Induce Receptor Down-regulation Research Articles

Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT2A-selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that β-arrestin-biased 5-HT2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.

Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin \u03b13 in human pancreatic cancer

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26.

MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.

Phase I dose-escalation trial of Sym004, an anti-EGFR antibody mixture, in Japanese patients with advanced solidtumors.

Sym004 is a 1:1 mixture of two antibodies targeting non‐overlapping epitopes of the epidermal growth factor receptor that antagonizes ligand binding and induces receptor downregulation. In preclinical models, it has superior antitumor activity to cetuximab and panitumumab. Japanese adults aged ≥20 years with an Eastern Cooperative Oncology Group status of 0/1 and life expectancy ≥3 months were eligible. Patients in Part A (dose escalation) had refractory or recurrent late‐stage solid tumors and received Sym004 6 mg/kg/wk (n = 3), 9 mg/kg loading/6 mg/kg/wk (n = 6), 12 mg/kg/wk (n = 6), or 18 mg/kg biweekly (n = 6). Patients in expansion Part B (n = 30) had esophageal squamous cell carcinoma and received Sym004 at the dose recommended from Part A. Fifty‐one patients received Sym004. No dose‐limiting toxicities were observed in Part A. A dose of 12 mg/kg/wk was selected for Part B. All patients in Part B experienced treatment‐related adverse events, most commonly dermatitis acneiform (76.7%). Eighteen grade ≥3 treatment‐related adverse events and five serious adverse events occurred (cardiac arrest, lung infection, interstitial lung disease, toxic skin eruption, blood creatinine increase). Two patients had treatment‐related adverse events resulting in death (cardiac arrest and blood creatinine increase). Five patients in Part B had a best overall response of partial response, 12 stable diseases and 12 disease progression (1 not evaluable). The objective response rate was 16.7% (95% CI: 5.6%‐34.7%). Sym004 therapy was well tolerated with no dose‐limiting toxicities at any dose studied. Evidence of antitumor activity was seen in patients with esophageal squamous cell carcinoma. ClinicalTrials.gov Identifier: NCT01955473.

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

Acinar-to-ductal metaplasia (ADM) results from pancreatic injury or KRAS activation, and is an early step in pancreatic cancer progression. In this issue of Cancer Cell, Ardito and colleagues and Navas and colleagues demonstrate that ADM- and KRAS-driven pancreatic cancer require EGFR signaling, revealing a mechanism for developmental reprogramming that primes tumorigenesis.

Antagonist Functional Selectivity: 5-HT2A Serotonin Receptor Antagonists Differentially Regulate 5-HT2A Receptor Protein Level In Vivo

Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

Endogenous Parathyroid Hormone-Related Protein Regulates the Expression of PTH Type 1 Receptor and Proliferation of Vascular Smooth Muscle Cells

The PTH type 1 receptor (PTH1R) and PTHrP are expressed in vessels, where they contribute to regulating vascular smooth muscle cell (VSMC) function. Elevated PTHrP levels in VSMC are often associated with hyperplasia. In contrast, exogenous PTHrP, acting through the PTH1R, inhibits VSMC proliferation. In this study, we investigated the regulation of PTH1R expression by endogenous PTHrP and the associated effects on VSMC proliferation. Blocking binding of secreted PTHrP fragments to the PTH1R by treatment with either an antagonist or an antibody against PTHrP, and inhibition of PTHrP expression by small interfering RNA significantly increased PTH1R expression. Interestingly, treatment of the cells with a PTHrP analog (Bpa(1)-PTHrP) that activates the PTH1R without inducing its internalization had the same effect on receptor expression. To examine the association between receptor expression and the antiproliferative effect of N-terminal fragments of PTHrP, VSMC were treated with exogenous PTHrP (1-36) acutely and chronically to induce receptor down-regulation. Stimulation of VSMC with exogenous PTHrP (1-36) significantly reduced cell proliferation during the first 18 h of treatment but was no longer effective after 3 d, a time when PTH1R was down-regulated. In contrast, treatment with the noninternalizing agonist Bpa(1)-PTHrP strongly inhibited cell proliferation at all time points. In conclusion, our study show that PTHrP, after its intracellular processing and secretion, promotes down-regulation of the PTH1R in VSMC, thereby regulating cell proliferation in an auto/paracrine fashion. This regulatory mechanism may have important implication during vascular remodeling, in particular in the development of neointima after arterial injury, where PTHrP overexpression occurs.

