125I-epidermal Growth Factor Binding Research Articles

Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactase dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 25I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependent (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number of available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immunodetection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. Conclusions: Transient hypoxia, which caused no increase in lactase dehydrogenase leakage but affected intracellular adenosine triphosphate levels, did, however, modulate the number of available epidermal growth factor receptors without affecting the receptor protein expression, and inhibit the epidermal growth factor-induced DNA synthesis of hepatocytes. This suggests that even transient nonlethal hypoxia affects the epidermal growth factor-induced DNA synthesis of rat hepatocytes through reversible changes in the epidermal growth factor receptor molecule, which depends on oxygen availability.

Time-dependent alteration of epidermal growth factor receptor in rat stomach by ethanol feeding

This study investigated the time-dependent effects of ethanol (EtOH) feeding on epidermal growth factor binding and epidermal growth factor-mediated functions in the stomach. Adult male rats were fed either an isocaloric control or EtOH-containing liquid diet (36% total calories as EtOH) for 2, 4 and 6 weeks. At the end of each feeding period, animals were sacrificed and the stomach was dissected for the sample preparation. EtOH caused a time-dependent alteration ( r = 0.89) of the 125I-epidermal growth factor binding to the gastric mucosal membrane (% control: week 2, 114%; week 4, 64% ∗ and week 6, 45% ∗, n = 5, ∗P < 0.05 ). Protein kinase analysis also showed that EtOH caused a time-dependent decrease of epidermal growth factor-stimulated autophosphorylation of epidermal growth factor receptor protein (180 kDa) during three feeding periods. Western blot analysis, using anti-tyrosine phosphorylated epidermal growth factor receptor (active form) antibody, revealed a major immunoreactive protein band (180 kDa) in all samples pre-incubated with 1 μM epidermal growth factor. Consistent with data from kinase analysis, treatment of EtOH decreased the immunoreactivity of the active form of epidermal growth factor receptor (180 kDa) in the stomach. In conclusion, EtOH feeding caused a time-dependent alteration of epidermal growth factor receptor in the stomach, which may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.

Inhibition of epidermal growth factor binding system by ionizing radiation in A431 human squamous carcinoma cells.

To elucidate the effect of ionizing radiation on the membrane anchored signal transduction, the binding of 125I epidermal growth factor (EGF) to its receptor (EGF-R) and the EGF-dependent EGF-R tyrosine phosphorylation were examined in a human squamous cell carcinoma cell line, A431. The significant suppression of 125I EGF binding to A431 cells was observed from 3-5 h after 10 Gy irradiation, whereas this inhibition was not observed both in non-irradiated and in 5 Gy-irradiated cells. This phenomenon was mediated by the protein kinase C pathway, because the inhibition was not observed in cells which had been pretreated with phorbol ester and treated with an inhibitor of the enzyme, H7. Scatchard analysis showed that the receptor affinity was decreased. In contrast, the level of EGF-dependent EGF-R-tyrosine phosphorylation was not decreased, compared with non-irradiated cells. These results suggest that ionizing radiation may modulate the function of EGF/EGF-R interaction through the direct activation of protein kinase C.

Putrescine As A Comitogen of Epidermal Growth Factor in Rat Liver Growth

Putrescine can stimulate regeneration of the remnant liver after partial hepatectomy in rats when exogenously administered, but its mitogenic action has not been shown in cultured hepatocytes. To find its action site(s) in the regulation of hepatocyte proliferation, we examined its effect on hepatocyte DNA synthesis in relation to mitogenic action of epidermal growth factor in vitro and in vivo. When putrescine was added to the medium of adult rat hepatocytes in primary culture 36 hr after plating, DNA synthesis at 50 hr induced by addition of epidermal growth factor at 24 hr was significantly enhanced. This enhancement disappeared by removal of epidermal growth factor at the time of putrescine addition. Putrescine added to the medium was taken up in a dose-related manner by hepatocytes, irrespective of the presence of epidermal growth factor, whereas 125I-epidermal growth factor binding to hepatocytes was not affected by addition of putrescine. When rats received epidermal growth factor at 2-hr intervals until 10 hr, 5-bromo-2'-deoxyuridine labeled and mitotic hepatocytes were increased in number at 48 hr with increased hepatic DNA content. These increases were not affected by concomitant administration of putrescine until 10 hr, but significantly enhanced by additional administration of putrescine and epidermal growth factor from 20 to 30 hr. We conclude that putrescine may stimulate proliferation of hepatocytes that have entered the G1-phase of the cell cycle as a comitogen of epidermal growth factor, probably through action at the molecular levels to enhance its mitogenic activity.

