor in human glioma and sarcoma cells suggests the expression of the cellular homology of the transforming protein of simian sarcoma virus. Biochem. Biophys. Res. Com mun., 777: 176-182,1983. Nistér, M., Heldin,C.-H.,Wasteson, A., and Westermark, B. A glioma-derived analogto platelet-derivedgrowth factor: demonstrationof receptor competing activity and immunological crossreactivity. Proc. Nati. Acad. Sci. USA, 87: 926-930,1984. Huang, J. S., Huang, S. S., Kennedy, B., and Deuel, T. F. Platelet-derived growth factor. Specific binding to target cells. J. Biol. Chem., 257: 81308136,1982. Bowen-Pope, D. F., and Ross, R. Platelet-derivedgrowth factor. II. Specific binding to cultured cells. J. Biol. Chem., 257: 5161-5171,1982. Heldin,C.-H., Westermark, B.. andWasteson,A. Specificreceptor for plateletderived growth factor on cells derived from connective tissue and glia. Proc. Nati. Acad. Sci. USA, 78: 3664-3668,1981. Ross, R., and Vogel, A. The platelet-derivedgrowth factor (review). Cell, 74: 203-210,1978. DiCorleto, P. E., and Bowen-Pope,D. F. Cultured endothelialcells produce a platelet-derived growth factor-like protein. Proc. Nati. Acad. Sci. USA, 80: 1919-1923,1983. Seifert, R. A., Schwartz, S. M.. and Bowen-Pope, D. F. Developmental^ regulated production of platelet-derivedgrowth factor-like molecules. Nature (Lond.),377: 669-671, 1984. Goustin, A. S., Betsholtz, C., Pfeifer-Ohlsson,S., Persson, H., Rydnert, J.. Bywater, M., Holmgren, G., Heldin, C.-H., Westermark, B., and Ohlsson, R. Coexpression of the sis and myc proto-oncogenes in developing human placenta suggests autocrine control of trophoblast growth. Cell, 47: 301312.1985. Beaconsfield,P., Birdwood, G., and Beaconsfield,R. The placenta. Sei. Am., 243:94-103, 1980. Ek, B., and Heldin,C.-H. Characterizationof a tyrosine-specifickinaseactivity in human fibroblast membranesstimulated by platelet-derivedgrowth factor. J. Biol. Chem., 257:10486-10492,1982. Williams, L. T., Tremble, P. M., Lavin, M. F., and Sunday, M. E. Plateletderived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies. J. Biol. Chem., 259: 5287-5294, 1984. Daniel, T. 0., Tremble, P. M., Frackelton, A. R., Jr., and Williams, L. T. Purification of the platelet-derivedgrowth factor receptor by using an antiphosphotyrosineantibody. Proc. Nati. Acad. Sci. USA,82:2684-2687,1985. Todaro, G. J., Fryling, C., and DeLarco, J. E. Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc. Nati. Acad. Sci. USA, 77: 52585262, 1980. Marquardt, H., Hunkapiller, M. W., Hood, L. E., and Todaro, G. J. Rat transforming growth factor type 1: structure and relationto epidermalgrowth factor. Science (Wash. DC), 223: 1079-1082, 1984. Todaro, G. J., DeLarco, J. E., and Cohen, S. Transformation by murine and feline sarcoma viruses specificallyblocks binding of epidermal growth factor to cells. Nature (Lond.), 264: 26-31. 1976. DeLarco, J. E., and Todaro, G. J. Growth factors from murinesarcoma virustransformed cells. Proc. Nati. Acad. Sci. USA, 75: 4001-4005,1978. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B., and DeLarco, J. E. Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type «and type ßtransforming growth factors. Proc. Nati. Acad. Sci. USA,80: 6264-6268,1983. Marquardt, H., Hunkapiller, M. W., Hood, L. E., Twardzik, D. R., DeLarco, J. E., Stephenson, J. R., and Todaro, G. J. Transforming growth factors produced by retrovirus-transformedrodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor. Proc. VOL. 46 MARCH 1986 1025 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH FACTORS Nati. Acad. Sci. USA, 80. 4684-4688, 1983. 81. Derynck, R., Roberts, A. B., Winkler, M. E., Chen, E. Y., and Goeddel, D. V. Human transforming growth factor-«: precursor structure and expression in E. co/i. Cell, 38: 287-297, 1984. 82. Stromberg, K., Pigott, D. A., Ranchalis, J. E., and Twardzik, D. R. Human term placenta contains transforming growth factors. Biochem. Biophys. Res Commun., 706:354-361, 1982. 83. Matrisian, L. M., Pathak, M., and Magun, B. E. Identification of an epidermal growth factor-related transforming growth factor from rat fetuses. Biochem. Biophys. Res. Commun., 707: 761-769, 1982. 84. Twardzik, D. R., Ranchalis, J. E., and Todaro, G. J. Mouse embryonic transforming growth factors related to those isolated from tumor cells. Cancer Res., 42.: 590-593.1982. 85. Tucker, R. F., Shipley, G. D., Moses, H. L., and Holley, R. W. Growth inhibitor from BSC-1 cells closely related to the platelet type ßtransforming growth factor. Science (Wash. DC), 226: 705-707, 1984. 86. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F., and Sporn, M. B. Type i¡transforming growth factor: a bifunctional regulator of cellular growth. Proc. Nati. Acad. Sci. USA, 82: 119-123, 1985. 87. Moses, H. L., Tucker, R. F., Leof, E. B., Coffey, R. J., Jr., Halper, J., and Shipley, G. D. Type (Jtransforming growth factor is a growth stimulator and a growth inhibitor. In: J. Feramisco, B. Ozanne, and C. Stiles (eds.). Growth Factors and Transformation. Cancer Cells 3, pp. 65-71. Cold Spring Harbor NY: Cold Spring Harbor Press, 1985. 