Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer.

De novo insensitivity and acquired resistance to the selective oestrogen receptor modulator tamoxifen and the pure anti-oestrogen fulvestrant (faslodex) severely limit their effectiveness in breast cancer patients. This is a major clinical problem, since each year upward of 1 million women are dispensed anti-oestrogenic drugs. In order to investigate the phenomenon of anti-oestrogen resistance and to rapidly screen drugs that target the resistance mechanism(s), we have previously established several in vitro breast cancer models that have acquired resistance to anti-hormones. Such cells commonly develop an ability to proliferate after approximately 3 months of exposure to 4-hydroxytamoxifen or fulvestrant, despite an initial endocrine-responsive (i.e. growth-suppressive) phase. The current paper explores the role that growth factor signalling plays in the transition of oestrogen receptor-positive endocrine-responsive breast cancer cells to anti-oestrogen resistance or insensitivity and how we might, in the future, most effectively use anti-growth factor therapies to treat or delay endocrine-resistant states.

Insulin-like growth factor-I receptor signaling in tamoxifen-resistant breast cancer: a supporting role to the epidermal growth factor receptor.

There is considerable evidence that the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) cross-talk in breast cancer cells. In the present study, we have examined whether EGFR/IGF-IR cross-talk exists in EGFR-positive tamoxifen-resistant variants of MCF-7 (Tam-R) and T47D (T47D-R) breast cancer cell lines. Although Tam-R cells expressed reduced IGF-IR protein levels compared with their wild-type MCF-7 counterparts, phosphorylated IGF-IR protein levels were equivalent in the two cell lines under basal growth conditions, possibly as a consequence of increased IGF-II expression in Tam-R cells. IGF-II activated both IGF-IR and EGFR in Tam-R cells, whereas only activation of IGF-IR was observed in wild-type cells. In contrast, epidermal growth factor rapidly induced EGFR, but not IGF-IR, phosphorylation in Tam-R cells. IGF-II promoted direct association of c-SRC with IGF-IR, phosphorylated c-SRC, and increased EGFR phosphorylation at tyrosine 845, a c-SRC-dependent phosphorylation site. Pretreatment with either AG1024 (IGF-IR-specific inhibitor) or an IGF-II neutralizing antibody inhibited basal IGF-IR, c-SRC, and EGFR phosphorylation, and AG1024 significantly reduced Tam-R basal cell growth. The c-SRC inhibitor SU6656 also inhibited growth, reduced basal and IGF-II-induced c-SRC and EGFR phosphorylation, and blocked EGFR activation by TGFalpha. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGFalpha-induced EGFR activity. These results suggest the existence of a unidirectional IGF-IR/EGFR cross-talk mechanism whereby IGF-II, acting through the IGF-IR, regulates basal and ligand-activated EGFR signaling and cell proliferation in a c-SRC-dependent manner in Tam-R cells. This cross-talk between IGF-IR and EGFR is not unique to Tam-R cells because this mechanism is also active in a tamoxifen-resistant T47D-R cell line.

Growth factor signalling networks in breast cancer and resistance to endocrine agents: new therapeutic strategies.

Recent evidence demonstrates that growth factor networks are highly interactive with the estrogen receptor (ER) in the control of breast cancer growth and development. As such, tumor responses to anti-hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents, with alterations/aberrations in growth factor signalling providing a mechanism for the development of anti-hormone resistance. In this light, the current article focuses on illustrating the relationship between growth factor signalling and anti-hormone failure in our in-house tumor models of breast cancer and describes how we are now beginning to successfully target their actions to improve the effects of anti-hormonal drugs and to block aggressive disease progression.

Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy.

There is an increasing body of evidence demonstrating that elevated growth signaling in breast cancer cells can promote forms of endocrine resistance in either an estrogen receptor-dependent or -independent manner. The current article reviews what is known about such growth factor signaling networks and resistance to estrogen withdrawal and considers the many novel therapeutic opportunities that stem from this knowledge.