Inhibition of PDGF receptor binding and PDGF-stimulated biological activity in vitro and of intimal lesion formation in vivo by 2-bromomethyl-5-chlorobenzene sulfonylphthalimide.

The proliferation of vascular smooth muscle cells (SMCs) is a key event in the development of atherosclerotic lesions and in the restenosis of arteries after angioplasty. Polypeptide growth factors are potent SMC mitogens in vitro and are believed to be involved in SMC proliferation in vivo. Strong data exist linking platelet-derived growth factor (PDGF) activity to human atherosclerosis. However, no low-molecular-weight antagonists of this growth factor have been discovered. We identified a compound, SCH 13929 (2-bromomethyl-5-chlorobenzene sulfonylphthalimide), which inhibits binding of 125I-PDGF BB to cell surface receptors with an IC50 of 0.13 mumol/L. This compound has a lesser effect on the binding of 125I-epidermal growth factor (EGF), 125I-basic fibroblast growth factor (bFGF), or 125I-endothelin to specific cell surface receptors. Exposure of cultured SMCs to SCH 13929 inhibits PDGF BB- and PDGF AA-stimulated DNA synthesis but not EGF- or bFGF-stimulated DNA synthesis. PDGF BB-stimulated SMC division is also inhibited by exposure to SCH 13929. Chemotaxis assays indicate that SCH 13929 inhibits PDGF-stimulated directional migration and suggest that the compound interacts with PDGF rather than with the receptor. Oral administration of SCH 13929 (100 mg/kg per day) to Sprague-Dawley rats or spontaneously hypertensive rats results in significant inhibition of lesion formation in the balloon catheter-deendothelialized carotid artery. These results suggest that SCH 13929 may be a useful tool for understanding the role of PDGF in intimal lesion formation.

Biphasic effects of suramin on 125I-epidermal growth factor binding to human meningiomas.

1. We studied the effects of suramin, a nonspecific growth factor antagonist, on epidermal growth factor (EGF) binding to cell surface receptors in surgically excised human meningiomas, using quantitative receptor autoradiographic methods with radioluminography. 2. High concentrations (10(-4) - 10(-2) M) of suramin inhibited 125I-EGF binding to meningioma sections with IC50's of 3.2 +/- 0.4 x 10(-4) M, whereas lower concentrations (10(-5) - 10(-4) M) of the drug significantly enhanced EGF binding to the tumor. Scatchard analysis of EGF binding profile revealed significant increases in binding affinity following incubation in the presence of 5 x 10(-5) M suramin, without significant alterations in maximal binding capacity. 3. The addition of 10(-3) M suramin to the incubation buffer rapidly dissociated 125I-EGF previously bound to meningioma tissues as a function of time (dissociation half-life, T1/2 = 12.4 min). 4. Preincubation in the presence of 5 x 10(-5) M suramin resulted in significant increases in the subsequent binding of 125I-EGF to meningiomas, compared to findings in the control. 5. Our data indicate that (a) suramin exerts biphasic effects on EGF binding to the tissue sections of meningiomas in vitro, depending on the concentration of the drug; and (b) low concentrations of suramin enhance the affinity of the EGF receptor in the tumor sections, probably by interacting with the EGF receptor molecule rather than with the EGF peptide. 6. The functional role of increased EGF receptor affinity in meningioma sections in the presence of lower concentrations of suramin remains to be determined.

Epidermal growth factor receptor expression in three different human endometrial cancer cell lines.

The aim of the current study was to establish the characteristics of the epidermal growth factor (EGF) receptor in endometrial cell lines to determine the possible relation of EGF to endometrial cancer. Three different cell lines were used: RL95-2 (derived from a moderately differentiated adenosquamous carcinoma), HEC-I-A (from a moderately differentiated adenocarcinoma), and KLE (from a poorly differentiated adenocarcinoma). The binding of (125-I) EGF to these cell lines and the stimulatory effect of EGF on (3H) thymidine incorporation into DNA were examined. EGF receptor was present in all three cell lines. The binding of 125-I-labeled EGF was saturable and of high affinity. Scatchard analysis of the competitive binding data for KLE, HEC-I-A, and RL95-2 revealed linear plots, indicating a single class of binding sites with almost identical equilibrium dissociation constants (0.34 nM, 0.23 nM, and 0.20 nM, respectively). Other peptides, such as insulin-like growth factor (IGF) I and II and insulin, did not compete for the receptor. RL95-2 cells bound significantly more EGF (P < 0.005) than did the HEC-I-A and KLE cell lines. EGF increased 3H-thymidine incorporation in all three cell lines. Because EGF receptors are expressed by all three cell lines at markedly different levels and because EGF stimulates 3H-thymidine incorporation into DNA in the three cell lines, the current study suggests that EGF may play a role in the promotion of growth endometrial adenocarcinoma.