88. Moses, H. L., Branum, E. L., Proper, J. A., and Robinson, R. A. Transforming growth factor production by chemically transformed cells. Cancer Res 412842-2848.1981. 89. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., and Sporn, M. B. New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc. Nati. Acad Sci USA 785339-5343,1981. 90. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., Frolik, C.A., Marquardt, H., Todaro, G. J., and Sporn. M. B. Isolation from murine sarcoma cells of a new class of transforming growth factors potentiated by epidermal growth factor. Nature (Lond.), 295: 417-419, 1982. 91. Tucker, R. F., Volkenant, M. E., Branum, E. L., and Moses, H. L. Comparison of intra- and extracellular transforming growth factors from nontransformed and chemically transformed mouse embryo cells. Cancer Res 43- 15811586,1983. 92. Moses, H. L., Shipley, G. D., Leof, E. B., Halper, J., Coffey, R. J., Jr., and Tucker, R. F. Transforming growth factors. In: A. L. Boynton and H. L. Leffert (eds.), Control of Animal Cell Proliferation. New York: Academic Press, in press, 1985. 93. Roberts, A. B., Anzano, M. A., Meyers, C. A., Wideman, J., Blacher, R., Pan, Y.-C. E., Stein, S., Lehrman, S. R., Smith, L. C., Lamb, L. C., and Sporn, M. B. Purification and properties of a type-fi transforming growth factor from bovine kidney. Biochemistry, 22: 5692-5698,1983. 94. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. M., and Sporn, M. B. Purification and initial characterization of a type-/} transforming growth factor from human placenta. Proc. Nati. Acad. Sci. USA, 80: 3676-3680,1983. 95. Assoian, R. K., Komoriya, A., Meyers, C. A., Miller, D. M., and Sporn, M. B. Transforming growth factor-fi in human platelets: identification of a major storage site, purification and characterization. J. Biol. Chem., 258- 71557160, 1983. 96. Massagué,J. Type fi transforming growth factor from feline sarcoma virustransformed rat cells. Isolation and biological properties. J. Biol. Chem 2599756-9761,1984. 97. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, D. V. Human transforming growth factor-fi cDNA sequence and expression in tumor cell lines Nature (Lond.), 376: 701-705, 1985. 98. Proper, J. A., Bjornson, C. L.. and Moses, H. L. Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture. J. Cell. Physiol 770-169-174 1982. 99. Nickell, K. A., Halper, J., and Moses, H. L. Transforming growth factors in solid human malignant neoplasms. Cancer Res., 43: 1966-1971,1983. 100. Lawrence, D. A., Pircher, R., Krycève-Martinerie, C., and Jullien, P. Normal embryo fibroblasts release transforming growth factors in a latent form J Cell. Physiol., 727: 184-188, 1984. 101. Pircher, R., Lawrence, D. A., and Jullien, P. Latent ¿-transforming growth factor in nontransformed and Kirsten sarcoma virus-transformed normal rat kidney cells, clone 49F. Cancer Res., 44: 5538-5543,1984. 102. Holley, R. W., Armour, R., Baldwin, J. H., and Greenfield, S. Preparation and properties of a growth inhibitor produced by kidney epithelial cells. Cell Biol Int. Rep., 7:525-526, 1983. 103. Holley, R. W., Bohlen, P., Fava, R., Baldwin, J. H., Kleeman, G., and Armour, R. Purification of kidney epithelial cell growth inhibitors. Proc. Nati. Acad Sci USA, 77:5989-5992, 1980. 104. Sporn, M. B., and Roberts, A. B. Autocrine growth factors and cancer. Nature (Lond.), 373: 745-747, 1985. 105. Tucker, R. F., Branum, E. L., Shipley, G. D., Ryan, R. J., and Moses, H. L. CANCER RESEARCH AND CANCER 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. VOL. Specific binding to cultured cells of 125l-labeled transforming growth factortype ßfrom human platelets. Proc. Nati. Acad. Sci. USA, 87- 6757-6761 1984. Frolik, C. A., Wakefield, L. M., Smith, D. M., and Sporn, M. B. Characterization of a membrane receptor for transforming growth factor-/} in normal rat kidney cells. J. Biol. Chem., 259: 10995-11000,1984. Massagué,J., and Like, B. Cellular receptors for type fi transforming growth factor: ligand binding and affinity labeling in human and rodent cell lines J Biol. Chem., 260: 2636-2645, 1985. Massagué,J. Subunit structure of a high-affinity receptor for type fi-transforming growth factor: evidence for a disulfide-linked glycosylated receptor complex. J. Biol. Chem., 260: 7059-7066,1985. Hirai, R., Yamaoka, K., and Mitsui, H. Isolation and partial purification of a new class of transforming growth factors from an avian sarcoma virustransformed rat cell line. Cancer Res., 43: 5742-5746,1983. Yamaoka, K., Hirai, R., Tsugita, A., and Mitsui, H. The purification of an acidand heat-labile transforming growth factor from an avian sarcoma virustransformed rat cell line. J. Cell. Physiol., 779: 307-314,1984. Halper, J., and Moses, H. L. Epithelial tissue-derived growth factor-like polypeptides. Cancer Res., 43:1972-1979, 1983. Salmon, W. D., Jr., and Daughaday, W. H. A hormonally controlled serum factor which stimulated sulfate incorporation by cartilage in vitro J Lab Clin Med., 49: 825-829, 1957. Blundell, T. L., and Humbel, R. E. Hormone