Effect of AMG 479 on anti-tumor effects of gemcitabine and erlotinib against pancreatic carcinoma xenograft models

4617 Background: Binding of IGF-1 or IGF-2 to the type I insulin-like growth factor receptor (IGF-1R) activates intracellular signals important in the growth and survival of pancreatic tumors. AMG 479 is a fully human monoclonal antibody against IGF-1R currently in phase II clinical trials. AMG 479 exerts its anti-tumor activity by blocking ligand binding and inducing receptor downregulation in vivo. Methods: IGF-1R and Insulin Receptor (IR) levels were determined using FACS analysis or Meso Scale Detection (MSD). Level of IGF-1R and AKT phosphorylation were measured using MSD. Animal experiments were performed by injecting 5 million MiaPaCa or BxPC-3 cells subcutaneously into female athymic nude mice. Treatment began when xenografts (n=10/group) were approximately 200 mm3 and continued for the length of the experiment. AMG 479 and gemcitabine were dosed 2x/week intraperitoneally. Erlotinib was dosed orally every day. Repeated measures ANOVA was used to compare the efficacy of the treatment groups. Results: The ratios of IGF-1R to IR were 60:1 and 1.5:1 in the BxPC-3 and MiaPaCa cell lines, respectively. In vitro, AMG 479 blocked IGF-1 induced IGF-1R and AKT activation in both cell lines. In vivo, AMG 479 blocked IGF-1 induced IGF-1R activation in both cell lines but could only block AKT activation in the BxPC-3 cell line. Dose- dependent tumor growth inhibition by AMG 479 was observed in both human pancreatic carcinoma xenograft models. Maximal inhibition (70%) was achieved with a 300 μg/dose, 2x/week schedule. Analysis of tumor xenografts following AMG 479 treatment revealed significant down- regulation of IGF-1R expression (>60%). Since the survival signals driven by IGF-1R have been shown to convey resistance to traditional therapies, we tested the ability of AMG 479 to enhance the efficacy of gemcitabine or erlotinib in vivo. Both gemcitabine and erlotinib were significantly more effective in combination with AMG 479. Furthermore, the triple combination of optimal doses of AMG 479, erlotinib and gemcitabine resulted in tumor stasis and regression. Conclusions: These results demonstrate that IGF-1R blockade by AMG 479 inhibits AKT signaling and enhances the in vivo anti-tumor effects of approved therapies for the treatment of pancreatic carcinoma. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amgen Amgen

Uncommon Endocytic and Trafficking Pathway of the Natural Killer Cell CD94/NKG2A Inhibitory Receptor

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic-like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1(+), Rab5(+) early endosomes. It does appear in Rab4(+) early/sorting endosome, but, in the time period examined, fails to reach Rab11(+) recycling or Rab7(+) late endosomes or lysosome-associated membrane protein-1(+) lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis.The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated.The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with naïve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-κB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K–NF-κB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.

Fertilization and Inositol 1,4,5-Trisphosphate (IP3)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs1

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.

A randomized phase I study of the humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody EMD 72000 in subjects with advanced gastrointestinal cancers