Modulation of epidermal growth factor receptor in endocrine-resistant, oestrogen receptor-positive breast cancer.

There is an increasing body of evidence demonstrating that growth factor networks are highly interactive with oestrogen receptor (ER) signalling in the control of breast cancer growth. As such, tumour responses to anti- hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents. The current article examines the modulation of growth factor networks during endocrine response, and presents in vitro and clinical evidence that epidermal growth factor receptor signalling, maintained in either an ER-dependent or -independent manner, is critical to anti- hormonal-resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor-selective tyrosine kinase inhibitor, ZD 1839 (Iressa; AstraZeneca) to efficiently treat, and perhaps even prevent, endocrine-resistant breast cancer is highlighted.

The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro.

Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.

Mechanotransduction through the endothelial cytoskeleton: mediation of flow- but not agonist-induced EDRF release.

1. We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of the endothelial microfilament and microtubule cytoskeleton to EDRF release evoked by time-averaged shear stress and by acetylcholine in rabbit abdominal aorta. 2. Cytochalasin B (1 microM) and phalloidin (100 nM) were used to depolymerize and stabilize, respectively, F-actin microfilaments. Colchicine (500 nM) was used to inhibit tubulin dimerization and thus disrupt the microtubule network. Experiments were performed before or 1 h after administration of agents to the donor perfusate or organ bath. 3. In cascade bioassay studies, time-averaged shear stress was manipulated with dextran (1-4% w/v, 80,000 MW), to increase perfusate viscosity. EDRF release induced by increased perfusate viscosity was significantly (P < 0.01) attenuated by cytochalasin B, phalloidin and colchicine. 4. Endothelium-dependent relaxations to acetylcholine (0.01-30 microM) in cascade bioassay and in isolated aortic ring preparations were unaffected by pretreatment with any of these agents both in terms of their EC50 and maximal responses. Endothelium-independent relaxations to sodium nitroprusside (0.001-10 microM) were similarly unaffected. 5. We conclude that the endothelial F-actin microfilament and microtubule networks are involved in the mechanotransduction pathway for flow-evoked EDRF release in rabbit abdominal aorta. However, these cytoskeletal elements appear to play no role in acetylcholine-induced EDRF release in this tissue.

Central role of intracellular calcium stores in acute flow- and agonist-evoked endothelial nitric oxide release.

1. We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca2+ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta. 2. Experiments were performed before and following incubation with either the endoplasmic reticulum Ca(2+)-ATPase inhibitors cyclopiazonic acid (CPA, 10 microM) and thapsigargin (TSG, 1 microM) or ryanodine (30, 100 microM) which binds to a specific endoplasmic reticulum Ca(2+)-release channel. 3. In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4 +/- 3.8%; TSG, 51.5 +/- 10.6%; ryanodine, 17.4 +/- 1.6%) which were significantly attenuated by preincubation of the donor with 100 microM NG-nitro-L-arginine methyl ester (L-NAME). However, in isolated rings, only CPA and TSG induced L-NAME-sensitive relaxations (CPA 52.7 +/- 6.5%; TSG 61.3 +/- 7%). 4. Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3 +/- 1.4%, n = 22). Prior administration of SOD attenuated relaxations to TSG (23.2 +/- 3.8% n = 4) and ryanodine (1.7 +/- 0.8%, n = 4), and pre-incubation with TSG and ryanodine blunted SOD-induced responses (4 +/- 1.5%, n = 4 and 8.9 +/- 1.1%, n = 4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant and did not modulate relaxations to CPA, TSG or ryanodine. 5. In cascade bioassay studies time-averaged shear stress was manipulated with dextran (1-4% w/v, 800000 MW) to increase perfusate viscosity. NO-dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P < 0.01; n = 6) and TSG (P < 0.05; n = 7), but not by ryanodine (n = 6). 6. Endothelium-dependent relaxations to acetylcholine (0.1-30 microM) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium-independent relaxations to sodium nitroprusside (0.001-10 microM), whereas TSG had no effect. 7. We conclude that release of Ca2+ from CPA- and TSG-sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca(2+)-induced Ca2+ release via the ryanodine-sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.

