Epidermal Growth Factor Receptor Blockers Research Articles

Effects of benzo- a-pyrene on oxytocin-induced Ca 2+ oscillations in myometrial cells

Benzo- a-pyrene (BaP) is a polycyclic aromatic hydrocarbon that exists as a major environmental pollutant. The effect of this carcinogen/mutagen upon myometrial Ca 2+ signaling in a human myometrial cell line (PHM1) was examined. Exposure of cells to BaP did not alter basal Ca 2+ levels or the inositol(1,4,5) trisphosphate-releasable Ca 2+ pool. However, BaP significantly decreased the initial oxytocin-induced Ca 2+ transient and the frequency of oxytocin-induced Ca 2+oscillations as well as delayed their onset. To determine the specific effects of BaP, pharmacologic agents that target intracellular Ca 2+ homeostasis mechanisms were used. Genistein (a non-specific tyrosine kinase inhibitor) and AG1478 (an epidermal growth factor receptor blocker) markedly reduced the oxytocin-induced Ca 2+ oscillations in control, but had no effect in BaP treated cells. Addition of epidermal growth factor or serum before or after oxytocin restored the Ca 2+ oscillations in BaP treated cells to a level similar to control cells, while the K + channel blocker tetraethylammonium chloride, partially restored the Ca 2+ response. These data suggest that the tyrosine kinase pathway, which is part of the G-protein coupled receptor pathway response to oxytocin in PHM1 cells, is a target of BaP action and that EGF or serum can restore the oxytocin-induced Ca 2+ oscillations.

Clinical experience with erlotinib in non-small-cell lung cancer (NSCLC)

Lung cancer is the leading cause of cancer death worldwide. Despite the introduction of more- effective chemotherapeutic agents, it appears that a survival plateau has been reached, so new treatment strategies are clearly needed. One innovative therapeutic cancer strategy is the introduction of biological agents that target specific intracellular pathways related to the distinctive properties of cancer cells. Among these agents, epidermal growth factor receptor (EGFR)-targeting agents have received particular attention in lung cancer. Numerous EGFR blockers have been evaluated, including monoclonal antibodies to the receptor and small-molecule tyrosine kinase inhibitors. The present review focuses on the tyrosine kinase inhibitor erlotinib. Preclinical studies have shown that erlotinib blocks the growth of human non-small-cell lung cancer (NSCLC) cell lines in vitro by inhibiting the receptor and the downstream protein phosphorylation. In a randomized study conducted by the National Cancer Institute of Canada (BR.21) in second- and third-line NSCLC treatment, erlotinib significantly prolonged overall survival and decreased symptoms compared with placebo. A crucial aspect of the clinical development of molecular-targeted therapies is to understand which patients will obtain clinical benefit from their use. Sensitivity to erlotinib has been associated with EGFR mutations, most commonly deletions of four to six amino acids in exon 19 or a point mutation (L858R) in exon 21. Increased EGFR gene copy number has also been pointed out as a good predictive marker for erlotinib response. Intense research activity is ongoing to validate known predictive markers and to discover new tools which maximize clinical benefit using erlotinib. However, there is no conclusive evidence, as yet, linking response to survival.

Simultaneous targeting of the epidermal growth factor receptor and cyclooxygenase-2 pathways for pancreatic cancer therapy

The aims of this study were to determine the effects of (a) combining the epidermal growth factor receptor (EGFR) blocker (erlotinib) and the cyclooxygenase-2 inhibitor (celecoxib) on cell growth and apoptosis in human pancreatic cancer cell lines, (b) baseline EGFR expression on the potentiation of erlotinib-induced apoptosis by celecoxib, and (c) the effects of the combination on the expression of the COX-2, EGFR, HER-2/neu, and nuclear factor-kappaB (NF-kappaB). Baseline expression of EGFR was determined by Western blot analysis in five human pancreatic cancer cell lines. BxPC-3, PANC-1, and HPAC had high EGFR and MIAPaCa had low EGFR. Cells were grown in culture and treated with erlotinib (1 and 10 micromol/L), celecoxib (1 and 10 micromol/L), and the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. Reverse transcriptase-PCR was used to evaluate COX-2 and EGFR mRNA. EGFR, COX-2, and HER-2/neu expression was determined by Western immunoblotting. Electrophoretic mobility shift assay was used to evaluate NF-kappaB activation. Growth inhibition and apoptosis were significantly (P < 0.05) higher in BxPC-3, HPAC, and PANC-1 cells treated with celecoxib and erlotinib than cells treated with either celecoxib or erlotinib. However, no potentiation in growth inhibition or apoptosis was observed in the MIAPaCa cell line with low expression of the EGFR. Significant down-regulation of COX-2 and EGFR expression was observed in the BxPC-3 and HPAC cells treated with the combination of erlotinib (1 micromol/L) and celecoxib (10 micromol/L) compared with celecoxib- or erlotinib-treated cells. Celecoxib significantly down-regulated HER-2/neu expression in BxPC-3 and HPAC cell lines. Significant inhibition of NF-kappaB activation was observed in BxPC-3 and HPAC cell lines treated with erlotinib and celecoxib. (a) Celecoxib can potentiate erlotinib-induced growth inhibition and apoptosis in pancreatic cell lines, (b) high baseline EGFR expression is a predictor of this potentiation, and (c) the down-regulation of EGFR, COX-2, and HER-2/neu expression and NF-kappaB inactivation contributes to the potentiation of erlotinib by celecoxib.

