The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila

Overexpression of many growth factor receptors, as well as growth factors, has been shown to confer varying degrees of estrogen-independent growth on estrogen receptor (ER) positive breast cancer cells. The proto-oncogene Raf-1 is a key intermediate in the signal transduction pathway of many of these growth factor receptors, and when constitutively activated in fibroblasts is transforming. To examine the effects of Raf-1 kinase activity on the estrogen-dependent growth of human breast cancer cells, ER + MCF-7 breast cancer cells were stably transfected with an expression construct directing the expression of an amino-truncated protein having constitutive kinase activity. Expression of constitutively activated Raf in MCF-7 cells is incompatible with growth in the presence of estrogen; that is, cells down-regulate expression of the transfected Raf. Constitutive Raf activity does allow for growth of the cells in the absence of estrogen, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions. In addition, the high levels of Raf activity induce apoptosis in cells grown under either condition. This is a novel activity for Raf, and may occur because the levels of the constitutive Raf are extremely high in these cells.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin-like growth factor I receptor (IGF-I R), erbB-type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF-7. To evaluate the function of 2 post-receptor signaling molecules, insulin receptor substrate-1 (IRS-1) (a major substrate of the IGF-IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF-7-derived cell clones in which the synthesis of either IRS-1 or SHC was blocked by antisense RNA. In MCF-7 cells, down-regulation of IRS-1 by 80-85% strongly suppressed anchorage-dependent and -independent growth and induced apoptotic cell death under growth factor- and estrogen-reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage-independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF-7/anti-SHC clones, but not in MCF-7/anti-IRS-1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF-7/anti-SHC clones. Our results suggest that in MCF-7 cells: 1) both IRS-1 and SHC are implicated in the control of monolayer and anchorage-independent growth; 2) IRS-1 is critical to support cell survival; 3) SHC is involved in EGF-dependent motility; and 4) normal levels of SHC, but not IRS-1, are necessary for the formation and maintenance of cell-cell interactions.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.     

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)     

Excessive activation of tyrosine kinases leads to inhibition of proliferation in a thyroid carcinoma cell line

Autocrine stimulation of growth is a hallmark of many tumor cell lines. In this work we investigated the synthesis and secretion of growth factors and the expression of their corresponding receptors in HTC-TSHr thyroid carcinoma cells. These cells synthesize epidermal growth factor (EGF) receptors and platelet-derived growth factor beta (PDGF beta) receptors and in addition transforming growth factor alpha (TGF alpha), PDGF-A and PDGF-B chains, respectively. Addition of EGF or PDGF-BB to the culture medium resulted in growth inhibition of HTC-TSHr cells. In contrast, treatment of the cells with low concentrations of neutralizing anti-TGF alpha antibodies or tyrosine kinase inhibitors led to stimulation of cell proliferation. Low concentrations of neutralizing anti-PDGF-B antibodies did not affect growth of the cells. As expected, cell proliferation was inhibited when high concentrations of either neutralizing anti-TGF alpha antibodies or anti-PDGF-B antibodies were applied. PDGF-AA did not influence growth of HTC-TSHr cells. We conclude that growth of HTC-TSHr thyroid carcinoma cells is influenced by two autocrine loops between TGF alpha and EGF receptors and between PDGF-B and PDGF beta receptors. However, our data suggest that excessive activation of tyrosine kinase receptors in these cells results in a relative inhibition rather than stimulation of growth.     

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.     

Cell type-specific modulation of cell growth by transforming growth factor beta 1 does not correlate with mitogen-activated protein kinase activation

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta 1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta 1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta 1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta 1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta 1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta 1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.     

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.     

Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation

Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.     

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.     

Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.     

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP-6 is involved in numerous developmental processes. We have demonstrated expression of BMP-6 in breast cancer cell lines by RT-PCR and immuno-histochemistry. The level of BMP-6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP-6 mRNA levels in a dose-dependent manner, with a maximum at 50 ng/ml EGF under serum-free conditions in hormone-sensitive (MCF-7) and in hormone-insensitive (SK-BR-3) breast cancer cell lines. The EGF-like growth factors transforming growth factor-alpha, amphiregulin and betacellulin were also able to elevate the BMP-6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG 1517 repressed the inductive effect of these growth factors, indicating an EGF receptor-mediated regulation of BMP-6 mRNA. In addition, BMP-6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor-free resection margins. In 12 of these 18 patients, at least a 10-fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor-free samples was observed. This suggests a putative relationship between EGF receptor and BMP-6 mRNA levels in breast cancer.     

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.     

Stearate inhibition of breast cancer cell proliferation. A mechanism involving epidermal growth factor receptor and G-proteins

Long chain saturated fatty acids are known to inhibit breast cancer cell proliferation; however, the mechanism of this inhibition is not known. Treatment of Hs578T breast cancer cells with long chain saturated fatty acids (0.15 mmol/L for 6 hours) before epidermal growth factor (EGF) treatment inhibited EGF-induced cell proliferation in a chain-length-dependent manner. Stearate (C:18) completely inhibited the EGF-induced cell proliferation, whereas palmitate (C:16) inhibited by 67 +/- 8% and myristate (C:14) had no effect. In contrast, stearate had little effect on insulin-like growth factor-1-stimulated cell proliferation. The inhibitory effect of stearate on cell proliferation was dose and time dependent and independent of EGF receptor (EGFR) tyrosine phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 24 hours) inhibited the EGF-induced cell growth by 50 +/- 8%, also independent of EGFR tyrosine phosphorylation. A pertussis-toxin-sensitive, 41-kd G-protein was specifically co-immunoprecipitated with the EGFR. Pretreatment of cells with 0.15 mmol/L stearate from 0 to 6 hours inhibits, in parallel, both the EGF-induced cell proliferation and pertussis-toxin-catalyzed ADP ribosylation of the G-protein associated with the EGFR. These studies suggest that long chain saturated fatty acids inhibit EGF-induced breast cancer cell growth via a mechanism involving an EGFR-G-protein signaling pathway.     

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.     

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.