families: pancreatic hormones and homologous growth factors. Nature-(Lond.), 287: 781-787, 1980. Svoboda, M. E., Van Wyk, J. J., Klapper, D. G., Fellows, R. E., Grissom, F. E., and Schlueter, R. J. Purification of somatomedin-C from human plasma: chemical and biological properties, partial sequence analysis, and relationship to other somatomedins. Biochemistry, 79: 790-797, 1980. Van Wyk, J. J., Underwood, L E., D'Ercole, A. J., Clemmons, D. R., Pledger, W. J., Wharton, W. R., and Leof, E. B. Role of somatomedin in cellular proliferation. In: E. R. Ritzen (ed.), The Biology of Normal Human Growth pp. 223-233. New York: Raven Press, 1981. Van Wyk, J. J., Underwood, L. E., Baseman, J. B., Hintz, R. L., Clemmons, D. R., and Marshall, R. N. Explorations of the insulin-like and growthpromoting properties of somatomedin by membrane assays. Adv Metab Disord.,8: 127-150, 1975. Furianetto, R. W. The somatomedin C binding protein: evidence for a heterologous subunit structure. J. Clin. Endocrin. Metab., 57: 12-19, 1980. Temin, H., Pierson, R. W., Jr., and Dulak, N. C. The role of serum in the control of multiplication of avian and mammalian cells in culture. In: G. H. Rothblatt and V. J. Cristofalo (eds.), Growth, Nutrition, and Metabolism of Cells in Culture, pp. 50-81. New York: Academic Press, 1972. Dulak, N. C., and Temin, H. M. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts. J. Cell. Physiol., 87: 153-160, 1973. Nissley, S. P., Short, P. A., Rechter, M. M., Podskalny, J. M., and Coon, H. G. Proliferation of Buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA). Cell 77- 441-446 1977. D'Ercole, A. J., Stiles, A. D., and Underwood, L. E. Tissue concentrations of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc. Nati. Acad. Sci. USA 87- 935-939 1984. Russell, W. E., Van Wyk, J. J., and Pledger, W. J. Inhibition of the mitogenic effect of plasma by a monoclonal antibody to somatomedin C. Proc Nati Acad. Sci. USA, 87: 2389-2392, 1984. Rinderknecht, E., and Humbel, R. E. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin J Biol Chem., 253: 2769-2776,1978. Van Wyk, J. J., Russell, W. E., and Li, C. H. Synthetic somatomedin C: comparison with natural hormone isolated from human plasma Proc Nati Acad. Sci. USA, 87: 740-742, 1984. Bell, G. I., Merryweather, J. P., Sánchez-Pescador, R., Stempien, M. M., Preistly, L., Scott, J., and Rail, L. B. Sequence of a cDNA clone encoding human preproinsulin-like growth factor II. Nature (Lond) 370- 775-777 1984. Bell, G. I., Gerhard, D. S., Fong, N. M., Sánchez-Pescador, R., and Rail, L. B. Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous. Proc. Nati Acad Sci USA 826450-6454,1985. Adams, S. 0., Nissley, S. P., Handwerger, S., and Rechter, M. M. Develop mental patterns of insulin-like growth factor-l and -II synthesis and regulation in rat fibroblasts. Nature (Lond.), 302: 150-153, 1983. Massagué, J., and Czech, M. P. The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J. Biol. Chem., 257: 5028-5045,1982. Pilch, P. F., and Czech, M. P. The subunit structure of the high-affinity insulin receptor. Evidence of disulfide-linked receptor complex in fat cell and liver plasma membranes. J. Biol. Chem., 255:1722-1731, 1980. Pilch, P. F., and Czech, M. P. Interaction of cross-linking agents with the 46 MARCH 1986 1026 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. FACTORS insulin effector system of isolated fat cells. J. Biol. Chem., 254: 3375-3381, 1979. Van Obberghen, E., Rossi, B., Kowalski, A., Cazzano, H., and Ronzio, G. Receptor-mediated phosphorylation of the hepatic insulin receptor: evidence that the M, 95,000 receptor subunit is its own kinase. Proc. Nati. Acad. Sci. USA, 80. 945-949,1983. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T.J., Gray, A., Coussens, L, Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature (Lond.), 3r3: 756-761, 1985. Kasuga, M., Van Obberghen, E., Nissley, S. P., and Rechter, M. M. Demon stration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J. Biol. Chem., 256: 5305-5308, 1981. Massagué,J. Type-t¡transforming growth factor receptors in cells chronically exposed to ligand. In: J. Feramisco, B. Ozanne, and C. Stiles (eds.), Growth Factors and Transformation, (Cancer Cells 3), pp. 73-78. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1985. Smith, K. A. Interleukin 2. Annu. Rev. Immunol., 2. 319-333,1984. Morgan, D. A., Ruscetti, F. W., and Gallo, R. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science (Wash. DC), 793: 1007-1008,1976. Taniguchi, T., Matsui, H., Takashi, F., Takaoka, C., Kashima, N., Yoshimoto, R., and Hamuro, J. Structure and expression of a cloned cDNA for human interleukin-2. Nature (Lond.), 302: 305-310, 1983. Robb, R. J., Kutny, R. M., and Chowdry, V. Purification and partial sequence analysis of human T-cell growth factor. Proc. Nati. Acad. Sci. USA, 80: 59905994, 1983. Holbrook, N. J., Smith, K. A., Fornace, A. J., Jr., Comeau, C. M., Wiskocil, R. L., and Crabtree, G. R. T-cell growth factor: complete nucleotide sequence and organization of the gene in normal and malignant cells. Proc. Nati. Acad. Sci. USA, 81: 1634-1638, 1984. Kashima, N., Nishi-Takaoka, C., Fujita, T., Taki, S., Yamada, G., Hamuro, J., and Taniguchi, T. Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature (Lond.), 373: 402-404,1985. Efrat, S., and Kaempfer, R. Control of biologically active interleukin-2 mes senger RNA formation induced human lymphocytes. Proc. Nati. Acad. Sci. USA, 81: 2601-2605, 1984. Kränke,M., Leonard, W. J., Depper, J. M., Arya, S. K., Wong-Staal, F., Gallo, R. C., Waldmann, T. A., and Greene, W. C. Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription. Proc. Nati. Acad. Sci. USA, 87: 5214-5218, 1984. Urdal, D. L., March, C. J., Gillis, S., Larsen, A., and Dower, S. K. Purification and chemical characterization of the receptor for interieukin 2 from activated human T lymphocytes and from a human T-cell lymphoma line. Proc. Nati. Acad. Sci. USA. 81: 6481-6485, 1984. Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kränke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T., and Greene, W. C. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature (Lond.), 377: 626-631,1985. Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigawara, K., Maeda, M.. Uchiyama, T., Yodoi, J., and Honjo, T. Molecular cloning of cDNA encoding human interieukin-2 receptor. Nature (Lond.), 377: 631-635, 1984. Smith, K. A., and Cantrell, D. A. Interleukin 2 regulates its own receptors. Proc. Nati. Acad. Sci. USA, 82: 864-868, 1985. Robb, R. J., Munck, A., and Smith, K. A. T cell growth factor receptors: quantitation, specificity, and relevance. J. Exp. Med., 754:1455-1474,1981. Larsson. E.-L., and Coutinho, A. The role of mitogenic lectins in T-cell triggering. Nature (Lond.), 280: 239-241, 1979. Gospodarowicz, D., Moran, M., Braun, D., and Birdwell, C. R. Clonal growth of bovine endothelial cells in tissue culture: fibroblastic growth factor as a survival agent. Proc. Nati. Acad. Sci. USA, 73: 4120-4124, 1976. Gospodarowicz, D. Purification of a fibroblast growth factor from bovine pituitary. J. Biol. Chem., 250: 2515-2520, 1975. Gospodarowicz, D., Bialecki, H., and Greenburg, G. Purification of the fibro blast growth factor activity from bovine brain. J. Biol. Chem., 253: 37363743,1978. Westall, F. C., Lennon, V. A., and Gospodarowicz, D. Brain-derived fibroblast growth factor: identity with a fragment of the basic protein of myelin. Proc. Nati. Acad. Sci. USA, 75: 4675-4678, 1978. Thomas, K. A., Riley, M. C., Lemmon, S. K., Baglan, N. C., and Bradshaw, R. A. Brain fibroblast growth factor. Nonidentity with myelin basic protein fragments. J. Biol. Chem., 255: 5517-5520,1980. Lemmon, S. K., and Bradshaw, R. A. Purification and partial characterization of bovine pituitary fibroblast growth factor. J. Cell. Biochem., 27: 195-208, 1983. Lemmon, S. K., Riley, M. C., Thomas, K. A., Hoover, G. A., Maciag, T., and Bradshaw, R. A. Bovine fibroblast growth factor: comparison of brain and pituitary preparations. J. Cell Biol., 95: 162-169, 1982. Thomas, K. A., Rios-Candelore, M., and Fitzpatrick, S. Purification and characterization of acidic fibroblast growth factor from bovine brain. Proc. Nati. Acad. Sci. USA, 87: 357-361, 1984. CANCER RESEARCH AND CANCER 157. Bohlen, P., Baird, A., Esch, F., Ling, N., and Gospodarowicz, D. Isolation and partial molecular characterization of pituitary fibroblast growth factor. Proc. Nati. Acad. Sci. USA, 87: 5364-5368,1984. 158. Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A., and Bohlent, P. Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography: identity with pituitary fibroblast growth factor. Proc. Nati. Acad. Sci. USA, 87: 6963-6967,1984. 159. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J., and Klagsbrun, M. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science (Wash. DC), 223: 1296-1299,1984. 160. Gospodarowicz, D., Massoglia, S., Chen, J., Lui, G.-M., and Bohlen, P. Isolation and pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization. J. Cell. Physiol., 722: 323-332,1985. 161. Shipley, G. D., O'Sullivan, D., Swanson, B. M., and Moses, H. L. Isolation of 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. VOL. pituitary-derived fibroblast growth factor (FGF) by heparin agarose chromatography and its effect on gene expression in mouse AKR-2B cells. Fed. Proc., 44. 626, 1985. Lobb, R. R., and Fett, J. W. Purification of two distinct growth factors from bovine neural tissue by heparin affinity chromatography. Biochemistry, 23: 6295-6299,1984. Klagsbrun, M., and Shing, Y. Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors. Proc. Nati. Acad. Sci. USA, 82: 805809,1985. Esch, F., Baird, A., üng,N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillemin, R. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the aminoterminal sequence of bovine brain acidic FGF. Proc. Nati. Acad. Sci. USA, 82:6507-6511,1985. Thomas, K. A., Rios-Candelore, M., Giménez-Gallego, G., DiSalvo, J., Ben nett, C., Rodkey, J., and Fitzpatrick, S. Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interieukin 1. Proc. Nati. Acad. Sci. USA, 82: 64096413.1985. Schreiber, A. B., Kenney, J., Kowalski, W. J., Friesel, R., Mehlman, T., and Maciag, T. Interaction of endothelial cell growth factor with heparin: charac terization by receptor and antibody recognition. Proc. Nati. Acad. Sci. USA, 82:6138-6142, 1985. Folkman, J. Angiogenesis: initiation and modulation. Symp. Fundam. Cancer Res.,36. 201-208,1983. Lillien, L. E., and Claude, P. Nerve growth factor is a mitogen for cultured chromaffin cells. Nature (Lond.), 377: 632-634,1985. Levi-Montalcini, R., and Hamburger, V. Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J. Exp. Zool., 776: 321-351, 1951. Cohen, S. Purification and metabolic effects of a nerve growth-promoting protein from snake venom. J. Biol. Chem., 234: 1129-1137,1959. Cohen, S. Purification of a nerve-growth promoting protein from the mouse saliva gland and neurocytotoxic antiserum. Proc. Nati. Acad. Sci. USA, 46: 302-311,1960. Bradshaw, R. A. Nerve growth factor. Annu. Rev. Biochem., 47: 191-216, 1978. Angeletti, R. J., and Bradshaw, R. A. Nerve growth factor from mouse submaxillary gland: amino acid sequence. Proc. Nati. Acad. Sci. USA, 68: 2417-2420,1971. Scott, J., Selby, M., Urdea, M., Quiroga, M., Bell, G., and Rutter, W. J. Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor. Nature (Lond.), 302: 538-540,1983. Greene, L. A., and Tischler, A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Nati. Acad. Sci. USA, 73: 2424-2428. 1976. Massagué,J., Guillette, B. J., Czech, M. P., Morgan, C. J., and Bradshaw, R. A. Identification of a NGF receptor protein in sympathetic ganglia mem branes by affinity chromatography. J. Biol. Chem., 256: 9419-9424,1981. Puma, P., Buxser, S. E., Watson, L., Kelleher, D. J., and Johnson, G. L. Purification of the receptor for NGF from A875 melanoma cells by affinity chromatography. J. Biol. Chem., 258: 3370-3375, 1983. Landreth, G. E., and Shooter, E. M. Nerve growth factor receptors on PC12 cells: ligand-induced conversion from low- to high-affinity states. Proc. Nati. Acad. Sci. USA, 77: 4751-4755, 1980. Costruii, N. V., and Bradshaw, R. A. Binding characteristics and apparent molecular size of detergent-solubilized nerve growth factor receptor of sym pathetic ganglia. Proc. Nati. Acad. Sci. USA, 76: 3242-3245.1979. Levi, A., Eldridge, J. D., and Paterson, B. M. Molecular cloning of a gene sequence regulated by nerve growth factor. Science (Wash. DC), 229: 393395.1985. Johnson, G. R., and Metcalf, R. Pure and mixed erythroid colony formation In vitro stimulated by spleen conditioned medium with no detectable erythropoietin conversion from low- to high-affinity states. Proc. Nati. Acad. Sci. USA, 74: 3879-3882,1977. Stanley, E. R., and Heard. P. M. Factors regulating macrophage production 46 MARCH 1986 1027 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH 183. 184. 185. 186. FACTORS and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells. J. Biol. Chem., 252: 4305-4312, 1977. Burgess, A. W., Camakaris, J., and Metcalf, D. Purification and properties of colony-stimulating factor from mouse lung conditioned medium. J. Biol. Chem., 252: 1998-2003,1977. Nicola, N. A., Metcalf, D., Matsumoto, M., and Johnson, G. R. Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells: identification as granulocyte colony-stimulating factor. J. Biol. Chem., 258: 9017-9023,1983. Ihle, J. N.. Keller. J., Henderson, L, Copeland, F., Fitch, M. B., Prystowsky, M. B., Goldwasser, E., Schrader, J. W., Palaszynski, E., Dy, M., and Lebel, B. Biological properties of homogeneous interteukin 3. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, P-cell stimu lating activity, colony stimulating factor activity, and histamine producing cell stimulating factor activity. J. Immunol., 737. 282-287, 1983. Clark-Lewis, I., and Schrader, J. W. P cell-stimulating factor: biochemical characterization of a new T cell-derived factor. J. Immunol., 727:1941-1951, 1981. 187. Yokota, T., Arai, N., Lee, F., Rennick, D., Mosmann, T., and Arai, K-I. Use of a cDNA expression vector for isolation of mouse interieukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells. Proc. Nati. Acad. Sci. USA, 82: 68-72, 1985. 188. Bartelmez, S. H., Sacca, R., and Stanley, E. R. Lineage specific receptors used to identify a growth factor for developmentally early hemopoietic cells: assay of hemopoietin-2. J. Cell. Physiol., 722: 362-369,1985. 189. Iscove, N. N., and Guilbert, L. J. Erythropoietin-independence of early erythropoiesis and a 2-regulator model of proliferative control in the hemopoietic system. In: M. J. Murphy (ed.). In Vitro Aspects of Erythropoietin, pp. 3-7. Berlin: Springer-Verlag, 1978. 190. Bazill, G. W., Haynes, M., Garland, J., and Dexter, T. M. Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium. Biochem. J., 270: 747-759, 1983. 191. Ihle, J. N., Pepersack, L., and Rebar, L. Regulation of T cell differentiation: in vitro induction of 20«-hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine. J. Immunol., 726: 2183-2189, 1981. 