Proteases as prognostic markers in cancer

Polyunsaturated fatty acids (PUFAs) have been implicated in tumour development and have been shown to influence cell proliferation in vitro. We report here that n-3 and n-6 PUFAs at concentration > 10 microM inhibited the proliferation of a human kidney epithelial cell line (21HKE), which has retained phenotypic characteristics of normal kidney epithelial cells. In contrast, the proliferation was stimulated by n-3 and n-6 PUFAs at concentrations < 10 microM under defined growth conditions. The stimulatory effect of n-3 and n-6 PUFAs was even more profound in the presence of EGF. In human kidney epithelial cell lines reflecting different stages of transformation (11HKE and 1THKEras), the stimulatory effect was abrogated both in the presence and absence of EGF. Saturated fatty acids did not show any stimulatory effect on cell growth. The tyrosine kinase inhibitors genistein and tyrphostin-47 inhibited EGF-induced protein tyrosine phosphorylation dose-dependently in the 21HKE cells, and abolished the growth stimulatory effect of docosahexaenoic acid (DHA). This indicates the involvement of EGF receptor tyrosine kinase activity in the observed increase in cell proliferation.     

Effect of a coffee lipid (cafestol) on cholesterol metabolism in human skin fibroblasts

The receptor (R) for epidermal growth factor (EGF) is expressed at high levels on human breast cancer cells and associates with ErbB2, ErbB3, and Src proto-oncogene family protein tyrosine kinases (PTKs) to form membrane-associated PTK complexes with pivotal signaling functions. Recombinant human EGF was conjugated to the soybean-derived PTK inhibitor genistein (Gen) to construct an EGF-R-directed cytotoxic agent with PTK inhibitory activity. The EGF-Gen conjugate was capable of binding to and entering EGF-R-positive MDA-MB-231 and BT-20 breast cancer cells (but not EGF-R-negative NALM-6 or HL-60 leukemia cells) via its EGF moiety, and it effectively competed with unconjugated EGF for target EGF-R molecules in ligand binding assays. EGF-Gen inhibited the EGF-R tyrosine kinase in breast cancer cells at nanomolar concentrations, whereas the IC50 for unconjugated Gen was >10 microM. Notably, EGF-Gen triggered a rapid apoptotic cell death in MDA-MB-231 as well as BT-20 breast cancer cells at nanomolar concentrations. The EGF-Gen-induced apoptosis was EGF-R-specific because cells treated with the control granulocyte-colony stimulating factor-Gen conjugate did not become apoptotic. Apoptosis was dependent both on the PTK inhibitory function of Gen and the targeting function of EGF, because cells treated with unconjugated Gen plus unconjugated EGF did not undergo apoptosis. The IC50s of EGF-Gen versus unconjugated Gen against MDA-MB-231 and BT-20 cells in clonogenic assays were 30 +/- 3 nM versus 120 +/- 18 microM (P < 0.001) and 30 +/- 10 nM versus 112 +/- 17 microM (P < 0.001), respectively. Thus, the EGF-Gen conjugate is a >100-fold more potent inhibitor of EGF-R tyrosine kinase activity in intact breast cancer cells than unconjugated Gen and a >100-fold more potent cytotoxic agent against EGF-R+ human breast cancer cells than unconjugated Gen. Taken together, these results indicate that the EGF-R-associated PTK complexes have vital antiapoptotic functions in human breast cancer cells and may therefore be used as therapeutic targets.     

PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner

PD153035 is reported to be a specific and potent inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase and, to a lesser degree, of the closely related HER2/neu receptor. We show that PD153035 inhibits EGF-dependent EGF receptor phosphorylation and suppresses the proliferation and clonogenicity of a wide panel of EGF receptor-overexpressing human cancer cell lines. EGF receptor autophosphorylation in response to exogenous EGF was completely inhibited at PD153035 concentrations of >75 nM in cells overexpressing the EGF receptor. In contrast, PD153035 only reduced heregulin-dependent tyrosine phosphorylation in HER2/neu-overexpressing cell lines at significantly higher concentrations (1400-2800 nM). PD153035 exposure did not affect the expression of either EGF receptors or HER2/neu. PD153035 caused a dose-dependent growth inhibition of EGF receptor-overexpressing cell lines at low micromolar concentrations, and the IC50 in monolayer cultures was less than 1 microM in most cell lines tested. At doses of up to 2.5 microM, the IC50 for HER2/neu-overexpressing cells was not reached. In colony-forming assays, the PD153035 growth-inhibitory activity in cultures driven by endogenous (autocrine) ligand was correlated with EGF receptor number, with higher activity in cells expressing higher numbers of EGF receptors and only minimal activity in cells expressing normal numbers of EGF receptors but high HER2/neu levels. PD153053 also abolished all growth effects mediated by the addition of exogenous EGF; this condition could be reversed upon removal of the compound. Cotreatment with C225, an anti-EGF receptor-blocking monoclonal antibody, further enhanced the antitumor activity of PD153035, suggesting mechanisms of action for C225 other than competition with ligand binding. This latter finding also suggests that combined anti-EGF receptor strategies may be of enhanced benefit against tumors with high levels of EGF receptor expression.     

Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines

In the course of the random screening of a pool of CIBA chemicals, the two pyrazolopyrimidines 1 and 2 have been identified as fairly potent inhibitors of the EGF-R tyrosine kinase. Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), the class of the pyrazolo[3,4-d]pyrimidines was then optimized in an interactive process leading to a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]-pyrimidines as highly potent inhibitors of the EGF-R tyrosine kinase. The most potent compounds 13, 14, 15, 17, 19, 22, 26, 28, and 30 of this series inhibited the EGF-R PTK with IC50 values below 10 nM. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC alpha, CDK1) was observed. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 15, 19, 22, and 23 at IC50 values below 50 nM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 microM, thus indicating high selectivity for the inhibition of the ligand-activated EGF-R signal transduction pathway. Compounds 15 and 19 inhibited proliferation of the EGF-dependent MK cell line with IC50 values below 0.5 microM. In addition, two compounds, 9 and 11, showing satisfactory oral bioavailability in mice after oral administration, exhibited good in vivo efficacy at doses of 12.5 and 50 mg/kg in a nude mouse tumor model using xenografts of the EGF-R overexpressing A431 cell line. From SAR studies, a binding mode for 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines, especially for compound 15, at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)-1H-pyrazolo[3,4-d]pyrimidines represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.     

Binding of erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris to the epidermal growth factor receptor inhibits receptor function in the human glioma cell line, U373 MG

Little is known about the role of the N-linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N-linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N-linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.     

Epidermal growth factor-dependent cell cycle progression is altered in mammary epithelial cells that overexpress c-myc

Amplification and overexpression of the c-myc gene are common in primary human breast cancers and have been correlated with highly proliferative tumors. Components of the epidermal growth factor (EGF) receptor signaling pathway are also often overexpressed and/or activated in human breast tumors, and transgenic mouse models have demonstrated that c-myc and transforming growth factor alpha (a member of the EGF family) strongly synergize to induce mammary tumors. These bitransgenic mammary tumors exhibit a higher proliferation rate than do tumors arising in single transgenics. We, therefore, chose to investigate EGF-dependent cell cycle progression in mouse and human mammary epithelial cells with constitutive c-myc expression. In both species, c-myc overexpression decreased the doubling time of mammary epithelial cells by approximately 6 h, compared to parental lines. The faster growth rate was not due to increased sensitivity to EGF but rather to a shortening of the G1 phase of the cell cycle following EGF-induced proliferation. In cells with exogenous c-myc expression, retinoblastoma (Rb) was constitutively hyperphosphorylated, regardless of whether the cells were growth-arrested by EGF withdrawal or were traversing the cell cycle following EGF stimulation. In contrast, the parental cells exhibited a typical Rb phosphorylation shift during G1 progression in response to EGF. The abnormal phosphorylation status of Rb in c-myc-overexpressing cells was associated with premature activation of cdk2 kinase activity, reduced p27 expression, and early onset of cyclin E expression. These results provide one explanation for the strong tumorigenic synergism between deregulated c-myc expression and EGF receptor signal transduction in the mammary tissue of transgenic mice. In addition, they suggest a possible tumorigenic mechanism for c-myc deregulation in human breast cancer.     

Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185neu and EGFR. Previous analysis of p185neu has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine882 by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine882 residue may be important for p185neu-mediated transformation by affecting the enzymatic kinase function of the p185neu receptor.     

Mitochondrial malic enzyme: purification from bovine brain, generation of an antiserum, and immunocytochemical localization in neurons of rat brain

The receptor kinase activity associated with the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor plays an important role in ligand-induced signaling events. The effect of specific, synthetic chemical inhibitors of PDGF- and EGF-mediated receptor tyrosine autophosphorylation on receptor signaling were examined in NIH 3T3 cells overexpressing PDGF or EGF receptors. Specific inhibition of ligand-dependent receptor autophosphorylation, PI3K activation, mitogen-activated protein kinase (MAPK) activation, cyclin E-associated kinase activity and cell proliferation was measured after treatment of cells with these inhibitors. A synthetic PDGF receptor kinase inhibitor exhibited specific inhibitory properties when tested for PDGF-induced receptor autophosphorylation, MAPK activity, PI3K activation, entry into S phase and cyclin E-associated kinase activity. A synthetic EGF receptor kinase inhibitor showed selective inhibitor properties when tested for EGF-induced receptor autophosphorylation, MAPK activation, PI3K activation, entry into S phase and cyclin E-associated kinase activity. In both cases, these compounds were found to be effective as inducers of growth arrest and accumulation of cells in the G1 phase of the cell cycle after ligand treatment. However, at high concentrations, the EGF receptor kinase inhibitor was observed to exhibit some nonspecific effects as demonstrated by attenuation of PDGF-induced receptor autophosphorylation and cell cycle progression. This demonstrates that it is critical to use the lowest concentration of such an inhibitor that will alter the response under investigation, to have confidence that the conclusions derived from the use of such inhibitor are valid. We conclude that these experimental parameters signify useful end points to measure the relative selectivity of tyrosine kinase inhibitors that affect receptor-mediated signal transduction.     

Inhibition of thrombin-induced contractile responses by protein kinase inhibitors in porcine pulmonary arteries

The clotting enzyme thrombin is known to cause receptor-mediated contractile effects in isolated blood vessels. In the present studies the influence of protein kinase inhibitors on the contractile response of porcine pulmonary arteries to thrombin (3 U/ml) was investigated. Endothelium-denuded rings (2-3 mm) from small arteries were placed in organ baths for isometric tension recording. The vessels were preincubated for 30 min with the inhibitors before inducing contractions. In the presence of the protein kinase C (PKC)-inhibitors staurosporine, BIM I (bisindolyl-maleimide I), chelerythrine and Ro 31-8220 (1 microM each), the contractile responses to the PKC activator phorbol 12,13-dibutyrate (PDBu; 50 nM) were diminished by 70-100%. However, for inhibition of thrombin-induced contractions generally higher concentrations of the inhibitors were required. Only staurosporine at 1 microM inhibited the thrombin effect by about 75%. The tyrosine kinase inhibitor erbstatin (30 microM) did not significantly alter the thrombin effect, whereas genistein at 10 microM caused a significant inhibition of contractile responses to both thrombin and PGF2alpha. At 100 microM genistein also inhibited the contractile effects of PdBu and KCl. These studies suggest that activation of both PKC and non-receptor tyrosine kinases seems to be involved in the signal transduction pathways of thrombin-induced contractile effects in isolated vessels.     