Epidermal growth factor receptor and lipid membrane components in human lung cancers.

The binding of 125I-epidermal growth factor (EGF) to the plasma membranes of 54 samples of human lung tumors was determined. These included 34 squamous cell carcinomas and 20 adenocarcinomas. Twenty samples of histologically normal lung excised surgically along with the tumors were used as controls. Most of the plasma membranes showed an EGF receptor level higher than that of normal tissue. A moderate increase in the amount of 125I-EGF bound (2-5 fold) was observed in the majority of the tumors. Only a few cases (5-10% of the total) showed a large increase (> than 10 fold). The binding of 125I-EGF was compared with clinical stages and grades of differentiation. No correlation between the stage of the tumor and 125I-EGF binding was observed. However, the highest levels of EGF receptor (EGF-R) were found in poorly differentiated squamous cell carcinomas. The total amount and the distribution pattern of gangliosides and phospholipids were analyzed in individual tumors. A decrease in GD1b, GD1a and sphingomyelin and an increase in GM1 and GM3 was observed. No correlation was detected when tumors with the highest or lowest levels of gangliosides or phospholipids were compared with tumors exhibiting the highest binding of 125I-EGF.

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses

Embryos recovered from sows on Days 9–13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater ( P < 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites ( K d = 7.0 ± 2.6 × 10 −11 M, B max = 6.2 ± 1.4 fmol/mg protein and K d = 3.4 ± 0.2 × 10 −8 M, B max = 420 ± 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.

Impairment of hepatic insulin receptors during chronic ethanol administration

Chronic alcoholism is frequently associated with impaired intermediary metabolism and insulin resistance. The cellular defects leading to insulin resistance have not been clearly defined but could result from reduced insulin binding or abnormalities in any one of several postreceptor steps. The purpose of the present studies was to measure 125I-insulin binding and internalization kinetics and postreceptor response of the enzyme activity of tyrosine aminotransferase in isolated cultured rat hepatocytes. Four weeks of alcohol ingestion significantly reduced to 47% of control the 125I-insulin binding sites measured either as surface or total (after digitonin permeabilization). In contrast, 125I-epidermal growth factor binding was not significantly changed. Internalization of surface-bound 125I-insulin was decreased, but degradation was not increased, indicating that altered kinetics did not account for the change. Ethanol ingestion markedly reduced in liver cytosol some enzymes regulated by insulin and involved in glucose homeostasis. Basal activities of tyrosine aminotransferase and glucokinase were reduced 51% (P less than 0.01) and 32% (P less than 0.01), respectively. In contrast, phosphoenolpyruvate carboxykinase was unchanged. In short-term cultured hepatocytes from ethanol-fed rats, the maximum response of tyrosine amino-transferase to insulin was reduced 40% (P less than 0.01) without a change in the concentration causing 50% of the maximum response (EC50) compared with controls. In contrast, dexamethasone increased tyrosine aminotransferase to similar maximal levels and with similar EC50, indicating that ethanol did not alter the intracellular response. In conclusion, chronic ethanol ingestion caused significant time-dependent and selective changes in cell surface binding of insulin that was associated with subsequent postreceptor events.

Increased cell surface EGF receptor expression during the butyrate-induced differentiation of human HCT-116 colon tumor cell clones

Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones was inhibited by butyrate (IC 50 values varied from 0.44 to 1.5 m M), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 × 10 4 EGF receptors/cell with a k d of 0.61 n M). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 m M butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 × 10 5 EGF receptors/cell ( k d of 3.2 n M). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-α) and transforming growth factor beta (TGF-β) was examined in butyrate-treated cells. While TGF-α mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-β mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.

Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.

Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.