3018 Background: Growth factor signaling through EGFR drives tumor proliferation, motility, angiogenic stimulation, and protects the cells from stress induced apoptosis. EMD 72000 is a humanized monoclonal anti-EGFR IgG1 antibody that blocks ligand binding, EGFR signaling, induces receptor downregulation, and potentially participates in antibody-dependent cell-mediated tumor cell killing. Methods: A phase I study was conducted to assess the safety and anti-tumor activity of EMD 72000 in 24 patients with advanced refractory gastrointestinal tumors. Antitumor activity was assessed using CT and 18FDG PET dual-modality tomography. Subjects were randomized in groups of 6 to receive absolute doses of 100, 200, 400, or 800 mg EMD 72000 weekly for 4 wks followed by treatment with 400 mg per wk until disease progression. EMD 72000 was administered by 1-hr IV infusion without routine premedication. CT and 18FDG PET images were obtained before treatment and in wks 4 and 8. Skin and tumor biopsies were obtained before treatment and at wks 4 (skin only) and 8. Blood samples for pharmacokinetics were obtained weekly for 8 wks. Results: Patient population: 21 colorectal, 2 cholangiocellular, and 1 gastric cancer; median age was 58 (range 29–70) years with a median of 3 prior chemotherapy protocols (range 0–6) and 2 sites of metastatic disease (range 1–4). Tumors were EGFR+ by immunohistochemistry. No NCI-CTC grade 3 or 4 drug-related toxicities were observed; grade 1/2 skin reaction was seen in > 80% of subjects with doses ≥ 200 mg. Assessment of metabolic response by PET-CT using EORTC criteria in 18 patients after 4 wks on study showed 1 CR (800 mg), 6 PR (2 each at 400 and 800 mg, 1 each at 100 and 200 mg), and 9 SD. One patient who did not show 18FDG uptake at baseline achieved a CR during follow up with magnetic resonance imaging. Median time to progression was 13 wks. Conclusions: In this study administration of EMD 72000 was associated with no grade 3 or 4 toxicities. Preliminary evidence of metabolic response by PET and antitumor activity by CT and MRI was seen in heavily pretreated subjects with gastrointestinal cancers. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck KGaA, Darmstadt, Germany Merck KGaA, Darmstadt, Germany

A randomized phase I study of the humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody EMD 72000 in subjects with advanced gastrointestinal cancers

3018 Background: Growth factor signaling through EGFR drives tumor proliferation, motility, angiogenic stimulation, and protects the cells from stress induced apoptosis. EMD 72000 is a humanized monoclonal anti-EGFR IgG1 antibody that blocks ligand binding, EGFR signaling, induces receptor downregulation, and potentially participates in antibody-dependent cell-mediated tumor cell killing. Methods: A phase I study was conducted to assess the safety and anti-tumor activity of EMD 72000 in 24 patients with advanced refractory gastrointestinal tumors. Antitumor activity was assessed using CT and 18FDG PET dual-modality tomography. Subjects were randomized in groups of 6 to receive absolute doses of 100, 200, 400, or 800 mg EMD 72000 weekly for 4 wks followed by treatment with 400 mg per wk until disease progression. EMD 72000 was administered by 1-hr IV infusion without routine premedication. CT and 18FDG PET images were obtained before treatment and in wks 4 and 8. Skin and tumor biopsies were obtained before treatment and at wks 4 (skin only) and 8. Blood samples for pharmacokinetics were obtained weekly for 8 wks. Results: Patient population: 21 colorectal, 2 cholangiocellular, and 1 gastric cancer; median age was 58 (range 29–70) years with a median of 3 prior chemotherapy protocols (range 0–6) and 2 sites of metastatic disease (range 1–4). Tumors were EGFR+ by immunohistochemistry. No NCI-CTC grade 3 or 4 drug-related toxicities were observed; grade 1/2 skin reaction was seen in > 80% of subjects with doses ≥ 200 mg. Assessment of metabolic response by PET-CT using EORTC criteria in 18 patients after 4 wks on study showed 1 CR (800 mg), 6 PR (2 each at 400 and 800 mg, 1 each at 100 and 200 mg), and 9 SD. One patient who did not show 18FDG uptake at baseline achieved a CR during follow up with magnetic resonance imaging. Median time to progression was 13 wks. Conclusions: In this study administration of EMD 72000 was associated with no grade 3 or 4 toxicities. Preliminary evidence of metabolic response by PET and antitumor activity by CT and MRI was seen in heavily pretreated subjects with gastrointestinal cancers. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck KGaA, Darmstadt, Germany Merck KGaA, Darmstadt, Germany

Epidermal Growth Factor Receptor Down-regulation Induced by UVA in Human Keratinocytes Does Not Require the Receptor Kinase Activity

