125I-labeled Epidermal Growth Factor Research Articles

Characterization and localization of receptors for epidermal growth factor in ovine skin.

Specific receptor sites for murine epidermal growth factor (EGF) have been characterized and their distribution determined in ovine skin. Binding of 125I-labelled EGF to skin membrane particles was temperature- and time-dependent, with equilibrium being reached within 1 h at 23 degrees C. Analysis of skin biopsies collected from ten castrated Merino sheep demonstrated the presence of a single class of saturable, high-affinity binding sites with a dissociation constant of 64 +/- 4 (S.E.M.) pmol/l and a binding capacity of 33.8 +/- 4.5 fmol/mg protein. Skin particle binding of 125I-labelled EGF was inhibited equipotently by mouse salivary gland EGF, EGF produced by recombinant DNA procedures and urogastrone. The EGF peptides 1-48, 6-53 and 7-53, derived from the native molecule by enzymatic cleavage, were much less potent. The relative binding potency of these molecules was correlated with their ability to induce precocious eyelid opening in mice and to inhibit wool follicle activity. Synthetic fragments representing the major structural domains of the EGF molecule (EGF(29-44), EGF(33-42) and EGF(3-31] were inactive in both the receptor and bioassays. Autoradiography of skin sections incubated with 125I-labelled EGF in vitro or of sections from skin which was perfused with 125I-labelled EGF in vivo demonstrated that EGF receptors were localized in undifferentiated cells of the epidermis and sebaceous glands, the inner and outer root sheath and bulb of wool follicles and in dermal arterioles. Differences in receptor concentration were observed between follicles following in-vivo perfusion of 125I-labelled EGF but not when the in-vitro labelling technique was used. The presence of receptors in these regions is consistent with the morphological changes in sheep skin in response to EGF administration which have been reported previously.

Keloid fibroblasts in culture: Abnormal growth behaviour and altered response to the epidermal growth factor

Keloid fibroblasts were propagated in culture and their proliferative behaviour and response to the Epidermal Growth Factor (EGF) were studied. Keloid fibroblasts grew at a rate which was approximately one-half that of normal age, sex and race matched control fibroblasts. Keloid fibroblasts were stimulated to grow in the presence of EGF (10ng/ml), but to a lesser degree than the normal control fibroblasts. Scatchard analysis of the binding data obtained using 125I-labeled EGF revealed no difference in binding affinity or receptor numbers between keloid and normal fibroblasts.

Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of [125I]EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of [125I]EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound [125I]EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, [125I]EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of [125I]EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of [125I]EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of [125I]EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in [125I]EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterologous regulation of EGF receptor function in cultured aortic smooth muscle cells

A10 cultured smooth muscle cells from rat embryonic thoracic aorta bound 125I-labelled epidermal growth factor ( 125I-EGF), and responded to EGF by an increase in DNA synthesis. Scatchard analysis of binding data obtained at 4° C showed curvilinearity consistent with there being two affinity classes of binding site. The amount of 125I-EGF that bound was decreased by treatment of the A10 cells at 37° C with [Arg 8]vasopressin or with 5-hydroxytryptamine (5-HT). Scatchard analysis of binding (at 4° C after pretreatment at 37° C) revealed this effect to be due to a loss of the high-affinity component of 125I-EGF binding, with no change in total receptor number. The presence of vasopressin or 5-HT raised the concentration of EGF required for the stimulation of DNA synthesis. Cultured A10 aortic smooth muscle cells therefore have receptors for EGF that mediate an increase in cell proliferation. EGF receptor function is modified by vasopressin and 5-HT, probably as a consequence of their effects on EGF receptor affinity.

IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism.

Abstract The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.

Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.

The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic kinase domain by a single transmembrane (TM) region. To explore the role of the TM region in the process of receptor activation, we have generated EGF-receptor mutants with altered TM regions by utilizing in vitro site-directed mutagenesis. The TM regions of two mutant receptors were either extended (designated i626-3) or shortened (designated d625.3) by three hydrophobic amino acid residues. In the other two mutant receptors, hydrophobic amino acids were substituted by charged residues--i.e., Val-627 was replaced by glutamic acid (designated V627E) or Leu-642 was replaced by an arginine residue (designated L642R). NIH 3T3 cells lacking endogenous EGF receptors were transfected with constructs encoding either wild-type or mutant receptors and shown to express the receptor molecules using 125I-labeled EGF binding and immunoprecipitation experiments. The mutant receptors were expressed on the cell surface as polypeptides of Mr 170,000 exhibiting typical high- and low-affinity binding sites for 125I-labeled EGF. Similar to its effect on wild-type receptors, phorbol 12-myristate 13-acetate abolished the mutant-receptor high-affinity binding sites for EGF. Moreover, EGF was able to stimulate the kinase activities of wild-type and mutant receptors both in vitro and in living cells. The mutant receptors were also able to undergo EGF-induced receptor dimerization as revealed by cross-linking experiments with a bifunctional covalent cross-linking agent. These results are compatible with an intermolecular allosteric oligomerization model for receptor activation rather than with a model based on an intramolecular mechanism for receptor activation.