Effect of epidermal growth factor on rat pleural mesothelial cell growth

We recently reported that the growth of normal rat pleural mesothelial cells (RPMCs) is inhibited by conditioned media from either in vivo or in vitro transformed RPMCs. In this study we report that the growth of normal RPMCs is inhibited by epidermal growth factor (EGF). This was demonstrated by using three methods of investigation. Two types of studies were carried out with growing cells. First, cell counts indicated that the number of cells was reduced in EGF-treated cultures when compared with untreated cultures. Second, the percentage of S cells detected by flow cytometry following treatment with EGF was lower than without EGF. In other experiments, incorporation of tritiated thymidine in confluent cells was decreased by EGF treatment, either in the presence or absence of fetal calf serum; these effects were dose dependent and were observed from 2 ng/ml EGF. Lower EGF concentrations did not significantly modify thymidine incorporation when compared with untreated cells. Analysis of 125I EGF binding experiments by the Scatchard method indicated that RPMCs posses EGF receptors (about 10(5) per cell) with low ligand binding affinity (Kd = 1.7 +/- 0.4 nM). These results indicate that EGF might modulate the growth of RPMCs.

Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin.

The results presented here demonstrate that bradykinin, acting through a B2 subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-2-loaded cells and by 45Ca2+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by one- and two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of 125I-epidermal growth factor binding or enhancement of cAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inositol phosphates in cells labeled with myo-[3H]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides.

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.

Transmodulation of epidermal growth factor binding by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate is not sodium-dependent in Balb/c/3T3 cells.

The addition of platelet-derived growth factor (PDGF) to many types of cells causes a rapid decrease in high affinity binding of 125I-epidermal growth factor (EGF), a process which has been termed transmodulation. Treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also results in the transmodulation of the EGF receptor in many cell types. PDGF can transmodulate EGF binding through a mechanism that is not dependent on protein kinase C activity. A recent report (Wattenberg, E. V., McNeil, P. L., Fujiki, H., and Rosner, M. R. (1989) J. Biol. Chem. 264, 213-219) described the requirement for a sodium ion influx in the down-modulation of the EGF receptor stimulated by a non-TPA-type tumor promoter, palytoxin, in Swiss 3T3 cells. We tested for a similar sodium requirement in Balb/c/3T3 and Swiss 3T3 cells stimulated by PDGF or TPA in Balb cells treated with TPA for prolonged periods to down-regulate protein kinase C activity. Our results clearly show that the PDGF- and TPA-stimulated transmodulation of the EGF receptor does not require external sodium nor is the process affected by amiloride. In each of these experiments, the loss of 125I-EGF binding occurred to a similar extent and at a similar rate in the presence or absence of sodium. Intracellular pH also did not appear to have a role in the response. The sodium ionophore, monensin, was previously shown to bring about the down-modulation of 125I-EGF binding in Swiss cells. However, our results indicate that monensin-induced transmodulation of the EGF receptor occurs with or without external sodium, suggesting that the loss of binding is not the result of a sodium ion influx. These findings demonstrate that an increase in intracellular sodium does not cause nor is it required for PDGF- or TPA-stimulated EGF receptor transmodulation.

Inhibition of 125 I-Epidermal Growth Factor Binding to Cultured Keratinocytes by Antiproliferative Molecules Gamma Interferon, Cyclosporin A, and Transforming Growth Factor-Beta

The growth of cultured human keratinocytes (KC) is inhibited by gamma interferon (IFN-gamma), cyclosporin A and transforming growth factor-beta, but not by tumor necrosis factor. When these antiproliferative molecules were added to KC they induced a concentration and time-dependent inhibition of 125I-epidermal growth factor (I-EGF) binding. These anti-proliferative molecules primarily reduced the number of binding sites by approximately 25%-50% without affecting the binding affinity. Tumor necrosis factor did not influence the ligand binding by I-EGF. In parallel with the ability of the antiproliferative molecules to inhibit I-EGF binding, there was an increase in transforming growth factor-alpha production. These results suggest that several different antiproliferative molecules may share a common mechanism to inhibit cell growth by reducing I-EGF binding to KC.

Regulation of thyroidal inducibility of Na,K‐ATPase and binding of epidermal growth factor in wild‐type and cold‐sensitive E1a mutant type 5 adenovirus‐transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Interferon inhibition of DNA synthesis in Swiss 3T3 cells: Dissociation from protein kinase C activation

We have examined the role of protein kinase C in the anti-proliferative effects of interferon in Swiss 3T3 cells. Treatment of these cells with interferon did not stimulate the phosphorylation of an acidic Mr 80,000 cellular protein which serves as a substrate for protein kinase C. In addition, interferon did not inhibit the binding of 125I-epidermal growth factor to specific receptors or induce the expression of the proto-oncogene, c-fos in Swiss 3T3 cells. Thus, interferon does not activate protein kinase C. Moreover, interferon can still inhibit DNA synthesis in protein kinase C down-regulated 3T3 cells, indicating that the presence of this phosphotransferase is not essential for the anti-proliferative effects of interferon.