Epidermal Growth Factor Binding Levels Research Articles

Alterations in epidermal growth factor binding to hepatic membranes following dietary exposure of rats to known hepatocarcinogens

Administration of the chemical carcinogen 2-acetylaminofluorene (2-AAF) has previously been shown to lower hepatic epidermal growth factor (EGF) binding levels during chemically induced hepatocarcinogenesis. To further characterize the specificity of this response, EGF binding levels for liver microsomes were determined after a 3-week administration of subacute doses of 2-AAF and five other known hepatocarcinogens: 3′-methyl-4-dimethylaminoa-zobenzene (3′Me-DAB), 2-AAF, aflatoxin B 1 (AFB1), thioacetamide (TA), ethionine, benzidine (Benz), as well as four non-hepatocarcinogeos: fluorene, p-aminoazobenzene, 4-acetylaminofluorene (4-AAF), and 3-methylcholanthrene. Five of six of the hepatocarcinogens tested (3′Me-DAB, 2-AAF, TA, AFB1 and Benz) caused significant lowering of EGF binding levels, and one of the four non-hepatocarcinogens (4-AAF) caused significant lowering of EGF binding levels. Paired feeding studies indicated that the decreases in EGF binding levels were not a result of differences in net diet consumption. These findings show that decreases in EGF binding capacity are caused by a diverse group of known hepatocarcinogenic compounds at an early stage in the carcinogenesis process.

Epidermal growth factor binding sites in the mouse exocrine and endocrine pancreas shown by in vivo quantitative microautoradiography and confocal laser scanning microscopy

Microautoradiography at 3, 6 and 15 min after intravenous injection of 125I-EGF was used to investigate the distribution of epidermal growth factor (EGF) binding sites in the pancreas of normal male mice. The autoradiographs were observed by confocal laser microscopy, which allows the quantification of silver grains. The results demonstrated that both endocrine and exocrine pancreatic cells exhibited substantial specific binding of 125I-EGF. The highest level of EGF binding was found in the duct cells of the exocrine pancreas followed by the acinar cells. The cells of the islets of Langerhans also showed substantial specific binding of 125I-EGF though the binding level was lower than that of the exocrine pancreas. In the control experiments, mice were injected with 125I-EGF and various amounts of unlabeled EGF.

Carcinogen-induced alteration in liver epidermal growth factor receptor distribution during the promotion stage of hepatocarcinogenesis in rat.

Changes in hepatic membrane binding capacity for epidermal growth factor (EGF) were assessed during 2-acetylaminofluorine (2-AAF)-induced hepatocarcinogenesis. An overall decrease in membrane EGF binding levels was observed throughout the period of 2-AAF administration. Immunochemical studies indicated the decreases in EGF binding levels were paralleled by decreases in tissue EGF receptor levels. Immunohistochemical studies showed that the losses in hepatic EGF receptor levels were not uniform throughout the liver during the early, promotion stage of cancer development. During the promotion stage, preneoplastic liver nodules were found to display a higher level of EGF receptor than surrounding hepatic tissue. This resistance to 2-AAF-mediated down-regulation of EGF receptor may confer a proliferative advantage to the developing nodule relative to the surrounding liver tissue. Similar immunochemical and immunohistochemical analysis of hepatocarcinomas at late stages of 2-AAF-induced hepatocarcinogenesis indicated a uniform loss of EGF receptor in tumor and surrounding tissue. These studies indicate that carcinogen-mediated changes in EGF binding levels are different during the multistage process of hepatocarcinogenesis, and that resistance to down-regulation of EGF receptor among preneoplastic nodules may have a role in providing a selective growth advantage to initiated cell populations. EGF receptor may be useful as a dynamic marker assessing the development of hepatic tumors.

Changes in the Binding Capacity of Hepatic Membranes for Epidermal Growth Factor during Multistage Hepatocarcinogenesis in Rats

To study changes in hepatic capacity for binding epidermal growth factor (EGF) during 2-acetylaminofluorene (2-AAF)-induced, multistage hepatocarcinogenesis, a 5 cycle protocol of discontinuous 2-AAF administration was used to produce hepatocarcinogenesis in rats. The hallmark of the 5 cycle protocol is that rats fed 1 to 3 cycles of 2-AAF are at low risk for cancer, while rats fed 2-AAF for 4 or 5 cycles are at high risk for cancer. EGF binding by liver membranes was found to be lowered to 20–25% of control throughout the 5 cycle regimen. When the persistence of lowered EGF binding was tested by returning rats fed 2-AAF for 1 to 3 cycles to diet without 2-AAF for 3 weeks, binding was found to recover to 80 to 90% of values for control rats. In contrast, for rats fed 2-AAF for 4 or 5 cycles, EGF binding capacity remained low, 30 to 40% of control, following placement of rats on diet without 2-AAF for 3 weeks. Immunochemical analysis indicated a close correspondence between changes in EGF receptor levels and changes in the above EGF binding levels. These studies show that during the 2-AAF protocol, the 2-AAF-mediated loss in hepatic EGF binding capacity and EGF receptor protein undergo a transition from a reversible loss to a persistent loss in binding capacity, and EGF receptor protein, as rats underwent a change from low to high risk for developing hepatocarcinomas. The persistent decrease in hepatic EGF binding level may be associated with the progression stage of hepatocarcinogenesis.

Light microscopic autoradiographical analysis of [125I]epidermal growth factor binding in basal cell epithelioma and squamous cell carcinoma of the skin.