Endosomes differ from plasma membranes in the phospholipid molecular species composition

125I-Labeled epidermal growth factor was incorporated into and highly concentrated in endosomes of Chinese hamster V79-UF cells during incubation at 37°C for 8 min after binding to its receptors on the cell surface at 4°C. From the labeled cells, endosomes were isolated by isopychic centrifugation on a Percoil density gradient and then sucrose density gradients. The isolated endosomes were mostly free from contamination by Golgi, endoplasmic reticulum, lysosome, plasma membrane and mitochondria. Endosome membranes were found to differ from plasma membranes in the phospholipid composition. Sphingomyelin and phosphatidylserine were enriched in endosomes, compared with plasma membranes. Diacylphosphatidylcholine and diacylphosphatidylethanolamine were major phospholipids of the membranes in both organelles. The contents of molecular species of diacylphosphatidylcholine and diacylphosphatidylethanolamine with two monoenoic fatty acids were lower in endosomes than in plasma membranes. The differences in the polar head group and molecular species compositions of phospholipids between endosomes and plasma membranes did not change, regardless of whether or not the proportions of phospholipid molecular species in plasma membranes changed. The significance of the lipids in endosomes is discussed.

Alterations in EGF binding to acini during pancreatic regeneration in the rat.

The binding of 125I-labeled epidermal growth factor (EGF) was compared in acini isolated from the regenerating remnant following 90% partial pancreatectomy (ppx) and from the pancreas of sham-pancreatectomized (sham-ppx) rats. Saturation binding studies with increasing amounts of unlabeled EGF revealed that cell-associated radioactivity was decreased in acini from the regenerating remnant by comparison to acini from sham-ppx rats. Analysis of these data indicated that binding was decreased by 35% and 27% at 3 and 7 days post-ppx, respectively. This alteration in EGF binding coincides with increased exocrine cell mitotic activity. EGF binding was normalized at 14 days post-ppx, at which time the exocrine cell mitotic activity is no longer increased (Brockenbrough et al. 1987, Diabetes). 125I-insulin binding was the same in ppx and sham-ppx acini at 3 days post-ppx. Furthermore, plasma EGF concentrations were the same in ppx and sham-ppx rats. These data indicate that EGF handling by the pancreatic acinar cell is altered during the proliferative response to ppx.

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of 125I-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [ln (inulin/EGF) versus time] of the dilution curve for EGF exhibited an upward straight line initially for a short period of time (approximately equal to 10 sec), whereas the ratio plot [ln (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer approximately equal to 30 micrograms) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kappa on), the dissociation rate constant of EGF from the EGF-receptor complex (kappa off), and the sequestration rate constant of the complex. The kinetic parameters [the dissociation constant (Kd = kappa off/kappa on) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones--cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.

Distribution of i.v. administered epidermal growth factor in the rat

The distribution of i.v. injected 125I-labeled epidermal growth factor (EGF) was examined in the rat. The uptake of radioactivity was examined for the following tissues: liver, kidney, skin, stomach, small intestine, colon, brain, submandibular gland, lung, spleen, and testis. 125I-EGF was cleared from the circulation within minutes. At 2.5 min after the injection only 7% of the label was left in the blood. Most of the label was found in the liver (52%), the kidneys (14%), the small intestine (11%) and the skin (7%). The other organs examined contained 1% or less of the radioactivity. The uptake of 125I-EGF per g tissue was markedly higher for the liver and kidneys than for the rest of the organs. By autoradiography 125I-EGF was found in the peripheral parts of the classical liver lobule, in the proximal tubules of the kidneys, in the surface epithelium of the stomach, and in the surface epithelium of the villi in the small intestine. In conclusion the present study showed that small doses of homologous EGF was cleared from the circulation of rats within minutes, mainly by the liver, the kidneys, and the small intestine.

Epidermal growth factor-mediated effects on equine vascular smooth muscle cells.

Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125I-labeled EGF, indicate the presence of a single class of high-affinity binding sites (apparent KD = 2.8 X 10(-11) M), with an estimated maximal binding capacity of 5,800 sites/cell. EGF stimulated [3H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 X 10(-11) M. Likewise, EGF-mediated cellular proliferation was dose dependent (50% effective dose = 5 X 10(-11) M) under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease.