192. Iscove, N. N., Roitsch, C. A., Williams, N., and Guilbert, L. J. Molecules stimulating early red cell, granulocyte, macrophage and megakaryocyte pre cursors in culture: similarity in size, hydrophobicity and charge. J. Cell. Physiol., 82: 65-78, 1982. 193. Dunn, A. R., Metcalf, D., Stanley, E., Grail, H., King, J., Nice, E. C., Burgess, A. W., and Gough, N. Biological characterization of regulators encoded by cloned hematopoietic growth-factor gene sequences. In: J. Feramisco, B. Ozanne, and C. Stiles (eds.), Growth Factors and Transformation, (Cancer Cells 3), pp. 227-234. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1985. 194. Das, S. K., and Stanley, E. R. Structure-function studies of a colony stimu lating factor (CSF-1). J. Biol. Chem., 257: 13679-13684,1982. 195. Stanley, E. R. Colony-stimulating factor (CSF) radioimmunoassay: detection of a CSF subclass stimulating macrophage production. Proc. Nati. Acad. Sci. USA, 76: 2969-2973,1979. 196. Stanley, E.R., and Guilbert, L. J. Methods for the purification, assay, char acterization and target cell binding of a colony stimulating factor (CSF-1). J. Immunol. Methods, 42: 253-284, 1981. 197. Kawasaki. E. S., Ladner, M. B., Wang, A. M., Van Arsdell, J., Warren, M. K., Coyne, M. Y., Schweickart, V. L., Lee, M. T., Wilson, K. J., Boosman, A., Stanley, E. R., Ralph, P., and Mark, D. F. Molecular cloning of a complemen tary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1 ). Science (Wash. DC), 230: 291 -296,1985. 198. Bartelmez, S. H., and Stanley, E. R. Synergism between hemopoietic growth factors (HGFs) detected by their effects on cells bearing receptors for a lineage specific HGF: assay of hemopoietin-1. J. Cell. Physiol., 722: 370378.1985. 199. Rettenmier, C. W., Chen, J. H., Roussel, M. F., and Sherr, C. J. The product of the c-fms proto-oncogene: a glycoprotein with associated tyrosine kinase activity. Science (Wash. DC), 228: 320-322, 1985. 200. Rettenmier, C. W., Roussel, M. F., Quinn, C. O., Kitchingman, G. R., Look, A. T., and Sherr, C. J. Transmembrane orientation of glycoproteins encoded by the v-fms oncogene. Cell, 40: 971-981, 1985. 201. Roussel, M. F., Rettenmier, C. W., Look, A. T., and Sherr, C. J. Cell surface expression of v-rms-coded glycoproteins is required for transformation. Mol. Cell. Biol., 4: 1999-2009, 1984. 202. Heisterkamp, N., Groffen, J., and Stephenson, J. R. Isolation of v-fms and in human cellular homology. Virology, 726: 248-258, 1983. 203. Sokal, G., Michaux, J. L., van den Berghe, H., Corbier, A., Rodhain, J., Ferrant, A., Moriam, M., de Bruyère, M., and Sonnet, J. A new hematopoietic syndrome with a distinct karyotype: the 5q chromosome. Blood, 46: 519533. 1975. 204. Wisniewski, L. P., and Hirschhorn, K. Acquired partial deletions of the long arm of chromosome 5 in hématologiedisorders. Am. J. Hematol., 75: 295310.1983. CANCER RESEARCH AND CANCER 205. Nienhuis, A. W., Bunn, H. F., Turner, P. H., Gopal, T. V., Nash, W. G., O'Brien, S. J., and Sherr, C. J. Expression of the human c-/ms proto oncogene in hematopoietic cells and its deletion in the 5q- syndrome. Cell, 42:421-428, 1985. 206. Metcalf, D. The granutocyte-macrophage colony-stimulating factors. Science (Wash. DC), 229:16-22,1985. 207. Wong, G. G., Witek, J. S., Temple, P. A., Wilkens, K. M., Leary, A. C., Luxenberg, D. P., Jones, S. S., Brown, E. L., Kay, R. M., Orr, E. C., Shoemaker, C., Golde, D. W., Kaufman, R. J., Hewick, R. M., Wang, E. A., and Clark, S. C. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science (Wash. DC), 228: 810-815,1985. 208. Gough, N. M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N. A., Burgess, A. W., and Dunn, A. R. Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor. Nature (Lond.), 309: 763-767, 1984. 209. Cantrell, M. A., Anderson, D., Cerretti, D. P., Price, V., McKereghan, K., Tushinski, R. J., Mochizuki, D. Y., Larsen, A., Grabstein, K., Gillis, S., and Cosman, D. Cloning, sequence, and expression of a human granulocyte/ macrophage colony-stimulating factor. Proc. Nati. Acad. Sci. USA, 82: 62506254,1985. 210. Gasson, J. C., Weisbart, R. H., Kaufman, S. E., Clark, S. C., Hewick, R. M., Wong, G. G., and Golde, D. W. Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils. Science (Wash. DC), 226:1339-1344, 1984. 211. Metcalf, D., and Nicola, N. A. Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells. J. Cell. Physiol., 776:198-206,1983. 212. Nicola, N. A., Metcalf, D., Matsumoto, M., and Johnson, G. R. Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. J. Biol. Chem., 258: 9017-9023,1983. 213. Hozumi, M., Umezawa, T., Takenaga, K., Ohno, T., Shikita, M., and Yamane, I. Characterization of factors stimulating differentiation of mouse myeloid leukemia cells from a Yoshida sarcoma cell line cultured in serum free medium. Cancer Res., 39: 5127-5131,1979. 214. Tomida, M., Yamato-Yamaguchi, Y., and Hozumi, M. Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J. Biol. Chem., 259: 10978-10982, 1984. 215. Lotem, J., Upton, J. M., and Sachs, L. Separation of different molecular forms of macrophage- and granulocyte-inducing proteins for normal and leukemic myeloid cells. Int. J. Cancer, 25: 763-771, 1980. 