Abolition of flow-dependent EDRF release before that evoked by agonists in hypercholesterolaemic rabbits.

1. We have used a pulsatile cascade bioassay system to investigate the effects of dietary-induced hypercholesterolaemia on EDRF release evoked by acetylcholine and by the oscillatory and time-averaged components of flow, in isolated segments of rabbit abdominal aorta. 2. Flow pulsatility (frequency range 0.1-10 Hz) was studied with constant flow (9 ml min-1) at a pulse pressure amplitude of 2 mmHg. Frequency-related EDRF release, maximal at 6 Hz, was slightly attenuated after 4 weeks and abolished after 8 weeks of cholesterol feeding. 3. Time-averaged shear stress was manipulated with dextran (1-4% w/v, 80000 mol. wt.), to increase perfusate viscosity. EDRF release induced by increased perfusate viscosity was unaffected after 4 weeks but abolished after 8 weeks of cholesterol feeding. 4. Endothelium-dependent relaxations to acetylcholine (0.1-10 microM) were not influenced after 4 weeks and only partially attenuated (by 60% of the maximal response, EC50 unchanged at 6.45 +/- 0.04 vs. 6.4 +/- 0.1 microM) after 8 weeks of cholesterol feeding. 5. Blood cholesterol levels were significantly (P < 0.001) increased after 4 weeks (26 +/- 3.6 vs 2.6 +/- 0.6 mmol l-1) and 8 weeks (56.2 +/- 3.8 vs 1.3 +/- 0.1 mmol l-1) of cholesterol feeding but after 8 weeks plasma L-arginine levels were not significantly different from the age-matched controls (0.2 +/- 0.05 vs. 0.19 +/- 0.04 mmol l-1). 6. We conclude that hypercholesterolaemia impairs flow-related (pulsatile- and time-averaged shear-induced) EDRF release earlier than acetylcholine-induced relaxation in rabbit aorta. This is consistent with the view that different transduction mechanisms mediate EDRF release in response to agonists and flow.

Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat.

1. The role of endogenous nitric oxide (NO) in maintaining intestinal vascular integrity following acute endotoxin (E. coli. lipopolysaccharide) challenge was investigated in the anaesthetized rat by use of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of NO synthesis. 2. L-NMMA (10-50 mg kg-1, i.v.) pretreatment enhanced both the macroscopic and histological intestinal damage and the increases in vascular permeability, measured as the leakage of [125I]-labelled human serum albumen, induced after 15 min by endotoxin (50 mg kg-1, i.v.). 3. The effects of L-NMMA (50 mg kg-1, i.v.) were enantiomer specific, as D-NMMA had no effect. Furthermore, these effects were reversed by L-arginine (300 mg kg-1, i.v.), the precursor of NO synthesis but not by D-arginine (300 mg kg-1, i.v.). 4. L-NMMA (10-50 mg kg-1, i.v.) increased mean systemic arterial blood pressure but this does not appear to be the mechanism by which endotoxin-induced intestinal damage was enhanced, since similar systemic pressor responses induced by phenylephrine (10 micrograms kg-1 min-1, i.v.), had no such effect. 5. The results suggest that synthesis of NO from L-arginine has a role in maintaining the microvascular integrity of the intestinal mucosa following acute endotoxin challenge.

Increased intestinal formation of Paf in endotoxin-induced damage in the rat.

Platelet-activating factor (Paf) has been proposed as a mediator of the gastrointestinal damage in endotoxic shock. The formation of Paf in rat jejunal tissue, determined following extraction and bioassay on rabbit washed platelets has therefore been correlated with the induction of damage following endotoxin administration. Intravenous injection of E. coli endotoxin led to a time-dependent increase in the jejunal formation of Paf, which after 20 min was twenty fold greater than the control level. There was a significant correlation between elevated Paf release and intestinal hyperaemia and haemorrhage, thus supporting a role for Paf as a mediator of such damage.