Inhibition of angiogenesis in cancer patients

Treatment with antiangiogenic agents as standard anticancer therapy with or without classical chemotherapy is rapidly approaching. The clinical efficacy of bevacizumab in colorectal cancer in combination with chemotherapy caused a revival of the antiangiogenic strategy. By combining this agent with a tyrosine kinase receptor epidermal growth factor receptor blocker (erlotinib), remarkable responses were seen in renal cell cancer. It has been thought that blocking these biological pathways would cause no drug-related toxicity, but a whole new pattern of relatively mild side effects compared with classical chemotherapy, including skin rash, fatigue and hypertension, has been observed. In combination with chemotherapy, other serious side effects, such as bleeding and thrombosis, also occur. Here, the preclinical and clinical data of antiangiogenic agents in clinical trials at this moment are summarised.

Epidermal Growth Factor Receptor Inhibition by Tyrphostin 51 Induces Apoptosis in Luteinized Granulosa Cells

Growth factors may be involved in the control of ovarian cell fate and could contribute to regulation of ovarian cell apoptosis. Our objective is to test the hypothesis that, in human luteinized granulosa cells, epidermal growth factor (EGF) works through the MAPK signaling pathway and inhibition of EGF receptor by a specific tyrosine kinase inhibitor, tyrphostin 51, will inhibit the activation of MAPK and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were treated as follows: 1) vehicle (dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. Blockage of EGF receptor by tyrphostin 51 reduced the MAPK activity and inhibited phosphorylation and nuclear translocation of activated MAPK. Blockage of EGF receptor also induced apoptosis as demonstrated by the activation of caspase-3, an executioner protease of the apoptotic pathway, and by an increased percentage of subdiploid apoptotic nuclei. These results support the hypothesis that in human luteinized granulosa cells, EGF works through the MAPK signaling pathway and that its inhibition by tyrphostin 51 inhibits MAPK phosphorylation and induces apoptotic nuclear changes. Our data thus provide additional information regarding regulation of apoptosis in luteinized granulosa cells.

Antiproliferative activity of new 1-aryl-4-amino-1 H-pyrazolo[3,4- d]pyrimidine derivatives toward the human epidermoid carcinoma A431 cell line

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1 H-pyrazolo[3,4- d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor (EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2.

Phase III evaluation of radiation with and without cetuximab for locoregionally advanced head and neck cancer

Phase III evaluation of radiation with and without cetuximab for locoregionally advanced head and neck cancer

Epidermal Growth Factor Receptor Inhibition Promotes Desmosome Assembly and Strengthens Intercellular Adhesion in Squamous Cell Carcinoma Cells

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.

Interleukin-1beta-induced mucin production in human airway epithelium is mediated by cyclooxygenase-2, prostaglandin E2 receptors, and cyclic AMP-protein kinase A signaling.