Activation of the epidermal growth factor (EGF) receptor by EGF, its ligand, results in receptor internalization and down-regulation, which requires receptor kinase activity, phosphorylation, and ubiquitination. In contrast, we have found here in human HaCaT keratinocytes that exposure to UVA induces EGF receptor internalization and down-regulation without receptor phosphorylation and ubiquitination. The presence of the receptor kinase activity inhibitor AG1478 increased UVA-induced receptor down-regulation, whereas it inhibited EGF-induced receptor down-regulation. These observations demonstrate that, in contrast to EGF, receptor kinase activity is not required for receptor down-regulation by UVA. Concurrent with receptor down-regulation, caspases were activated by UVA exposure. The presence of caspase inhibitors blocked receptor down-regulation in a pattern similar to poly(ADP)-ribose polymerase cleavage. Much more receptor down-regulation was observed after UVA exposure in apoptotic detached cells in which caspase is activated completely. These results indicate that UVA-induced receptor down-regulation is dependent on caspase activation. Similar to UVA, both UVB and UVC induced receptor down-regulation, in which receptor kinase activity is not required, whereas caspase activation is involved. Inhibition of EGF receptor down-regulation increased receptor activation and activation of its downstream survival signaling ERK and AKT after UVA exposure. Preventing the activation of each of these pathways enhanced apoptosis induced by UVA. These findings suggest that EGF receptor down-regulation by UVA may play an important role in the execution of the cell suicide program by attenuating its anti-apoptotic function and thereby preventing cell transformation and tumorigenesis in vivo.

Overview of epidermal growth factor receptor biology and its role as a therapeutic target in human neoplasia.

The ability of the epidermal growth factor receptor (EGFR) to transform epithelial cells, the overexpression of EGFR and its ligands in several human carcinomas, and the causal association of the receptor network with accelerated tumor progression provided a rationale for targeting this signaling system with tumor-selective strategies. Two of these antireceptor approaches, both based on the known structure/function of the EGFR, will be discussed. The first strategy involved the development of humanized monoclonal antibodies against the nonconserved receptor's extracellular domain. These antibodies block ligand binding and can induce receptor downregulation. The second approach was the generation of adenosine triphosphate-mimetics that compete with adenosine triphosphate for binding to the receptor's kinase pocket and disable the ability of the EGFR to transduce intracellular signals. Early clinical studies already suggest that both of these approaches, either alone or in combination with standard anticancer therapies, alter the natural history of EGFR-expressing cancers with little toxicity to the tumor-bearing host.

Overview of epidermal growth factor receptor biology and its role as a therapeutic target in human neoplasia

The ability of the epidermal growth factor receptor (EGFR) to transform epithelial cells, the overexpression of EGFR and its ligands in several human carcinomas, and the causal association of the receptor network with accelerated tumor progression provided a rationale for targeting this signaling system with tumor-selective strategies. Two of these antireceptor approaches, both based on the known structure/function of the EGFR, will be discussed. The first strategy involved the development of humanized monoclonal antibodies against the nonconserved receptor's extracellular domain. These antibodies block ligand binding and can induce receptor downregulation. The second approach was the generation of adenosine triphosphate-mimetics that compete with adenosine triphosphate for binding to the receptor's kinase pocket and disable the ability of the EGFR to transduce intracellular signals. Early clinical studies already suggest that both of these approaches, either alone or in combination with standard anticancer therapies, alter the natural history of EGFR-expressing cancers with little toxicity to the tumor-bearing host. Semin Oncol 29 (suppl 14):3-9. Copyright 2002, Elsevier Science (USA). All rights reserved.

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor.

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.

Biological effects of encapsulated insulin on transfected Chinese hamster ovary cells

Oral administration of insulin incorporated into the wall of isobutylcyanoacrylate nanocapsule to diabetic rats induces a long-lasting normalization of their fasting glycaemia. In this study, we examined the biological action of encapsulated insulin on DNA and glycogen syntheses in Chinese hamster ovary cells transfected with the human insulin receptor gene. In the 10(-11) mol/l-10(-9) mol/l concentration range, encapsulated insulin elicited responses comparable to those induced by native insulin: at 10(-9) mol/l, the rates of glycogen and DNA synthesis were enhanced by factors 3 and 2.5, respectively. Encapsulated insulin at 10(-7) mol/l evoked receptor desensitization although it did not induce receptor down-regulation and did not alter receptor recycling for up to 6 h. Chloroquine decreased the action of native insulin on glycogen synthesis, but did not affect the dose-response characteristics of encapsulated insulin. Acid-washing of the cells after 1 h of stimulation decreased maximal insulin responsiveness and provoked a dose response curve for encapsulated insulin similar to that of the native hormone. Direct measurement of effective insulin binding activity showed that encapsulated insulin (at 10(-8) and 10(-7) mol/l) was withdrawn from the incubation medium 5-8 times less efficiently than native insulin. These data are in agreement with previous results showing that the polymeric wall protects encapsulated insulin from degradation. Persistence of intact encapsulated insulin inside and outside the cell may result in modifying signalling events and thus be responsible for the observed cellular desensitization.

