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.     

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.     

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.     

Relation of epidermal growth factor receptor concentration to growth of human esophageal cancer cell lines

The relation between the concentration of epidermal growth factor (EGF) receptor and the effects of EGF on cell proliferation were studied using 16 newly established human esophageal cancer cell lines. According to 125I-EGF binding assay, the amount of EGF receptor was found to vary from 6 x 10(4) to 1.2 x 10(7) (sites/cell). Changes in EGF-stimulated tyrosine-specific protein kinase activity almost paralleled changes in the number of EGF receptors per cell. Amplification of EGF receptor gene was detected in only one cell line. Under monolayer culture conditions, we found three types of growth responses of esophageal cell lines to EGF; growth in 5 cell lines was inhibited and that in 4 cell lines was stimulated while that in the other 7 cell lines remained unaffected. Relation was observed between the number of EGF receptors per cell and the growth response to EGF. On the other hand, cell lines whose growth was inhibited by EGF in monolayer culture were stimulated by EGF in soft agar culture, though the opposite was not necessarily true.     

Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli

Conventional chemotherapy produces varying degrees of response in patients with many advanced cancers and has significant side effects. Receptors for luteinizing hormone-releasing hormone (LH-RH) are present in a high percentage of human ovarian, prostatic, breast and endometrial tumors and targeted chemotherapy based on cytotoxic analogs of LH-RH might yield better results. The present study was undertaken to determine whether human cancer cell lines express mRNA for LH-RH receptors and epidermal growth factor (EGF) receptors. Using radioligand binding studies, we showed the presence of high-affinity binding sites for LH-RH and EGF in the membranes of human ovarian, prostatic, breast and endometrial cancer cell lines as well as in the JAR choriocarcinoma cell line. The expression of the mRNA for LH-RH receptors and EGF receptors in these cell lines was demonstrated by RT-PCR using specific primers and by subsequent Southern blot analysis. The PCR products obtained were of the expected size, 319 bp for LH-RH receptors and 400 bp for EGF receptors. These findings support the view that cytotoxic analogs of LH-RH could be used for targeted chemotherapy of these cancers. Moreover, the results suggest that these human cancer cell lines might have local regulatory systems for their proliferation based on LH-RH and EGF. Further investigations are required to elucidate the signal transduction pathways involved in the effects of cytotoxic LH-RH analogs on human tumors.     

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.     

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)     

Cloning and sequencing of the pyrG encoding cytidine 5''-triphosphate synthetase from Mycobacterium bovis BCG

Treatment of estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced formation of a nuclear aryl hydrocarbon (Ah) receptor complex as determined by ligand-binding and gel electrophoretic mobility shift assays. TCDD also induced CYP1A1-dependent activity in MDA-MB-468 cells, which represents the first ER-negative Ah receptor-positive human breast cancer cell line that has been identified. Treatment of this cell line with TCDD and related compounds also caused a 50% inhibition of cell growth, which resembled the growth inhibitory effects previously reported for epidermal growth factor (EGF). However, EGF expression is minimal in this cell line and is not induced by TCDD; moreover, EGF and TCDD induced a different pattern of oncogene expression and apoptosis in MDA-MB-468 cells. In contrast, TCDD caused a rapid and sustained induction of transforming growth factor alpha (TGF alpha) gene expression and secreted protein (nearly 2-fold); moreover, the growth-inhibitory effects of TCDD could be blocked by antibodies to the EGF receptor. In a separate experiment, it was shown that TGF alpha also inhibited growth of MDA-MB-468 cells. The results of this study indicate that the mechanism of growth inhibition of MDA-MB-468 cells by TCDD is due to induction of TGF alpha, which is a potent antimitogen in this cell breast cancer line.     

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.     

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.     

Peptide and lipid growth factors decrease cis-diamminedichloroplatinum-induced cell death in human ovarian cancer cells

Growth factors have been demonstrated to regulate the proliferation and viability of a number of cell lineages. Because most drugs used in chemotherapy kill cells through programmed cell death, by the process of apoptosis, we determined whether growth factors, specifically epidermal growth factor (EGF) and lysophosphatidic acid (LPA), which we have demonstrated recently to be a potent growth factor for ovarian cancer cells, would alter the ability of cis-diamminedichloroplatinum (cis-DDP), the most effective chemotherapeutic agent for ovarian cancer, to kill the HEY ovarian cancer cell line. We demonstrate that both EGF and LPA decrease the ability of cis-DDP to kill HEY ovarian cancer cells as assessed by colony-forming cell activity and dye reduction. Morphological changes, DNA release, and electron microscopy suggested that LPA and EGF protect ovarian cancer cells from programmed cell death induced by cis-DDP. Because LPA is present in high levels in ascitic fluid from ovarian cancer patients, and the EGF receptor is expressed by tumor cells from a significant portion of patients where it correlates with prognosis, growth factor modulation of cis-DDP-induced apoptosis may play a role in the poor prognosis associated with ovarian cancer.     

Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis

BACKGROUND: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro. METHODS: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgen-independent), and LNCaP (androgen-dependent). We studied the expression of the HGF receptor c-met proto-oncogene (c-met) by Western blot analysis, and also determined the effects of HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay. RESULTS: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells. CONCLUSION: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Differential inhibition by hyperammonemia of the electron transport chain enzymes in synaptosomes and non-synaptic mitochondria in ornithine transcarbamylase-deficient spf-mice: restoration by acetyl-L-carnitine

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

Interleukin 6 acts as a paracrine growth factor in human mammary carcinoma cell lines

The effect of interleukin 6 (IL-6) on normal and human mammary carcinoma epithelial cells was studied. IL-6 inhibited the growth of estrogen receptor-positive [ER(+)] breast cancer cell lines, which underwent apoptosis with prolonged treatment. In contrast, ER(-) breast cancer cell lines were resistant to IL-6-mediated growth inhibition. By examining the components of the IL-6 receptor (IL-6R) system, we found that ER(+) breast cancer cells expressed predominantly soluble IL-6Ralpha, whereas the ER(-) breast cancer cells expressed primarily the transmembrane form of the IL-6R, gp130. In addition, detectable levels of IL-6 were secreted into the medium by ER(-) but not ER(+) breast cancer cells. Furthermore, the supernatant obtained from IL-6-secreting, ER(-) cells suppressed the growth of IL-6-sensitive, ER(+) breast cancer cells in a paracrine fashion. Although IL-6 is secreted by ER(-) breast cancer cells, this cytokine does not seem to stimulate the proliferation of these cells in an autocrine fashion. These studies indicate that IL-6 can regulate the growth of normal and transformed human mammary epithelial cells differentially, and that IL-6 secretion by some ER(-) breast cancer cells can function as a paracrine growth factor, suppressing the growth of ER(+) breast cancer cells in vitro.     

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.     

Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells

Several independent lines of evidence have implicated decorin, a small leucine-rich proteoglycan, in the inhibition of cell proliferation. However, the mechanism by which decorin mediates its effect on cell proliferation is unclear. Here we report, for the first time, decorin-mediated increases in intracellular Ca2+ levels of single A431 cells. The effects of decorin persisted in the absence of extracellular Ca2+ but were blocked by AG1478, an epidermal growth factor (EGF)-specific tyrosine kinase inhibitor, and by down-regulation of the EGF receptor. The effects of decorin were not mimicked by the structurally homologous protein, biglycan. Our results indicate a novel action of decorin on the EGF receptor, which results in mobilization of intracellular Ca2+ providing a possible mechanism by which decorin causes growth suppression.     

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.