We reported recently that interleukin (IL)-1beta exposure resulted in a prolonged increase in MUC5AC mucin production in normal, well differentiated, human tracheobronchial epithelial (NHTBE) cell cultures, without significantly increasing MUC5AC mRNA (Am J Physiol 286:L320-L330, 2004). The goal of the present study was to elucidate the signaling pathways involved in IL-1beta-induced MUC5AC production. We found that IL-1beta increased cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin (PG) E(2) production and that the COX-2 inhibitor celecoxib suppressed IL-1beta-induced MUC5AC production. Addition of exogenous PGE(2) to NHTBE cultures also increased MUC5AC production and IL-1beta-induced Muc5ac hypersecretion in tracheas from wild-type but not from COX-2-/- mice. NHTBE cells expressed all four E-prostanoid (EP) receptor subtypes and misoprostol, an EP2 and EP4 agonist, increased MUC5AC production, whereas sulprostone, an EP1 and EP3 agonist, did not. Furthermore, specific protein kinase A (PKA) inhibitors blocked IL-1beta and PGE(2)-induced MUC5AC production. However, neither inhibition of epidermal growth factor receptor (EGFR) activation with the tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline HCl (AG-1478) or EGFR blocking antibody nor inhibition of extracellular signal-regulated kinase/P-38 mitogen activated protein kinases with specific inhibitors blocked IL-1beta stimulation of MUC5AC mucin production. We also observed that tumor necrosis factor (TNF)-alpha, platelet activating factor (PAF), and lipopolysaccharide (LPS) induced COX-2 and increased MUC5AC production that was blocked by celecoxib, suggesting a common signaling pathway of inflammatory mediator-induced MUC5AC production in NHTBE cells. We conclude that the induction of MUC5AC by IL-1beta, TNF-alpha, PAF, and LPS involves COX-2- generated PGE(2), activation of EP2 and/or EP4 receptor(s), and cAMP-PKA-mediated signaling.

Cetuximab prolongs survival in patients with locoregionally advanced squamous cell carcinoma of head and neck: A phase III study of high dose radiation therapy with or without cetuximab

5507 Background: The majority of head and neck cancers overexpress the epidermal growth factor receptor (EGFR), which is associated with aggressive tumor behavior and poor clinical outcome. Preclinical in vitro and in vivo model systems demonstrate radiosensitization following molecular inhibition of EGFR signaling. Promising phase I/II clinical results were achieved when radiation was combined with the EGFR blocking antibody, cetuximab, in patients with locoregionally advanced squamous cell carcinoma (SCC) of the head and neck (H&N). This phase III international trial was designed to examine the impact of combining cetuximab with high dose radiation on locoregional disease control and survival in patients with locally advanced H&N cancer. Methods: Patients with locoregionally advanced SCC of the oropharynx, hypopharynx or larynx with measurable disease were eligible to enroll. Patients were stratified by Karnofsky score 60–80 vs. 90–100, node positive vs. negative, T1–3 vs. T4, radiation fractionation regimen (once-a-day vs. twice-a-day vs. concomitant boost), and were randomized to receive either radiation alone for 6–7 weeks, or radiation plus weekly cetuximab. Following completion of treatment, patients were followed by physical examination and radiographic imaging every 4 months for 2 years, and then every 6 months up to 5 years. Results: From 04/99 to 03/02, 424 patients were randomized on this trial in the USA (61%), Europe (28%), and other countries. Median age was 57; 80% were male; 69% had KPS 90–100; 60% had oropharyngeal, 25% laryngeal, and 15% hypopharyngeal primary tumors. The two treatment arms were well balanced with regard to patient characteristics and stratification factors. The two arms were also well balanced with respect to radiation dose and fractions received, use of post-radiotherapy neck dissection, and secondary cancer therapy. Minimum and median durations of follow-up were 24 and 38 months, respectively. Median survival times, two and three-year survival rates derive from Kaplan-Meier estimates. Survival and selected safety results are as follows: The overall toxicity profile was dominated by classic known effects of high dose H&N radiation, although some additional toxicity was attributed to cetuximab. Locoregional control assessment is ongoing. Conclusions: The addition of cetuximab to high dose radiation in patients with locoregionally advanced SCC of H&N demonstrated a statistically significant prolongation in overall survival. This clinical benefit was achieved with minimal enhancement in the overall toxicity profile associated with curative-intent radiation therapy. These results corroborate the promising preclinical and earlier phase clinical results supporting the use of EGFR blockade in combination with radiation for advanced SCC of H&N. Furthermore, this combination treatment should be explored in other common epithelial malignancies demonstrating EGFR overexpression. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ImClone Systems ImClone Systems ImClone Systems ImClone Systems

The AG1478 tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth.

