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Query: UNIPROT:Q9UL75 (A431)
 5,640 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polyamines--putrescine, spermidine, and spermine--are ubiquitous cellular components that play an important role in cell growth and differentiation. Using A431 cells, a cell line that overexpresses the epidermal growth factor (EGF) receptor, we found that polyamines modulate EGF-mediated growth inhibition. The natural polyamine, putrescine, was the most effective, followed by diamines containing lower and higher methylene bridging between the amino groups. To understand the mechanism, we examined the effects of polyamines on EGF-mediated signal transduction in A431 cells. All three polyamines partially inhibited EGF-receptor tyrosine kinase activity in a dose-dependent manner. The maximal inhibition was 75% with spermidine. Polyamine effects were exerted 12-16 h after treatment, although HPLC analysis revealed uptake of polyamines within 1 h. Homologues of putrescine had no significant effect on tyrosine kinase activity, indicating structural specificity of naturally occurring polyamines in this process. Amine oxidase inhibitors did not alter spermidine and spermine-mediated effects, suggesting that the inhibition of tyrosine kinase activity was not a consequence of the oxidative metabolism of polyamines. Difluoromethylornithine, a specific inhibitor of polyamine biosynthesis, did not affect EGF receptor tyrosine kinase activity. Polyamines also had no effect on EGF receptor levels or EGF-EGF receptor high-affinity binding, indicating that they are not competitive inhibitors of the EGF receptor tyrosine kinase. Our results suggest that polyamine action in A431 cells involves modulation of EGF receptor signal transduction pathways.
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PMID:Inhibition of epidermal growth factor-stimulated EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma cells by polyamines. 775 70

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.
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PMID:Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. 779 Mar 53

Modulation of protein kinase FA/GSK-3 alpha by tyrosine phosphorylation in A431 cells was investigated. Kinase FA/GSK-3 alpha was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in a concentration-dependent manner by 50-400 microM genistein (a specific tyrosine kinase inhibitor), as demonstrated by metabolic 32P-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase FA/GSK-3 alpha immunoprecipitate kinase assay. Taken together, the results provide evidence that kinase FA/GSK-3 alpha may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can be tyrosine-dephosphorylated and inactivated by extracellular stimulus and that tyrosine kinase(s) and/or tyrosine phosphatase(s) may play a role in the modulation of kinase FA/GSK-3 alpha activity in cells.
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PMID:Tyrosine dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3 alpha by genistein in A431 cells. 780 86

Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide with a wide range of biological activities. Recent data suggest that functional VIP receptors are expressed on various tumor cells. Somatostatin (SST) and its long-acting analogue octreotide (OCT) are potent inhibitors of tumor cell growth and secretion. In the present study, the interactions between VIP and SST/OCT on primary tumors (insulinomas, n = 3; VIPomas, n = 2; intestinal adenocarcinomas, n = 5; neuroblastomas, n = 5; papillary thyroid cancers, n = 7; carcinoids, n = 5; ductal breast cancers, n = 8; small cell lung cancers, n = 3; ACTH-producing hypophyseal adenomas, n = 5; pheochromocytomas, n = 5) as well as on tumor cell lines (A431, HT29, PANC1, COLO320, HMC1, and KU812 cells) were analyzed by use of 123I-labeled VIP and 123I-labeled Tyr-3-OCT. Cross-competition between VIP and SST/OCT for binding to tumor cells was observed. The rank-order of potency for displacement of 123I-labeled VIP binding to intact A431 cells was VIP [concentration causing half-maximal inhibition (IC50) = 2.9 +/- 1.9 (SD) nM] > OCT (IC50 = 9.3 +/- 1.7 nM) = SST > substance P = secretin (IC50 = 1 microM). Binding of 123I-labeled Tyr-3-OCT to A431 cells, in turn, was inhibited by OCT = Tyr-3-OCT (IC50 = 1.5 +/- 0.3 nM) = SST > VIP (IC50 = 4.9 +/- 1.1 nM). This rank-order of potency was also obtained for primary tumors and tumor cell lines. Furthermore, SST and OCT inhibited VIP-induced [3H]thymidine incorporation, cyclic AMP formation, and tyrosine kinase activity with IC50 values < 10 nM. Together, these data provide evidence for functional interactions between SST and VIP on various tumor cells. These interactions may involve peptide cross-competition at cellular binding sites and may have implications for the biology and pathophysiology of respective cells and disease states.
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PMID:Cross-competition between vasoactive intestinal peptide and somatostatin for binding to tumor cell membrane receptors. 790 85