216. Olsson, I., Samgadharan, M. G., Bretman, T. R., and Gallo, R. C. Isolation and characterization of a T lymphocyte-derived differentiation-inducing factor for the myeloid leukemic cell line HL-60. Blood, 63: 510-517, 1984. 217. Dulak, N. C., and Temin, H. M. Multiplication-stimulating activity for chicken embryo fibroblasts from rat liver cell conditioned medium: a family of small polypeptides. J. Cell. Physiol., 87: 161-170,1973. 218. Sporn, M. B., and Todaro, G. J. Autocrine secretion and malignant transfor mation of cells. N. Engl. J. Med., 303: 878-880,1980. 219. Moses, H. L., and Robinson, R. A. Growth factors, growth factor receptors and cell cycle control mechanisms in chemically transformed cells. Fed. Proc., 47:3008-3011,1982. 220. Clemmons, D. R., and Van Wyk, J. J. Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J. Clin. Invest., 75: 1914-1918, 1985. 221. Betsholtz, C., Westermark, B., Ek, B., and Heldin, C. H. Co-expression of a PDGF-like growth factor and PDGF receptors in a human osteosarcoma cell line: implications for autocrine receptor activation. Cell, 39: 447-457,1984. 222. Moses, H. L., Childs, C. B., Halper, J., Shipley, G. D., and Tucker, R. F. In: C. M. Veneziale (ed.), Control of Cell Growth and Proliferation, pp. 147-167. New York: Van Nostrand Reinhold Co., 1984. 223. Meuer, S. C., Hussey, R. E., Cantrell, D. A., Hodgdon, J. C., Schlossman, S. F., Smith, K. A., and Reinherz, E. Triggering of the T3-TÃŒ antigen-receptor complex results in clonal T-cell proliferation through an interieukin-2-dependent autocrine pathway. Proc. Nati. Acad. Sci. USA, 87:1509-1513,1984. 224. Smith, K. A. T-cell growth factor and glucocorticoids: opposing regulatory hormones in neoplastic T-cell growth. Immunobiology, 767:157-173,1982. 225. Arya, S. K., Wong-Staal, F., and Gallo. R. C. T-Cell growth factor gene: lack of expression in human T-cell leukemia-lymphoma virus-infected cells. Sci ence (Wash. DC), 223: 1086-1087, 1984. 226. Reddy, G. P. V., and Pardee, A. B. Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc. Nati. Acad. Sci. USA, 77: 3312-3316,1980. 227. Pastan, I. H., and Willingham, M. C. Journey to the center of the cell: role of the receptosome. Science (Wash. DC), 274: 504-509,1981. 228. Fava, R. A., and Cohen, S. Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells. J. Biol. Chem., 259: 2636-2645,1984. 229. Radke, K., and Martin, G. S. Transformation by Rous sarcoma virus: effects VOL. 46 MARCH 1986 1028 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. GROWTH 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. FACTORS of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Proc. Nati. Acad. Sci. USA, 76: 5212-5216, 1979. Erikson, E., and Erikson, R. L. Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product. Cell, 27:829-836,1980. Greenberg, M. E., and Edelman, G. M. Comparison of the 34,000-dalton pp60src substrate and a 38,000-dalton phosphoprotein identified by mono clonal antibodies. J. Biol. Chem., 258: 8497-8502, 1983. Cooper, J. A., and Hunter, T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol. Cell. Biol., 1: 165170,1981. Berridge, M. J., and Irvine, R. F. Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature (Lond.). 312: 315-321, 1984. Sefton, B. M., Hunter, T., Ball, E. H., and Singer, S. J. Vinculin:a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell, 24:165-174, 1981. Cooper, J. A., Reiss, N. A., Schwartz, R. J., and Hunter, T. Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sar coma virus. Nature (Lond.), 302: 218-223, 1983. Raines, M. A., Lewis, W. G., Crittenden, L. B.. and Kung, H.-J. c-eroB activation in avian leukosis virus-induced erythroblastosis: clustered integra tion sites and the arrangement of provirus in the c-eri>BalÃ-eles. Proc. Nati. Acad. Sci. USA, 82: 2287-2291,1985. Bum, P., Rotman, A., Meyer, R. K., and Burker, M. M. Diacylglycerolin large n-actinin/actin complexes and in the cytoskeleton of activated platelets. Nature (Lond.),374: 469-472, 1985. Lassing, I., and Undberg, U. Specificinteraction between phosphatkjylinositol 4,5-bisphosphate and profilactin. Nature (Lond.), 314: 472-474, 1985. Mellström,K., Hoglund, A.-S.,Nister, M., Heldin, C.-H., Westermark, B., and Lindberg, U. The effect of platelet-derivedgrowth factor on morphology and motility of human glial cells. J. Muscle Res. Cell Motil., 4: 589-609,1983. Herman, B., and Pledger, W. J. Platelet-derivedgrowth factor-induced alter ations in vinculinand actin distribution in BALB/C-3T3 cells. J. Cell Biol., 700: 1031-1040,1985. Chinkers, M. J., McKanna, J. A., and Cohen, S. Rapid induction of morpho logical changes in human carcinoma cell line A-431 by epidermal growth factor. J. Cell Biol., 83: 260-265,1979. Stacey, D. W., and Kung, H.-F. Transformation of NIH 3T3 cells by microin jection of Ha-ras p21 protein. Nature (Lond.),370: 508-511, 1984. Feramisco,J. R.. Clark, R., Wong, G., Amheim, N., Milley,R., and McCormick, F. Transient reversionof ras oncogen-inducedcell transformation by antibod ies specific for amino acid 12 of ras protein. Nature (Lond.), 374: 639-642, 1985. Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M., and Sweet, R. W. Microinjectionof the oncogeneform of the human H-ras(T-24)protein results in rapid proliferation of quiescent cells. Cell, 38: 109-117,1984. Blanchard, J-M., Piechaczyk, M., Dani. C., Chambard, J-C., Franchi, A., Pouyssegur, J., and Jeanteur, P. c-myc gene is transcribed at high rate in Go-arrestedfibroblasts and is post-transcriptionally regulated in response to growth factors. Nature (Lond.), 377: 443-445,1985. Elder, P. K., Schmidt, L. J., Ono, T., and Getz, M. J. Specific stimulation of actin gene transcription by epidermalgrowth factor and cycloheximide.Proc. Nati. Acad. Sci. USA, 87: 7476-7480, 1984. Cochran, B. H., Zullo, J., Verma, I. M., and Stiles, C. D. Expression of the cfos gene and of an tos-related gene is stimulated by platelet-derivedgrowth factor. Science (Wash. DC), 226:1080-1082,1984. Cochran, B. H., Reffel, A. C., and Stiles, C. D. Molecular cloning of gene CANCER RESEARCH AND CANCER 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. 267. sequences regulated by platelet-derived growth factor. Cell, 33: 939-947, 1983. Leof, E. B., Proper, J. A., Getz, M. J., and Moses, H. L. Transforming growth factor type-d regulation of actin mRNA. J. Cell. Physiol., in press, 1986. Thomas, G., Martin-Perez, J., Siegmann, M., and Otto, A. M. The effect of serum, EOF, PGF2«and insulin of S6 phosphorylation and the initiation of protein and DNA synthesis. Cell, 30: 235-242,1982. Persson, H., and Leder, P. Nuclear localization and DNA binding properties of a protein expressed by human c-myc oncogene. Science (Lond.), 225: 718-721,1984. Curran, T., Miller, A. D., Zokas, L., and Verma, I. M. Viral and cellular fos proteins: a comparative analysis. Cell, 36: 259-268,1984. Abrams, H. D., Rohrschneider,L. R., and Eisen, R. N. Nuclear location of the putative transforming protein of avian myelocytomatosisvirus. Cell, 29:417439,1982. Alitalo, K., Ramsay, G., Bishop, J. M., Pfeiffer, S. O., Colby, W. W., and Levinson,A. D. Identificationof nuclear proteins encoded by viral and cellular myc oncogenes. Nature (Lond.),306: 274-277, 1983. Eisenman, R. N., Tachibana, C. Y., Abrams, H. D., and Hann, S. R. v-mycand c-myc-encoded proteins are associated with the nuclear matrix. Mol. Cell. Biol., 5. 114-126, 1985. Olashaw, N. E., and Pledger, W. J. Association of platelet-derived growth factor-induced protein with nuclear material. Nature (Lond.), 306: 272-274, 1983. Pfeifer-Ohlsson,S., Goustin, A. S., Rydnert, J., Wahlström,T., Bjersing, L., Stehelin, D., and Ohlsson, R. Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embry onic cell proliferation. Cell. 38: 585-596.1984. Campisi,J., Gray, H. E., Pardee,A. B., Dean, M., and Sonenshein,G. E. Cellcycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell, 36: 241-247, 1984. Klein, G. Specific chromosomal translocations and the genesis of B-cell derived tumors in mice and men. Cell, 32: 311-315, 1983. Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D., Galto, R. C., and Croce, C. M. Humanc-myc one gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc. Nati. Acad. Sci. USA, 79: 7824-7827, 1982. Erikson, J., Ar-Rushdi, A., Drwinga, H. L., Nowell, P. C., and Croce, C. M. Transcriptional activation of the translocated c-myc oncogene in Burkitt lymphoma. Proc. Nati. Acad. Sci. USA, 80: 820-824,1983. Land, H., Parada, J. F., and Weinberg, R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature (Lond.),304: 596-602,1983. Armelin, H. A., Armelin, M. C. S., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. Functional role of c-myc in mitogenic response to platelet-derivedgrowth factor. Nature (Lond.), 370: 655-660,1984. Kaczmarek, L., Hyland, J. K., Watt, R., Rosenberg, M., and Baserga, R. Microinjected c-myc as a competence factor. Science (Wash. DC), 228: 1313-1315,1985. Smeland, E., Godai, T., Ruud, E., Beiske, K., Funderud, S., Clark, E. A., Pfeifer-Ohlsson, S., and Ohlsson, R. The specific induction of myc proto oncogene expression in normal human B cells is not a sufficient event for acquisition of competence to proliferate. Proc. Nati. Acad. Sci. USA, 82: 6255-6259,1985. Rapp, U. R., Cleveland, J. L., Brightman, K., Scott, A., and Ihte, J. N. Abrogation of IL-3 and IL-2 dependenceby recombinant murine retroviruses expressing v-myc oncogenes. Nature (Lond.), 377: 434-438,1985. Hann, S. R., Thompson, C. B., and Eisenman,R. N. c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells. Nature (Lond.),374:366-369, 1985. VOL. 46 MARCH 1986 1029 Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research. Growth Factors and Cancer Anton Scott Goustin, Edward B. Leof, Gary D. Shipley, et al. Cancer Res 1986;46:1015-1029. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/46/3/1015 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/46/3/1015. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. Downloaded from cancerres.aacrjournals.org on August 16, 2018. © 1986 American Association for Cancer Research.