Uterine leiomyomas are the most common benign smooth muscle cell tumours in women. Formation of leiomyomas, still not completely understood, is viewed as a multistep process, with involvement of ovarian steroid hormones, cytokines and growth factors. Our study aimed to identify tyrosine kinase inhibitors as potential 'signal transduction therapeutics' for leiomyomas, underlying the effect of ovarian steroidal hormones. The selective epidermal growth factor (EGF) receptor blocker AG1478 was evaluated as a potential target, since EGF has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth. Paired cultures of leiomyoma and normal myometrium samples were established and the suppressive effect of AG1478 on the cells prior and subsequent to steroidal hormone treatment was examined: cell proliferation, recovery after treatment, cell cycle analysis and immunochemical analysis of relevant proteins. Leiomyoma cell growth is effectively blocked by AG1478 and is unaffected by the presence of physiological concentrations of progesterone and estradiol. AG1478 (10 microM) completely suppressed proliferation and the cells did not recover after cessation of treatment. The growth-arresting properties of AG1478, unaffected by ovarian steroidal hormones, identify it as a potential lead agent for the non-surgical management of uterine leiomyomas.

Signaling and distribution of NPR-Bi, the human splice form of the natriuretic peptide receptor type B.

Recently, we described a splice variant of the human natriuretic peptide receptor type B (NPR-Bi) in human proximal tubule cells [immortalized human kidney epithelial cells (IHKE-1) that lacks a functional guanylate cyclase domain (Hirsch JR, Meyer M, Mägert HJ, Forssmann WG, Mollerup S, Herter P, Weber G, Cermak R, Ankorina-Stark I, Schlatter E, and Kruhøffer M. J Am Soc Nephrol 10: 472-480, 1999). Its signaling pathway does not include cGMP, cAMP, or Ca2+ but leads to inhibition of K+ channels. In patch-clamp experiments, effects of tyrosine kinase receptor blockers on C-type natriuretic peptide (CNP)-mediated depolarizations of membrane voltages (Vm) of IHKE-1 cells were tested. The epidermal growth factor (EGF) receptor blocker genistein (10 microM) abolished the effect of CNP (0.2 +/- 0.4 mV, n = 7), and comparable results were obtained with 10 microM daidzein (n = 8). Aminogenistein (10 microM, n = 5) and tyrphostin AG1295 (10 microM, n = 5) had no significant effects. EGF (1 nM) hyperpolarized cells by -5.3 +/- 0.8 mV (n = 5). This effect was completely blocked by genistein or daidzein. The Cl- channel blocker NPPB (10 microM, n = 5) inhibited the EGF-mediated hyperpolarization. mRNA expression of NPR-B and NPR-Bi shows reversed patterns along the human nephron. NPR-B is highly expressed in glomeruli and proximal tubules, whereas NPR-Bi shows strong signals in the distal nephron. Expression of NPR-Bi in the cortical collecting duct was also confirmed with immunohistochemistry. In other human tissues, NPR-Bi shows strongest expression in pancreas and lung, whereas in the heart and liver NPR-B is the dominating receptor. In conclusion, CNP inhibits an apical K+ channel in IHKE-1 cells independently of cGMP and so far this effect can only be blocked by genistein and daidzein. Tyrosine phosphorylation might be the missing link in the signaling pathway of CNP/NPR-Bi.

Novel therapeutics for head and neck cancer.

Head and neck squamous cell carcinoma is a clinically challenging disease that resulted in more than 500,000 cases worldwide in 2001. In the United States, head and neck squamous cell carcinoma has accounted for approximately 40,000 cases with 12,000 deaths reported in 2001, which makes it the fifth leading cause of cancer incidence and the sixth leading cause of cancer-related deaths. Patients with recurrent or metastatic disease have a median survival rate of approximately 6 months; and there is little evidence from randomized trials that survival advantages have been achieved over the community standard of cisplatin and infusional 5-fluorouracil combination. Despite significant improvements in diagnosis, local management, and chemotherapy of head and neck cancer, there has been no significant increase in long-term survival rates over the past 30 years. As little progress has been made, new management approaches are required. Novel biologic agents have been developed to target multiple specific regions of the cancer cell. Epidermal growth factor receptor blockers have been investigated, such as anti epidermal growth factor receptor monoclonal antibodies, tyrosine kinase inhibitors, ligand conjugates, immunoconjugates, and antisense oligonucleotides. Farnesyl transferase inhibitors are a class of compounds that inhibit a critical enzymatic step in the constitutive expression of mutated ras genes, which are present in more than 27% of oral cancers. Strategies targeting the p53 gene and protein may halt or reverse the process of tumorigenesis and metastasis as p53 mutations occur in 45 to 70% of head and neck squamous cell carcinoma patients. Continued development of these novel agents may help improve the overall response and survival rate of patients with head and neck cancer.

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor.

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.

Expression of TGFalpha autocrine activity in human colon carcinoma CBS cells is autoregulated and independent of exogenous epidermal growth factor.