Iodinated radiographic contrast media inhibit shear stress- and agonist-evoked release of NO by the endothelium.

1 We have used isolated arterial preparations from the rabbit and dog to investigate whether non-ionic iodinated radiographic contrast media (IRCM) modulate nitric oxide (NO) release. The tri-iodinated monomers iopromide and iohexol were compared with the hexa-iodinated dimer iodixanol. 2 The vasodilator effects of iohexol (300 mg ml-1) and iodixanol (320 mg ml-1) were assessed in cascade bioassay. Increasing concentrations of iohexol or iodixanol caused concentration-dependent relaxations of the detector tissue which were insensitive to 100 microM NG-nitro L-arginine methyl ester (L-NAME) and 10 microM indomethacin, whereas viscosity-associated relaxations induced by the 'inert' agent dextran (MW 80,000; 1-4%) were attenuated by inhibition of NO synthesis. 3 Relaxations of endothelium-intact rings to acetylcholine (ACh) were attenuated by preincubation with iohexol or iodixanol, whereas relaxations to sodium nitroprusside (SNP) in endothelium-denuded rings were unaffected. Inhibitory activity did not correlate with either molarity or iodine concentration. Mannitol caused inhibition of both ACh- and SNP-induced responses. 4 In isolated perfused arteries the depressor responses to iodixanol (320 mg ml-1) and iopromide (300 mg ml-1) administered as close arterial bolus attained a plateau with maximal dilatations of approximately 25% and approximately 60%, respectively. Addition of 100 microM NG-nitro L-arginine (L-NOARG) and/or 10 microM indomethacin to the perfusate had no effect on the responses to either agent. 5 We conclude that IRCM exert direct effects on the endothelium that inhibit NO production rather than its action on vascular smooth muscle. Shear stress-induced stimulation of NO production by IRCM is unlikely to contribute to their vasodilator activity in vivo when administered during angiography despite high intrinsic viscosity.

Comparison of glycyrrhetinic acid isoforms and carbenoxolone as inhibitors of EDHF-type relaxations mediated via gap junctions.

The vascular actions of the lipophilic gap junction inhibitors 18alpha-glycyrrhetinic acid (18alpha-GA), 18beta-glycyrrhetinic acid (18beta-GA) and the water-soluble hemisuccinate derivative of 18beta-GA, carbenoxolone, were investigated in preconstricted rings of rabbit superior mesenteric artery. EDHF-type relaxations to acetylcholine (ACh), observed in the presence of 300 microM NG-nitro-L-arginine methyl ester (L-NAME) and 10 microM indomethacin, were attenuated by preincubation with 18alpha-GA (to 100 microM), 18A-GA (to 10 microM) or carbenoxolone (to 300 microM) in a concentration-dependent fashion. By contrast, none of these agents affected responses to sodium nitroprusside, an exogeneous source of NO, and relaxations evoked by ACh in the absence of L-NAME were attenuated by only approximately 20%. 18alpha-GA exerted no direct effect on vessel tone, whereas 18beta-GA and carbenoxolone caused relaxations which were maximal at approximately 1 and approximately 10 mM, respectively. Relaxations to carbenoxolone were attenuated by endothelial denudation and by incubation with L-NAME, whereas those to 18beta-GA were unaffected. In conclusion, all three agents inhibit EDHF-type relaxations evoked by ACh, providing further evidence for the involvement of gap junctions in such responses. Unlike 18alpha-GA, carbenoxolone and 18beta-GA possess intrinsic vasorelaxant activity which in the case of carbenoxolone involves functional enhancement of NO activity in addition to direct effects on vascular smooth muscle.

Modulation of epidermal growth factor receptor in endocrine-resistant, estrogen-receptor-positive breast cancer.