The epidermal growth factor (EGF) receptor is overexpressed in squamous cell carcinoma and the EGF receptor has been proposed as a potential target for new therapeutic agents in this tumour type. We have utilized a tyrphostintype inhibitor of the EGF receptor tyrosine kinase domain (RG50864) to study EGF-dependent proliferation and phosphorylation in two human squamous cell carcinoma cell lines. There were selected on the basis that whereas both cell lines have a large number of EGF receptors, one is growth inhibited by EGF (A431) while the proliferation of the other cell line (B2A4) is stimulated by EGF. EGF induced receptor autophosphorylation in each of the two cell lines; however, the level of phosphorylation was greater in the A431 cells than in the B2A4 cells. The pattern of proteins phosphorylated in response to EGF was different in the two squamous cell lines. RG50864 antagonized the EGF-dependent proliferation of B2A4 cells, but was unable to reverse the inhibitory effect of EGF on A431 cell growth. RG50864 partially inhibited EGF receptor autophosphorylation in both cell lines and completely inhibited the EGF-dependent phosphorylation of other cellular proteins, one of which co-migrated with MAP2kinase in both cell lines. Moreover, different dose-response relationships for the inhibition of phosphorylation of various proteins were observed in A431 versus B2A4 cells. As a substrate competitive inhibitor of the EGF receptor tyrosine kinase, the primary mode of action of RG50864 may be to prevent the association and/or phosphorylation of multiple specific substrates of the receptor in a fashion which may be cell line dependent. The precise relationship of these phosphorylation events to tyrphostin sensitivity remains to be established.
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PMID:Alterations in EGF-dependent proliferative and phosphorylation events in squamous cell carcinoma cell lines by a tyrosine kinase inhibitor. 791 99

We previously reported that anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 can block receptor activation and inhibit proliferation of tumor cells bearing EGF receptors. To further explore the mechanism of mAb-mediated growth inhibition, we compared the capacities of bivalent 225 mAb and 225 F(ab')2, and monovalent 225 Fab' fragment to block ligand binding to EGF receptors, inhibit activation of receptor tyrosine kinase by exogenous and endogenous ligand, produce receptor dimerization, down-regulate receptors, and inhibit proliferation of cultured A431 squamous carcinoma cells. Unlike 225 mAb and 225 F(ab')2, 225 Fab' fragment was a poor inhibitor of A431 cell proliferation. The weak antiproliferative capacity of 225 Fab' was not due to depletion of active fragment from cultures. When cells were exposed to exogenous EGF, monovalent 225 Fab' remaining in conditioned culture medium could act as well as the bivalent forms of mAb to block binding and tyrosine kinase activation by exogenous EGF. However, unlike the bivalent forms, 225 Fab' fragment was unable to induce receptor dimerization and down-regulation, and it lacked the capacity to block autocrine activation of EGF receptors by endogenous ligand. These deficiencies were corrected by addition of rabbit anti-mouse IgG antibody, which also enabled 225 Fab' fragment to inhibit cell proliferation. We conclude that in A431 cells, inhibition of autocrine-stimulated proliferation by anti-EGF receptor mAbs requires antibody bivalency, which provides the capacity to produce EGF receptor dimerization accompanied by receptor down-regulation. These properties may explain the greater efficacy of bivalent mAb and F(ab')2, compared with monovalent Fab' fragment, in inhibiting proliferation of a variety of malignant and nonmalignant cultured cell lines.
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PMID:Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. 796 76

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.
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PMID:Conversion of epidermal growth factor (EGF) into a stimulatory ligand for A431-cell growth by herbimycin A by decreasing the level of expression of EGF receptor. 803 91

