Growth Factor-related Proteins Research Articles

P241 Systemic redox status associates with disease activity and clinical phenotypes in Inflammatory Bowel Disease

Abstract Background Oxidative stress is a key pathophysiological mechanism in inflammatory bowel diseases (IBD). Systemic levels of oxidative stress are reflected by reduced levels of free thiols (FT) in circulating proteins, especially those in albumin, which associates with disease activity in IBD. Yet, clinal value of circulating FT level as biomarkers of disease and therapy response remains largely unexplored. Here we investigated the association between plasma FT levels and clinical parameters, the plasma inflammatory proteome and medication use. Methods Plasma samples from 1,028 patients with IBD (567 Crohn's disease [CD] and 461 ulcerative colitis [UC]), and 500 healthy controls (HCs) participating in the 1000IBD and LifeLines projects were profiled for free thiols (FT), uric acid, bilirubin and 92 inflammation-related proteins (Olink Inflammation panelÒ). All biomarkers were associated with clinical phenotypes using general linear models adjusting for age, sex, body mass index, smoking and medication use. Results Plasma FT levels were significantly lower in IBD compared to HCs (p<0.05) (Figure 1A), with patients with UC showing even lower levels than patients with CD (p<0.05). Patients with UC on induction therapy had lower FT levels compared to patients on maintenance therapy (p<0.05). Furthermore, FT levels of patients with IBD were strongly associated with systemic inflammation (C-reactive protein [CRP], p<0.05) (Figure 1B). In both patients withCD and UC, reduced FT levels were associated with elevated levels of inflammation-, apoptosis-, and growth factor-related proteins, including C-X-C motif chemokine 9 (CXCL9) (Figure 1C-D), CUB domain-containing protein 1 (CDCP1) and caspase-8 (CASP8), proteins previous associated with preclinical IBD1-3. Furthermore, specifically for patients with UC, reduced FT levels were associated with elevated eotaxin-1 (CCL11) (Figure 1C), monocyte chemotactic protein-1 (MCP-1), and several cytokine biomarkers like Interleukine-6 (IL6) and Interleukin-17A (IL17A). Conclusion Systemic FT levels associate with inflammatory disease activity and clinical therapy and may offer potential utility in clinical assessments. This study highlights the intricate involvement of oxidative stress in various inflammatory pathways and components, particularly in innate/adaptive immune balance, cell damage, and apoptosis. These findings contribute to a deeper understanding of the interplay between oxidative stress, inflammation, and IBD. Funding: This study was supported by Janssen Research & Development LLC.

High-Throughput Proteomic Analysis of Human Dermal Fibroblast Response to Different Blood Derivatives: Autologous Topical Serum Derived from Plasma Rich in Growth Factors (PRGF) versus Leukocyte- and Platelet-Rich Plasma (L-PRP).

Platelet-rich plasma (PRP) is nowadays used in the treatment of different types of cutaneous lesions. However, different compositions can influence clinical outcomes. Among them, the inclusion of leukocytes is controversial. High-throughput proteomics techniques were used to analyze the proteins that are differentially expressed in human dermal fibroblasts (HDFs) after treatment for 24 h with two PRP types, autologous topical serum (Endoret serum—ES) derived from plasma rich in growth factors (PRGF) and leukocyte- and platelet-rich plasma (L-PRP). The identified proteins were then classified by both Gene Ontology and Ingenuity Pathway Analysis. The obtained results show that the compositions of ES and L-PRP differ in such a way that they induce different responses in HDFs. ES-treated HDFs overexpress growth factor-related proteins, leading to protein synthesis, cell proliferation and migration. By contrast, L-PRP treatment induces a response similar to that caused by proinflammatory molecules. These data could explain the contradictory clinical results obtained for the different types of PRP, especially with respect to their leukocyte contents.

Changes in Endocan and Dermatan Sulfate Are Associated with Biomechanical Properties of Abdominal Aortic Wall during Aneurysm Expansion and Rupture.

The study aimed to assess the potential of proteoglycans (PGs) and collagens as serological biomarkers in the abdominal aortic aneurysm (AAA). Furthermore, we investigated the underlying mechano-biological interactions and signaling pathways. Tissue and serum samples from patients with ruptured AAA (rAAA; n = 29), elective AAA (eAAA; n = 78), and healthy individuals (n = 8) were evaluated by histology, immunohistochemistry, and enzyme-linked immunosorbent assay, and mechanical properties were assessed by tensile tests. Regulatory pathways were determined by membrane-based sandwich immunoassay. In AAA samples, collagen type I and III (Col1 and Col3), chondroitin sulfate, and dermatan sulfate (DS) were significantly increased compared with controls (3.0-, 3.2-, 1.3-, and 53-fold; p < 0.01). Col1 and endocan were also elevated in the serum of AAA patients (3.6- and 6.0-fold; p < 0.01), while DS was significantly decreased (2.5-fold; p < 0.01). Histological scoring showed increased total PGs and focal accumulation in rAAA compared with eAAA. Tissue β-stiffness was higher in rAAA compared with eAAA (2.0-fold, p = 0.02). Serum Col1 correlated with maximum tensile force and failure tension (r = 0.448 and 0.333; p < 0.01, and r = 0.02), tissue endocan correlated with α-stiffness (r = 0.340; p < 0.01). Signaling pathways in AAA were associated with extracellular matrix synthesis and vascular smooth muscle cell proliferation. In particular, Src family kinases and platelet-derived growth factor- and epidermal growth factor-related proteins seem to be involved. Our findings reveal a structural association between collagen and PGs and their response to changes in mechanical loads in AAA. Particularly Col1 and endocan reflect the mechano-biological conditions of the aortic wall also in the patient's serum and might serve for AAA risk stratification.