An increasing body of evidence demonstrates that growth factor networks are highly interactive with estrogen receptor signaling in the control of breast cancer growth. As such, tumor responses to antihormones are likely to be a composite of the estrogen receptor and growth factor inhibitory activity of these agents. The modulation of growth factor networks during endocrine response is examined, and in vitro and clinical evidence is presented that epidermal growth factor receptor signaling, maintained in either an estrogen receptor-dependent or a receptor-independent manner, is critical to antihormone-resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor-selective tyrosine kinase inhibitor Iressa (ZD 1839) to efficiently treat, and perhaps even prevent, endocrine-resistant breast cancer is highlighted.

Role of phospholipase A(2) and myoendothelial gap junctions in melittin-induced arterial relaxation.

We have used preconstricted rings of rabbit superior mesenteric artery to investigate the contribution of phospholipase A(2) and gap junctional communication to endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by melittin, a polypeptide toxin known to mobilize arachidonic acid from the cell membrane. Arachidonyl trifluoromethyl ketone (30 microM), an inhibitor of the Ca(2+)-dependent phospholipase A(2), and Gap 27 (300 microM), a connexin-mimetic peptide which attenuates intercellular communication via gap junctions, both abolished the endothelium-dependent component of EDHF-type responses evoked by melittin in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) and the cyclooxygenase inhibitor indomethacin (10 microM). By contrast, the sulfhydryl agent thimerosal (300 nM), which amplifies EDHF activity, potentiated nitric oxide (NO)/prostanoid-independent relaxations induced by melittin. Neither arachidonyl trifluoromethyl ketone nor thimerosal modulated relaxations evoked by the peptide toxin in the absence of L-NAME and indomethacin. We conclude that melittin evokes EDHF-type relaxations through activation of the endothelial Ca(2+)-dependent phospholipase A(2) followed by the transmission of a chemical and/or electrical signal via myoendothelial gap junctions. This mechanism of vasorelaxation may be negatively regulated by NO.

Protective effect of S-nitroso-N-acetyl-penicillamine in endotoxin-induced acute intestinal damage in the rat.

Macroscopic jejunal damage and plasma leakage induced within 15 min by E. coli lipopolysaccharide (LPS 50 mg kg-1 i.v.) in the rat was enhanced by the inhibitor of nitric oxide (NO) formation, NG-monomethyl-L-arginine (L-NMMA 50 mg kg-1 i.v.). The nitro-vasodilator, S-nitroso-N-acetyl-penicillamine (SNAP; 10 micrograms kg-1 min-1 i.v.), which generates NO, attenuated both LPS-induced intestinal damage and the enhancement of such damage and plasma leakage produced by L-NMMA. Endogenous NO may thus have a protective role in the intestinal vasculature that can be mimicked by generators of NO.

Angiographic contrast media relax isolated rabbit aorta through an endothelium-independent mechanism that may not depend on the presence of the iodine atom.

Systemically administered iodinated angiographic contrast media evoke vasodilatation through mechanisms that are at present poorly understood. In the current investigation we have evaluated the role of the vascular endothelium in responses to an iso-osmolar formation of the non-ionic dimer iodixanol and a hyperosmolar formulation of the non-ionic monomer iopromide. Isolated rabbit aortic ring preparations with endothelium intact or removed by gentle abrasion were mounted in organ baths containing oxygenated Holman's solution, and cumulative concentration-response curves for relaxation to the contrast media were constructed after pre-constriction by phenylephrine (300 nM) in the presence of indomethacin to inhibit prostaglandin synthesis. Endothelial denudation did not influence the ability of either iodixanol or iopromide to relax the aortic ring preparations. Iopromide was significantly more potent than iodixanol when expressed in terms of iodine concentration (mg I ml-1), but both agents were equipotent when expressed in terms of molarity (mM). We conclude that relaxation of isolated rabbit aortic rings to iodixanol and iopromide under conditions where there is no fluid flow is endothelium-independent, and therefore not mediated by release of the potent endogeneous nitrovasodilator endothelium-derived relaxing factor (EDRF). Furthermore, their relaxant activity under the in vitro experimental conditions employed is attributable to a direct action on vascular smooth muscle by factors in addition to osmolality, and may depend on features that are not specifically associated with the presence of the iodine atom.