Modulation of brainstem 5-HT 1C receptors by serotonergic drugs in the rat

1. The sparse population of brainstem 5-hydroxytryptamine 1C (5-HT 1C) (also called 5-HT 2C) receptors has received little attention despite its possible role in the serotonin syndrome and 5-HT-mediated shaking behavior. We characterized [ 3H]mesulergine binding in rat brainstem and, to determine if brainstem 5-HT 1C sites respond to serotonergic manipulations, performed saturation studies of [ 3H]mesulergine binding in brainstem from rats treated chronically with 11 different 5-HT 1C/2 agonists and antagonists. 2. In competition studies in vitro, the rank order of drug potency was most compatible with a 5-HT 1C receptor binding site: mianserin, 5-HT, cinanserin, 1-(3-chlorophenyl)piperazine ( m-CPP), 1-(2-5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), MDL 100,907, RU 24969, 5-carboxamido-tryptamine (5-CT), 8-OH-DPAT, MDL 72,222. 3. Chronic treatment with the agonists quipazine and trifluoromethylphenylpiperazine (TFMPP) and the antagonists ritanserin and methiothepin significantly down-regulated brainstem 5-HT 1C sites, which were 65% of [ 3H]mesulergine-labeled sites in brainstem. Only metergoline and ritanserin significantly increased pK D. 4. Chronic treatment in vivo with DOI, m-CPP, mianserin, methysergide, spiperone, cyproheptadine, and metergoline had no significant effect on B MAX at the dose studied. 5. These data suggest similarities in the regulation of 5-HT 1C and 5-HT 2 sites at which both 5-HT 1C/2 agonists and antagonists also induce receptor down-regulation. 6. 5-HT 1C/2 agonists and antagonists that did not down-regulate brainstem 5-HT 1C sites may be more active in vivo at 5-HT 2 sites, at 5-HT 1C sites in other brain regions, have effects on 5-HT 1C receptors not detectable at the recognition site, or differ for pharmacokinetic reasons.

Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes.

Binding of GH to its cell surface receptors is thought to result in the formation of a complex comprised of one molecule of hormone per two molecules of receptor. It has been proposed that this hormone-induced receptor dimerization is important for the mechanism of signal transduction. We have developed a mathematical model for quantitative evaluation of the biological responses associated with sequential receptor dimerization. Based on these predictions, we have investigated whether GH-induced receptor dimerization plays a role in two classical effects of GH, i.e. stimulation of lipogenesis in primary rat adipocytes and GH receptor down-regulation in cultured human IM-9 lymphocytes. Model predictions of biological responses linked to dimer formation yielded a bell-shaped pattern, with self-antagonism at high GH concentrations when monomeric GH-receptor complexes become predominant. The GH lipogenic bioactivity curve was indeed biphasic and first increased in a concentration-dependent manner between 10(-10)-10(-8) M GH (ED50, 0.5 nM), up to a maximum of 1.7-fold stimulation above basal. Then, the response decreased continuously above 5 x 10(-8) M GH, returning to basal levels around 10(-5) M GH. Incubation of IM-9 cells with wild-type human GH resulted in a dose-dependent loss of their surface receptors. In contrast, a human GH analog (G120R), mutated in the second binding surface of the hormone and, therefore, unable to induce GH receptor dimerization, failed to induce receptor down-regulation in the IM-9 cells. Furthermore, when added together with wild-type human GH, human GH(G120R) inhibited, in a concentration-dependent manner, the down-regulation induced by wild-type human GH. Taken together, these data support the hypothesis that receptor dimerization is critical for the stimulation by GH of both lipogenesis in primary rat adipocytes and receptor down-regulation in cultured human IM-9 lymphocytes.