Regulation of prostaglandin H synthase-2 expression in cerebromicrovascular smooth muscle by serum and epidermal growth factor

Growth factors may play a role in the formation of prostaglandins (PG) by cerebral blood vessels during development or reaction to injury. In smooth muscle cultures isolated from murine cerebral microvessels PG production was induced with either serum or epidermal growth factor (EGF). Prostaglandin H synthase (PGHS) activity peaked at 6 h after the addition of 10% serum or 50 ng/ml EGF. Increases in expression of PGHS-1 mRNA were small (7- to 10-fold) compared with PGHS-2 (30- to 120-fold), and the induction patterns were different for serum and EGF. An increase in PGHS-2 message was detected by 0.5 h of adding either agent, but peak induction occurred earlier for EGF than for serum, 1 h vs. 3 h, respectively. The response to either stimulus had returned to prestimulation levels by 12 h. The induction of PGHS-2 protein was also transient, but followed a more delayed time course (peak levels at 6 h). Induction of activity, message, and protein by either agent was blocked by 1 microM dexamethasone and attenuated by genistein (100 microM), a nonspecific tyrosine kinase inhibitor. Tyrphostin 47, a more selective EGF receptor tyrosine kinase inhibitor, dose-dependently inhibited EGF-stimulated PGHS activity, completely abolishing PG production at 100 microM. However, this inhibitor had no effect on serum-stimulated PG production. Curiously, 100 microM tyrphostin 47 enhanced EGF-induced PGHS-2 mRNA and protein expression. These data suggest that EGF induces the expression of PGHS-2 in cerebromicrovascular smooth muscle by a mechanism that requires tyrosine kinase activity and that is distinct from serum.     

Biosynthesis of the proteoglycan decorin--transient 2-phosphorylation of xylose during formation of the trisaccharide linkage region

Genistein, an isoflavone inhibitor of tyrosine-specific protein kinases, was shown to specifically block the 22Na+ influx through voltage-sensitive Na+ channels in cultured rat brain neurons, whereas other tyrosine kinase antagonists such as lavendustin A, compound 5, tyrphostin A47 and an erbstatin analog were inactive at concentrations known to block kinase activity in other neuronal systems. Dose-response curves for genistein indicated a half-maximum effect at 60 microM. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on the 22Na+ influx with a half-maximum effect at 195 microM. The time course of genistein action was rapid, because maximum effect on 22Na+ influx was obtained in less than 20 s at 100 microM. Analysis of Na+ currents by the whole-cell recording technique showed that 20 microM genistein reduced the sodium current and shifted the voltage dependence of both activation and inactivation curves. No competition with [3H]saxitoxin binding was observed, whereas the binding of [3H]batrachotoxinin A 20-alpha-benzoate to rat brain synaptosomal membranes was partially inhibited, which suggested a direct or allosteric interaction with neurotoxin binding site 2. These data taken together clearly indicate that the inhibition of voltage-sensitive sodium channels by genistein is not mediated by tyrosine kinase inhibition.     

From famine to feast: the role of methylglyoxal production in Escherichia coli

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.     

Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle

1. Effects on the pinacidil-induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch-clamp method in smooth muscle cells of the rabbit portal vein. 2. A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil-induced current in a concentration-dependent manner with an IC50 of 5.5 microM. Superfusion of Ca2+-free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage-dependent Ba2+ and K+ currents with IC50 values of > 100 microM and 75 microM respectively. Tyrphostin B46 (30 microM), a tyrosine kinase inhibitor, also inhibited the pinacidil-induced current by 70% of the control. 3. Sodium orthovanadate (100 microM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil-induced and delayed rectifier K+ currents. Daidzein (100 microM), an inactive analogue of genistein, did not inhibit these currents. 4. Neither herbimycin A (1 microM), lavendustin A (30 microM), tyrphostin 23 (10 microM), which are also tyrosine kinase inhibitors, nor wortmannin (10 microM), a phosphatidylinositol 3-kinase inhibitor, had an effect on either the pinacidil-induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 microg ml(-1)) did not induce an outward current or enhance the pinacidil-induced current. 5. Pinacidil alone, in the cell-attached configuration, or pinacidil with GDP, in the inside-out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 microM) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (-30 to +20 mV). 6. In the inside-out configuration, genistein at 30 microM did not change the mean channel open time, but reduced the burst duration. At 100 microM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 microM genistein acted on the unitary current of the ATP-sensitive K+ channel were similar to those seen in the whole-cell voltage-clamp configuration. 7. Although direct inhibitory actions of genistein on the ATP-sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP-sensitive K+ channel activity in the rabbit portal vein.     

Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion

We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 microM) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 microM), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 microM). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nM), but was not altered by the PKC inhibitor GF 109203X (1 microM). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 microM), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84 cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated.     

Enhanced ligand-induced activation of EGF-receptor and overall tyrosine kinase and phospholipase C in colonocytes isolated from azoxymethane-treated rats

BACKGROUND/AIMS: In order to evaluate the role of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in colorectal cancer, the present investigation examines changes in EGF and TGF-alpha-mediated activation of overall and EGF receptor (EGF-R) associated tyrosine kinase activity in isolated rat colonocytes after administration of the colonic carcinogen azoxymethane. METHODOLOGY: Five days after a single injection of azoxymethane (20 mg/kg) or saline solution to 3-4 month old Fischer-344 rats, colonocytes were isolated, exposed for 2 minutes to 1 x 10(8) M EGF and TGF-alpha, and assessed for overall and EGF-R associated tyrosine kinase and phospholipase C activity. RESULTS: In colonocytes isolated from control animals, incubation with EGF and TGF-alpha resulted in a small (21-35%) increase in overall tyr-k. However, a marked (113-127%) rise of this enzyme occurred in colonocytes from AOM-treated rats, when compared with the corresponding basal levels. These differences were even more pronounced in colonocytes isolated from the distal part of the colon, as regards to the proximal part. In addition, EGF and TGF-alpha activated EGF-R tyr-k by 40-60% in controls and by 84-85% in AOM-treated animals. Incubation of colonocytes with these growth factors also stimulated PLC activity (in controls by 120-150% and in AOM injected rats by 204-271%) when compared with corresponding basal values. CONCLUSIONS: We conclude that AOM enhances the responsiveness of colonocytes to EGF and TGF-alpha, which may be one of the mechanisms involved in colorectal carcinogenesis.     

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor in labial salivary glands in Sj?gren's syndrome

OBJECTIVE: Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) affect cells through binding to a shared EGF receptor (EGF-R), which is a transmembrane protein with tyrosine kinase activity. They exert trophic effects on vascular endothelial, salivary acinar, and ductal and mucosal epithelial cells. In Sj?gren's syndrome (SS) focal sialadenitis leads to salivary gland tissue damage, diminished salivary flow, and changes in the oral epithelium, a complex referred to as xerostomia. We compared the localization of EGF, TGF-alpha, and EGF-R in labial salivary glands in SS and in healthy controls. METHODS: Labial salivary gland tissues of 12 patients with SS and 7 healthy controls were stained with the immunohistochemical peroxidase-antiperoxidase method for EGF, TGF-alpha, and EGF-R. RESULTS: Immunoreactivity for both EGF and TGF-alpha was found in endothelial cells of blood vessels and in some ductal epithelial cells. TGF-alpha, but not EGF, was also found in some acinar cells. EGF-R was found in endothelial, acinar, and salivary duct epithelial cells. There was no difference in the expression of EGF-R between diseased and healthy specimens, but both EGF and TGF-alpha were diminished in SS. CONCLUSION: The interrelated localization of EGF-R and its ligands, EGF and TGF-alpha, suggests an autocrine, juxtacrine, and paracrine mitogenic/trophic role for them and thus a role in the maintenance of the secretory and excretory cells of the normal salivary glands. The trophic effects on acinar cells seem not to be mediated by EGF, but more likely by TGF-alpha. The diminished expression of EGF and TGF-alpha indicates a failure of this trophic system in SS, which may contribute to the acinar atrophy and secondary changes thereof, including atrophy of the oral mucosa.     

Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies

We have previously described anti-epidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) which can block binding of transforming growth factor alpha (TGF-alpha) and EGF to receptors and inhibit activation of receptor tyrosine kinase. Studies with these MAbs involving cell cultures and nude mouse xenografts demonstrated their capacity to inhibit the growth of a variety of tumor cell lines, which express EGF receptors and TGF-alpha and appear to depend upon receptor activation for cell proliferation. To explore the mechanism(s) by which anti-EGF receptor 225 MAb inhibits cell proliferation, we have compared the activity of native 225 MAb with the response to bivalent 225 F(ab')2 and monovalent 225 Fab' fragments. Both native 225 MAb and its fragments could inhibit the binding of 125I-EGF to EGF receptors. Scatchard analysis revealed that the Kd of 225 F(ab')2 is comparable to that of 225 MAb (1 nM), whereas the Kd of 225 Fab' is 5 nM. Both bivalent 225 MAb and 225 F(ab')2 and monovalent 225 Fab' were able to completely inhibit TGF-alpha-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on MCF10A nonmalignant human mammary cells, MDA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endogenous (autocrine) TGF-alpha in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-alpha in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-alpha upon MDA468 and A431 cell cultures. Monovalent 225 Fab' had weaker inhibitory effects upon the proliferation of these cell lines. To determine whether the in vivo antiproliferative activity of anti-EGF receptor MAb can occur without the participation of the Fc portion of MAb, the capacities of 225 F(ab')2 and native 225 MAb to inhibit growth of s.c. A431 cell xenografts were compared. Equimolar amounts of either 225 MAb or 225 F(ab')2 were administered at intervals equivalent to the half-lives of the molecules, to attempt to maintain comparable plasma levels. Both 225 MAb and 225 F(ab')2 inhibited A431 cell xenograft growth in a dose-dependent manner, with a more sustained response in the case of the intact antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduction in numerical synapse density in chick (Gallus domesticus) dorsal hippocampus following transient cerebral ischaemia

BACKGROUND: The epidermal growth factor (EGF) signal transduction pathway, frequently activated in pancreatic cancer, is an important regulator of cellular growth and transformation. This study examined whether activation of the cyclic adenosine monophosphate protein kinase A pathway may inhibit the EGF signal transduction pathway in pancreatic cancer cell lines. METHODS: Human pancreatic cancer lines BxPC-3 and AsPC-1 were stimulated with EGF, forskolin, or both. Forskolin is a compound that increases cyclic adenosine monophosphate levels. Assays of cell lines were then obtained for cellular growth (MTT assay), anchorage-independent growth (soft agar), and EGF-induced mitogen-activated protein kinase activation as measured by an in-gel kinase assay. RESULTS: Treatment with forskolin resulted in inhibition of EGF-induced activation of mitogen-activated protein kinase activity (BxPC-3 78% inhibition and AsPC-1 70% inhibition, p < 0.005), diminished cellular proliferation (BxPC-3 92% inhibition and AsPC-1 86% inhibition, p < 0.001), and formation of colonies in soft agar (BxPC-3 98% inhibition and AsPC-1 76% inhibition, p < 0.001). Forskolin did not inhibit EGF receptor autophosphorylation or tyrosine kinase signaling in response to EGF. CONCLUSIONS: Forskolin-induced inhibition of mitogen-activated protein kinase is associated with diminished pancreatic cancer cell proliferation in vitro. Use of strategies to increase cyclic adenosine monophosphate levels may have therapeutic application in pancreatic cancer.