Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor

The capacity of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr = 125,000 amino-terminally truncated degradation product from the mature EGF receptor (EGF-dependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGF-alpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [125I]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [125I]-EGF binding after TGF-alpha treatment did not appear to require protein synthesis. Tyrosine phosphorylation of EGF receptor from cells treated with TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpha allows receptor recycling prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing.     

大鼠精核蛋白的纯化

用大鼠睾丸制备长形精子细胞核及用附睾制备的附睾精子核,用盐酸胍法提取总碱性蛋白（Ｔｏｔａｌ　ｂａｓｉｃ　ｐｒｏｔｅｉｎ,ＴＢＰ）。通过Ｓｅｐｈａｄｅｘ　Ｇ－１００分子筛层析,其第三峰用５％ＴＣＡ沉淀,得到纯大鼠精核蛋白（Ｒａｔ　ｐｒｏｔａｍｉｎｅ,ＲＰ）。ＲＰ也可用ＴＢＰ通过ＨＰＬＣ反相柱得到纯化,ＲＰ位于２６％－２８％乙腈梯度。     

Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.     

Differential effects of epidermal growth factor, transforming growth factor-alpha, and vaccinia virus growth factor in the positive regulation of IFN-gamma production

We have recently shown that epidermal growth factor (EGF) is capable of positive regulation of IFN-gamma production, thus establishing a functional relationship between nonhemopoietic growth factors and the immune system. In order to study this relationship further, EGF and the EGF-related growth factors transforming growth factor-alpha (TGF-alpha) and vaccinia virus growth factor (VGF), which stimulate cellular proliferation via binding to the EGF receptor, were studied for their functional and physicochemical effects on IFN-gamma production. In contrast to the positive signal of purified murine EGF and recombinant human EGF (both at 1 nM), neither synthetic TGF alpha nor recombinant VGF were capable of restoring competence for IFN-gamma production by Th cell-depleted spleen cell cultures. TGF-alpha and VGF, in molar excess, also failed to block the helper signal of EGF for IFN-gamma production. Thus TGF-alpha and VGF failed to functionally compete for the EGF receptor in the murine spleen cell system. Both TGF-alpha and VGF stimulated murine 3T3 cell proliferation at concentrations similar to those of EGF, and thus their failure to provide help for IFN-gamma production was not due to a general lack of biologic activity. Binding studies with 125I-EGF suggest that the EGF receptor on murine lymphocytes is not constitutively expressed, but inducible by the T cell mitogen staphylococcal enterotoxin A. TGF-alpha did not compete with 125I-EGF for the induced receptor. The data suggest that lymphocytes express a novel inducible EGF receptor that differs from that expressed on cells such as 3T3 fibroblasts.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Detection of endogenous epidermal growth factor-like activity in the developing chick embryo

Epidermal growth factor-like activity has been detected by radioreceptor assay and radioimmunoassay in the developing chick embryo. Very little activity could be detected prior to Day 8 of embryonic life (hatching is at Day 21). A peak of EGF activity was detectable by both assays over Days 10 to 12. The EGF activity then fell to virtually undetectable levels during Days 14 to 17. A later rise in RRA detectable EGF like activity was then observed over Days 18-20. The EGF activity from a Day 11 embryo chromatographed on high-performance liquid chromatography as a single peak, with very high recovery of activity, at a later elution position than mouse EGF or human EGF.     

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis

Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.     

Assessment of biological activity of synthetic fragments of transforming growth factor-alpha

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.     

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-alpha, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-alpha mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-alpha protein by DLD-1 and CoLo320DM cells was confirmed by TGF-alpha specific monoclonal antibody binding assay. The spontaneous 3H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-alpha monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-alpha produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

Urinary TGFs1 in neoplasia: immunoreactive TGF-alpha in the urine of patients with disseminated breast carcinoma

A tumor-associated growth factor was identified in a 22-liter pool of urine from patients with disseminated breast cancer using an isolation scheme which separates transforming growth factor-alpha (TGF-alpha) from the high level of human epidermal growth factor (hEGF) normally present in urine. Concentration of urinary proteins by adsorption onto methyl bonded microparticulate silica, selective elution by acetonitrile, and subsequent gel permeation, cation exchange, and high performance liquid chromatography, resolved an EGF-related growth factor which generated a competitive binding curve similar to that of synthetic rat TGF-alpha in radioimmunoassay. A 26-liter pool of urine from normal subjects, evaluated in the same manner as a part of another study, did not contain measureable quantities of this factor.     

Epidermal growth factor-like transforming growth factor. I. Isolation, chemical characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells

An acid-stable transforming growth factor (TGF) that interacts with epidermal growth factor (EGF) receptors and is structurally related to EGF was isolated from serum-free culture fluids of Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells. Purification of this EGF-like TGF (eTGF) was achieved by molecular filtration chromatography and successive reverse-phase high pressure liquid chromatography steps on octadecyl support eluted with acetonitrile and 1-propanol gradients, respectively. Rat eTGF consists of a 7.4-kD single polypeptide chain that co-migrates with biological activity in dodecyl sulfate-polyacrylamide electrophoresis gels. Like preparations of a related TGF from human melanoma cells (Marquardt, H., and Todaro, G.J. (1982) J. Biol. Chem. 257, 5220-5225), but unlike EGF from rat, human, or mouse, rat eTGF has phenylalanine and lacks methionine. However, the sequence of the first 30 amino acid residues in rat eTGF is H2N-Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly - Thr-(x)-Arg-Phe-Leu-Val-Gln-Glu-Glu-(Lys)-(Lys)-, which is significantly (20% and 28%) homologous to the NH2-terminal region of mouse EGF and human EGF, respectively. In addition to eTGF, molecular filtration chromatography of acid-soluble extracts from medium conditioned by FeSV-Fre cells resolved a 14-kD transforming factor(s) apparently devoid of intrinsic mitogenic activity but able to elicit a strong anchorage-independent growth response in the presence of eTGF or EGF. These results show that: 1) a 7.4-kDa TGF structurally and functionally related to EGF has been isolated from FeSV-Fre cells and 2) the full anchorage-independent growth-promoting activity of medium conditioned by FeSV-Fre cells is due to the coordinate action of at least two types of factors, the 7.4-kDa eTGF and a second 14-kDa transforming factor(s).