The pp60v-src tyrosine kinase desensitizes epidermal growth factor binding to 3T3 fibroblasts by two distinct protein kinase C-independent mechanisms.

Protein kinase C (Ca2+/phospholipid-dependent enzyme) is known to phosphorylate the epidermal growth factor receptor and reduce its affinity for epidermal growth factor. Transformation of 3T3 fibroblasts by the oncogenic tyrosine kinase pp60v-src is accompanied by an elevation of cellular diacylglycerol and partial activation of protein kinase C (Wolfman, A., Wingrove, T. G., Blackshear, P. J., and Macara, I. G. (1987) J. Biol. Chem. 262, 16546-16552). We therefore asked whether pp60v-src can down-modulate the epidermal growth factor receptor. We report that within 15 min of activating temperature-sensitive pp60v-src, binding of 125I-labeled epidermal growth factor to 3T3 cells falls at least 50%. Two distinct processes control the down-modulation by pp60v-src. The first is rapid and transient, while the second requires protein synthesis and persists long after inactivation of pp60v-src. Surprisingly, both mechanisms seem to be protein kinase C-independent. Both operate by decreasing the affinity of the epidermal growth factor receptor for its ligand.

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor ( 125I-EGF) to Swiss 3T3 cells by 15–20% at 37°C, but not at 4°C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.

Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling.

Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor.

Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling.

Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor.

Decreased autophosphorylation of EGF receptor in insulin-deficient diabetic rats.

We have previously reported that despite an increase in receptor concentration, there is a decrease in autophosphorylation and tyrosine kinase activity of the insulin receptor in insulin-deficient diabetic rats. To determine if other tyrosine kinases might be altered, we have studied the epidermal growth factor (EGF) receptor kinase in wheat germ agglutinin-purified, Triton X-100-solubilized liver membranes from streptozotocin (STZ)-induced diabetic rats and the insulin-deficient BB rat. We find that autophosphorylation of EGF receptor is decreased in proportion to the severity of the diabetic state in STZ rats with a maximal decrease of 67% (P less than 0.01). A similar decrease in autophosphorylation was observed in diabetic BB rats that was partially normalized by insulin treatment. Separation of tryptic phosphopeptides by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. A parallel decrease in EGF receptor phosphorylation was also found by immunoblotting with an anti-phosphotyrosine antibody. EGF receptor concentration, determined by Scatchard analysis of 125I-labeled EGF binding, was decreased by 39% in the STZ rat (P less than 0.05) and 27% in the diabetic BB rat (not significant). Thus autophosphorylation of EGF receptor, like that of the insulin receptor, is decreased in insulin-deficient rat liver. In the case of EGF receptor, this is due in part to a decrease in receptor number and in part to a decrease in the specific activity of the kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [ 3H]concanavalin A and 5′-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor ( 124I-EGF) with a K d = 4.7 nM and B max = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [γ- 32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170 000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.

Specific receptors for epidermal growth factor in rat intestinal microvillus membranes.

Epidermal growth factor (EGF) is present in high concentrations in milk, salivary, and pancreaticobiliary secretions. EGF, delivered to the intestinal lumen by these fluids, appears to influence intestinal proliferation. Because EGF exerts its mitogenic effect through binding to specific membrane-bound receptors, binding studies of 125I-labeled EGF to purified microvillus membrane (MVM) preparations from fetal, newborn, and adult rat small intestine were performed. Using the membrane filter technique, binding of 125I-EGF to adult MVM was specific, saturable, and reversible. Adult and fetal MVM binding was rapid and reached a plateau after 30 min at both 20 and 37 degrees C. No binding was detected at 4 degrees C. Specific binding increased linearly from 0 to 75 micrograms MVM protein. Scatchard analysis revealed a single class of receptors in fetal and adult MVM with an association constant of 1.0 +/- 0.35 X 10(9) and 2.3 +/- 1.6 X 10(9) M-1, respectively. Binding capacity was 435.0 +/- 89 and 97.7 +/- 41.3 fmol 125I-EGF bound/mg MVM protein for fetal and adult MVM, respectively. Newborn MVM binding was negligible. After binding, cross-linking utilizing disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography revealed a 170-kDa receptor. These data demonstrate specific receptors for EGF on MVM of rat small intestine and, thus, suggest a mechanism for the intraluminal regulation of enterocyte proliferation by EGF.

Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras

We studied the effects of simultaneous treatment with 0.1 mM N6, O2'-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.