Tumor necrosis factor (TNF) induces dose-dependent, but incomplete cytotoxicity in ME-180 cervical carcinoma cells resulting in a significant reduction in cell viability. In this cell line there exists a characteristic residual tumor cell population that appears to be resistant to TNF. In order to investigate tumor cell heterogeneity and characteristics that correlate with their escape from TNF-induced cytotoxicity, TNF-resistant ME-180 cell variants (ME-180R) were isolated from a population of ME-180 cervical carcinoma cells (ME-180 parental). Incubation of ME-180 parental cells with TNF resulted in measurable changes in tumor cell DNA structural integrity and dose-dependent cytotoxicity, whereas ME-180R cell growth and DNA integrity were not effected by incubation with TNF. Binding of 125I-labeled TNF to a TNF-specific cell-surface receptor was measurable and equivalent on both ME-180R and ME-180 parental cells and both cell lines predominantly expressed the p55 form of the TNF receptor based upon flow cytometric analysis. Although both cell lines shared similar doubling times, intrinsic EGF receptor tyrosine kinase activity in ME-180R cells was found to be > 3-fold higher than that isolated from ME-180 parental cells. These results suggest that TNF-responsiveness may be mediated at a point subsequent to TNF binding and may be regulated, in part, by the expression of tyrosine kinase activity. To further explore this hypothesis, A431 vulvular carcinoma cells that express resistance to TNF were cloned and variants were isolated that escaped EGF-induced growth inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Positive and negative selection for tumor necrosis factor responsiveness reveals an inhibitory role for EGF receptor in TNF-induced antiproliferation. 818 23

A series of polyhydroxylated 2-phenylbenzothiazoles 3 has been prepared by demethylation of the precursor methoxylated 2-phenylbenzothiazoles 9. The key step in the construction of the benzothiazole nucleus involves a Jacobson cyclization of methoxylated thiobenzanilides 8. The target compounds inhibit WiDr human colon tumor cells and MCF-7 human mammary tumor cells in vitro with IC50 values in the low micromolar range, but the activity against MCF-7 cells is not related to estrogen receptor-binding affinity. None of the compounds showed selective cytotoxicity against Abelson virus-transformed ANN-1 cells encoded with the pp120gag-abl tyrosine kinase compared with the parental 3T3 line. Compounds were only marginally inhibitory to the EGF receptor-associated protein tyrosine kinase from a membrane preparation of A431 cells. The most active compound was 4,6-dihydroxy-2-(4-hydroxyphenyl)benzothiazole (3b) which has the same overall hydroxyl substitution pattern as genistein (1a). The compounds were weakly cytotoxic for an EGF receptor, overexpressing cell line HN5, but when tested for differential toxicity against the EGF receptor tyrosine kinase or the PDGF receptor tyrosine kinase in a standard mitogenesis assay utilizing human fibroblasts, no discrimination was observed. In this assay, the compounds inhibited DNA synthesis when added to cells during S phase. This suggests that inhibition could not be interpreted in terms of tyrosine kinase inactivation but more likely as a relatively broad specificity for the ATP-binding domain of other kinases such as thymidine kinase.
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PMID:Structural studies on bioactive compounds. 23. Synthesis of polyhydroxylated 2-phenylbenzothiazoles and a comparison of their cytotoxicities and pharmacological properties with genistein and quercetin. 820 3

In this report, we describe the applicability of fluorescein-labeled EGF (FITC-EGF) in monitoring the interaction between EGF and its cellular receptor in real time. This work takes advantage of previous studies that demonstrated that EGF may be labeled at its amino terminus with FITC without significant deleterious effects on the binding of the growth factor to its receptor or on the ability of the growth factor to activate the intrinsic tyrosine kinase activity of the receptor. When suspended human epidermoid carcinoma (A431) cells were treated with FITC-EGF, a biphasic quenching of the FITC fluorescence was observed. Both phases were blocked when the experiments were performed in the presence of excess unlabeled EGF. The first phase, in which the FITC emission was quenched by 8-10%, was complete within 30 s. This rapid quenching was attributed to changes in the rotational mobility of the EGF molecule that accompany its binding to receptors. The slower phase required 20-30 min for completion and resulted in the further quenching of the FITC fluorescence by 30-40%. This slower phase appeared to reflect the internalization of the receptor and its routing to acidic intracellular compartments. The rapid fluorescence decay phase was used to determine the rate constants (k(on) and k-off)) for the interaction of FITC-EGF with receptors on the surface of cells.
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PMID:Fluorescent-labeled growth factor molecules serve as probes for receptor binding and endocytosis. 821 81


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