Autocrine transforming growth factor alpha (TGFalpha) activity and control mechanisms for its expression were examined in a representative clonal isolate (CBS4) of a well-differentiated human colon carcinoma cell line designated CBS. CBS4 cells expressed TGFalpha and its receptor, epidermal growth factor receptor (EGFr). Blockade of EGFr and TGFalpha by neutralizing antibodies inhibited clonal growth and the initiation of DNA synthesis from quiescence in CBS4 cells. Therefore, TGFalpha is an autocrine growth factor for CBS4 cells. Several studies have indicated that activation of the EGFr by exogenous EGF stimulates TGFalpha expression. However, in CBS4 cells EGF did not induce TGFalpha mRNA expression, indicating that EGF does not affect TGFalpha transcription in these cells. Exogenous treatment of exponentially growing cells with either EGF or EGFr blocking antibody enhanced release of TGFalpha protein into the conditioned medium. This indicated that the release of TGFalpha into the conditioned medium by exogenous EGF was at least partially due to the displacement of TGFalpha from the TGFalpha/EGFr complexes. Similarly to exponentially growing cells, the EGFr blocking antibody and EGF also enhanced TGFalpha release into the medium of CBS4 cells after release from quiescence. These results indicated that exogenous EGF had little if any effect on TGFalpha expression in these cells and suggested that TGFalpha expression might be under endogenous TGFalpha control. Blockade of the autocrine TGFalpha loop by TGFalpha neutralizing antibody suppressed TGFalpha mRNA both in exponentially growing and quiescent cells, demonstrating that autocrine TGFalpha is autoregulatory in this system.

Inhibition of tyrosine kinase activity decreases expression of surfactant protein A in a human lung adenocarcinoma cell line independent of epidermal growth factor receptor

Epidermal growth factor (EGF) enhances fetal lung development in vivo and in vitro. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase initiating a signal transduction cascade. We hypothesized that blocking EGF receptor function with tyrosine kinase inhibitors would decrease the expression of surfactant protein A in human pulmonary epithelial cells. Human pulmonary adenocarcinoma cells (NCI-H441) were exposed to genistein (a broad range inhibitor of tyrosine kinases) and tyrphostin AG1478 (a specific inhibitor of EGF receptor tyrosine kinase). Genistein significantly decreased surfactant protein A (SP-A) and SP-A mRNA levels in H441 cells without affecting cell viability. The inhibitory effect of genistein on SP-A content was reversible. In contrast, tyrphostin AG1478 had no effect on SP-A levels despite a greater inhibitory effect than genistein on EGF receptor tyrosine autophosphorylation. Furthermore, treatment of H441 cells with exogenous EGF did not increase SP-A content or mRNA levels beyond baseline. We conclude that inhibition of tyrosine kinase activity other than the EGF receptor decreases the expression of surfactant protein A at a pretranslational level in human pulmonary adenocarcinoma cells. These results suggest the importance of tyrosine kinases in modulating human SP-A synthesis.

Relative expression of epidermal growth factor receptor in placental cytotrophoblasts and choriocarcinoma cell lines

The role of transforming growth factor-α (TGF-α)-epidermal growth factor receptor (EGFR) interactions in regulating benign and malignant trophoblast proliferation were examined. Benign cytotrophoblast (CT) demonstrated mitogenic stimulation in response to TGF-α; BeWo and JAr choriocarcinoma cell lines failed to respond. EGFR levels in BeWo and JAr were determined by enzyme linked immunoassay (ELISA) to be at least 10-fold higher than those in benign CT. EGFR isolated from BeWo and JAr also demonstrated functional tyrosine kinase activity. Using a combination of immunoperoxidase (IP) and ELISA techniques, choriocarcinoma cells were found to produce significant quantities of TGF-α that were comparable with those reported previously by this laboratory for benign CT, and were felt to be stimulating their own proliferation in an autocrine fashion. EGFR blocking and TGF-α neutralizing antibodies inhibited JAr proliferation whereas an EGF neutralizing antibody did not. The data presented here and in our previous report indicate that a TGF-α-EGFR autocrine loop may regulate normal and malignant CT proliferation. Choriocarcinoma cells may be proliferating at a maximal rate due, in part, to EGFR overexpression and are therefore unable to respond further to exogenous growth factor. Thus, EGFR overexpression may contribute to the uncontrolled proliferation of choriocarcinoma cells in general.

Regulation of estrogen receptor concentration and activity by an erbB/HER ligand in breast carcinoma cell lines.

Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.