Levels of epidermal growth factor (EGF) receptor expression were investigated in five basal cell epitheliomas (BCEs) and 10 primary lesions from squamous cell carcinoma (SCC) of the skin, using light microscopic autoradiography with [125I]EGF. All of the BCEs were clinically the pigmented type and histologically the solid type. All of them showed an EGF binding level similar to that of the basal and suprabasal layers of the normal epidermis. The SCCs included one case of Bowen's disease (SCC in situ), five of the well differentiated type, three of the moderately differentiated type and one of the poorly differentiated type. Eight of the 10 SCCs showed an EGF binding level similar to that of the normal epidermal basal and suprabasal cell layers. One of the two remaining SCCs, a moderately differentiated type, showed highly increased EGF binding. The other one, a poorly differentiated type, showed very little EGF binding in a large region consisting of poorly differentiated cells, although a small area composed of more differentiated, nest-forming cells had an EGF binding level similar to that of the basal and suprabasal cells. Metastasis was found in three of these 10 SCCs after surgery. Two of the three SCCs with metastasis showed increased or decreased EGF binding levels in primary lesions as described above; in contrast, primary lesions of the seven SCCs without metastasis had EGF binding levels similar to those of the normal epidermal basal and suprabasal cells. Abnormally increased or decreased EGF binding level in SCC of the skin may be indicative of a poor prognosis, although it is necessary to examine more SCCs to confirm this assumption.

Binding of epidermal growth factor by human normal, hypertrophic, and carcinomatous prostate

Saturable binding sites for radioiodinated epidermal growth factor (EGF) have been quantified in surgical specimens of benign prostate hypertrophy (BPH), histologically normal (HN) prostate, and prostate cancer. Values for EGF binding did not differ significantly between HN prostate and prostate cancer, although dedifferentiated samples tended to higher levels. These were coincident with lower comparative levels of androgen receptors. Unfractionated BPH tissue contained lower levels of EGF binding than either HN or carcinomatous prostate, but in separated epithelial cells EGF binding fell into the same range. Saturation analyses showed two affinity classes of binding in all except dedifferentiated tissues.

Growth hormone enhances hepatic epidermal growth factor receptor concentration in mice.

The effect of growth hormone (GH) on binding of epidermal growth factor (EGF) to liver membrane preparations was investigated in hypophysectomized mice and partially GH-deficient, genetic mutant "little" (lit/lit) mice. The EGF binding of normal male mice and testosterone-treated females was higher than in normal females. Due to diminished receptor concentration, hepatic EGF binding was decreased in male and female lit/lit mice to a level that was unaffected by gender or androgen treatment. GH replacement therapy by intermittent injections and continuous infusion restored the EGF binding of hypophysectomized mice to normal male and female levels, respectively, suggesting a role for the more pulsatile GH secretion in normal males. In lit/lit mice, however, both continuous and intermittent GH resulted in EGF binding levels comparable to those in normal females. In normal males continuous GH suppressed EGF binding. In conclusion, endogenous GH secretion induces EGF receptors in mice and this effect may be modulated by sex differences in GH secretion.

Evidence that 2,3,7,8-tetrachlorodibenzo- p-dioxin and thyroid hormones act through different mechanisms in human keratinocytes

It has been proposed [ J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41–53] that 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T 4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [ L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361–1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T 3) and T 4 on human target cells. Keratinocytes were treated simultaneously with T 3 and T 4 in a 4:1 molar ratio ( T 3 T 4 ; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P 1-450 induction) were measured. T 3 T 4 (at concentrations up to 10 μ m T 3/2.5 μ m T 4) and T 3 or T 4 alone (0.1 to 10 μ m) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T 3 T 4 was not due to metabolism of these hormones by the keratinocytes. T 3 and T 4 did not compete with [ 3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [ 125I]T 3 or [ 125I]T 4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.

Cell type-specificity of epidermal growth factor (EGF) binding in primary cultures of early postnatal mouse cerebellum

Binding of 125I-labeled epidermal growth factor (EGF) to cells of cultured early postnatal mouse cerebellar cells was investigated by autoradiography in conjuction with cell type-specific immunolabeling of neurons, astrocytes and oligodendrocytes. By use of tetanus toxin for recognition of neurons and glial fibrilaary acidic (GFA) protein and O4 antigen as markers for astrocytes and oligodendrocytes, respectively, it could be shown that oligodendrocytes do not express receptors for EGF within the limits of sensitivity of the autoradiographic method, and that less than 1% of all small neurons (mostly granule cells) and 50–90% of all GFA protein-positive astrocytes show detectable levels of EGF binding.

Effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor

In this study the effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor (EGF) in mouse fibroblast Balb/c 3T6 cells are further examined. Retinoic acid treatment of 3T6 cells results in a sixfold enhancement of 125I-labeled mouse EGF binding when assayed at 37 degrees C. In both retinoic acid-treated and control cells, cell-associated 125I-EGF is rapidly internalized, degraded, and secreted. Retinoic acid treatment does not seem to have a significant effect on the rate of internalization and degradation of EGF. At 0 degrees C, internalization of EGF is strongly inhibited in both retinoic acid-treated and control cells. Under these conditions retinoic acid-treated cells still exhibit a tenfold higher level of EGF binding compared to control cells. When exposed to high concentrations of EGF both retinoic acid-treated and control cells "down-regulate" their EGF receptors. And although the growth rate of retinoic acid-treated cells is about half that of control cells, the rate at which EGF binding capacity is restored after down-regulation is about three times as fast as in control cells. No direct antagonism on EGF binding was observed between the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid. EGF is a potent mitogen for 3T6 cells in serum-free medium; retinoic acid inhibits the mitogenic activity of EGF even though it increases EGF binding. Retinoic acid also inhibits cell proliferation induced by sarcoma growth factor (SGF) and insulin.

Epithelioid and fibroblastic rat kidney cell clones: Epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 x 10(5) molecules per cell for the epithelioid clone and 1.3 x 10(4) molecules per cell for the fibroblastic clone) was due to a greater number or receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.