Growth factor signalling in endocrine and anti-growth factor resistant breast cancer.

Growth factors provide powerful mitogenic and survival signals to breast cancer cells and it is therefore not surprising that they are able to subvert inhibitory responses to anti-hormonal drugs. In this review we discuss several mechanisms by which this may be achieved and expand our observations to encompass recently emerging anti-growth factor treatments. The information presented is underpinned by inhibitor studies that show the targeting of such mechanisms in advance of anti-hormone or anti-growth factor resistance development is able to substantially delay this event, thus pointing the way forward to intelligent combination therapies relevant to the future management of breast cancer.

Bidirectional cross talk between ERalpha and EGFR signalling pathways regulates tamoxifen-resistant growth.

We have previously demonstrated that oestrogen receptor alpha (ERalpha) modulates epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signalling efficiency in a tamoxifen-resistant MCF-7 breast cancer cell line (Tam-R). In the present study we have investigated whether this cross-talk between EGFR/MAPK and ERalpha signalling pathways is bidirectional by examining the effects of EGFR/MAPK activity on ER functionality in the same cell line. Elevated expression levels of phosphorylated serine 118 (S118) ERalpha were observed in the Tam-R compared to the parental wild type MCF-7 cell line (WT-MCF-7) under basal growth conditions. Phosphorylation of ERalpha at S118 was regulated by the EGFR/MAPK pathway in Tam-R cells being increased in response to amphiregulin (AR) and inhibited by the selective EGFR tyrosine kinase inhibitor, gefitinib and the MEK1/2 inhibitor, PD184352. Recruitment of the co-activators p68 RNA helicase and SRC1 to ERalpha, oestrogen response element (ERE) activity and Tam-R cell growth were similarly EGFR/MAPK-regulated. Chromatin immunoprecipitation (ChIP) studies revealed that in Tam-R cells the ERalpha assembled on the AR gene promoter and this was associated with elevated basal expression of AR mRNA. Furthermore, AR mRNA expression was under the regulation of the EGFR/MAPK and ERalpha signalling pathways. Neutralising antibodies to AR inhibited EGFR/ERK1/2 activity, reduced S118 ERalpha phosphorylation and reduced AR mRNA expression in TAM-R cells. These findings suggest that ERalpha function in Tam-R cells is maintained as a consequence of EGFR/MAPK-mediated phosphorylation at serine residue 118 resulting in the generation of a self-propogating autocrine growth-regulatory loop through the ERalpha-mediated production of AR.

Release of endothelium-derived relaxing factor is modulated both by frequency and amplitude of pulsatile flow.

We have dissociated the effects of frequency and amplitude of pulsatile flow on flow-induced release of endothelium-derived relaxing factor (EDRF) using cascade bioassay. Rat aortic segments were buffer perfused with a peristaltic pump at a constant mean flow rate of 9 ml/min. EDRF activity in effluent was measured by relaxation of endothelium-denuded rabbit aortic rings preconstricted by phenylephrine. Pulse frequency was varied over the range 0.1-12 Hz at a constant amplitude of 2 mmHg; pulse amplitude was varied over the range 2-16 mmHg at a constant frequency of 0.1 Hz. Relaxation of the detector vessel depended on frequency of flow through the donor; peak response occurred between 4.2 and 6 Hz and was approximately three times greater than that induced at lower or higher frequencies. In contrast, increases in pulse pressure amplitude (maximum 16 mmHg) monotonically augmented constriction of partially preconstricted detector tissue by up to 10%. Incubation of the donor vessel with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, or removal of its endothelium by rubbing, abolished both the frequency- and the amplitude-dependent effects observed in the detector tissue, indicating that these were mediated by changes in EDRF release. Increasing the amplitude of the pressure pulse also reduced mean perfusion pressure (by up to 50%), implying distension of donor vessel since mean flow rate was constant. This fall in pressure was not affected by incubation with L-NAME or removal of endothelium, indicating that it was not dependent on EDRF activity.(ABSTRACT TRUNCATED AT 250 WORDS)