High-Efficient Production of Adipose-Derived Stem Cell (ADSC) Secretome Through Maturation Process and Its Non-scarring Wound Healing Applications

Recently, the stem cell-derived secretome, which is the set of proteins expressed by stem cells and secreted into the extracellular space, has been demonstrated as a critical contributor for tissue repair. In this study, we have produced two sets of high concentration secretomes from adipose-derived mesenchymal stem cells (ADSCs) that contain bovine serum or free of exogenous molecules. Through proteomic analysis, we elucidated that proteins related to extracellular matrix organization and growth factor-related proteins are highly secreted by ADSCs. Additionally, the application of ADSC secretome to full skin defect showed accelerated wound closure, enhanced angiogenic response, and complete regeneration of epithelial gaps. Furthermore, the ADSC secretome was capable of reducing scar formation. Finally, we show high-dose injection of ADSC secretome via intraperitoneal or transdermal delivery demonstrated no detectable pathological conditions in various tissues/organs, which supports the notion that ADSC secretome can be safely utilized for tissue repair and regeneration.

Snake Venom Proteins Isolated from Tunisian Vipers: Pharmacological and Therapeutic Overview

: The venoms of Tunisian wildlife snakes are complex mixtures containing proteins/ peptides and non-protein molecules. Proteins and peptides are the most abundant compounds responsible for the biological effects of venoms. Snake venoms proteins have enzymatic or nonenzymatic activities, which are grouped into different families, including C-type lectin proteins, disintegrins (long, medium and short disintegrins), Kunitz-type serine protease inhibitors, natriuretic- like peptides, vascular endothelial growth factor-related proteins, L-amino acid oxidases, phospholipases A2 and serine proteinases. With technological advancements, the toxic effects of venoms were turned into potential benefits for clinical diagnosis, basic research and development of new research tools and drugs of potential clinical use. Our research team has shown that Macrovipera lebetina and Cerastes cerastes venom components of Tunisian wildlife snakes had great potential for the development of new drugs for the treatment of cancer, angiogenesis disorders or cardiovascular diseases. This review is an overview of snake venom proteins from Macrovipera lebetina and Cerastes cerastes and their biochemical, pharmacological and molecular characterization and their importance as protein resources with therapeutic potential.

Vitamin E Derivative with Modified Side Chain Induced Apoptosis by Modulating the Cellular Lipids and Membrane Dynamics in MCF7 Cells.

The vitamin E derivative with side chain modification (TC6OAc) has been shown to possess anticancer activity in our earlier in vivo studies. It was hypothesized that, as Vitamin E (VE) and VE derivative are fat soluble lipophilic molecules, they exert their function by modulating the lipid metabolism and related pathways. This study aimed to evaluate the cellular impact of this VE derivative (2,5,7,8-Tetramethyl-2-(4'-Methyl-3'-Pentenyl)-6-Acetoxy Chromane-TC6OH), using α-tocopherol as a reference compound throughout the experiments. Their effects on the cellular metabolism, the biophysical properties of cellular lipids and the functional characteristics of cells were monitored in human estrogen receptor (ER) positive breast cancer cells. It has been documented that TC6OH treatment induces tumor cell apoptosis by dissipating the mitochondrial membrane potential, modulating the lipid, transportation and degradation as well as downregulating certain anti-apoptotic and growth factor related proteins. Due to resistance of ER positive cells to the established therapies, the findings of this study are of translational value.

Bioinformatic analysis of cytokine expression in the proximal and distal nerve stumps after peripheral nerve injury.

In our previous study, we investigated the dynamic expression of cytokines in the distal nerve stumps after peripheral nerve injury using microarray analysis, which can characterize the dynamic expression of proteins. In the present study, we used a rat model of right sciatic nerve transection to examine changes in the expression of cytokines at 1, 7, 14 and 28 days after injury using protein microarray analysis. Interleukins were increased in the distal nerve stumps at 1-14 days post nerve transection. However, growth factors and growth factor-related proteins were mainly upregulated in the proximal nerve stumps. The P-values of the inflammatory response, apoptotic response and cell-cell adhesion in the distal stumps were higher than those in the proximal nerve stumps, but the opposite was observed for angiogenesis. The number of cytokines related to axons in the distal stumps was greater than that in the proximal stumps, while the percentage of cytokines related to axons in the distal stumps was lower than that in the proximal nerve stumps. Visualization of the results revealed the specific expression patterns and differences in cytokines in and between the proximal and distal nerve stumps. Our findings offer potential therapeutic targets and should help advance the development of clinical treatments for peripheral nerve injury. Approval for animal use in this study was obtained from the Animal Ethics Committee of the Chinese PLA General Hospital on September 7, 2016 (approval No. 2016-x9-07).

On the Modulatory Roles of Neuregulins/ErbB Signaling on Synaptic Plasticity.

Neuregulins (NRGs) are a family of epidermal growth factor-related proteins, acting on tyrosine kinase receptors of the ErbB family. NRGs play an essential role in the development of the nervous system, since they orchestrate vital functions such as cell differentiation, axonal growth, myelination, and synapse formation. They are also crucially involved in the functioning of adult brain, by directly modulating neuronal excitability, neurotransmission, and synaptic plasticity. Here, we provide a review of the literature documenting the roles of NRGs/ErbB signaling in the modulation of synaptic plasticity, focusing on evidence reported in the hippocampus and midbrain dopamine (DA) nuclei. The emerging picture shows multifaceted roles of NRGs/ErbB receptors, which critically modulate different forms of synaptic plasticity (LTP, LTD, and depotentiation) affecting glutamatergic, GABAergic, and DAergic synapses, by various mechanisms. Further, we discuss the relevance of NRGs/ErbB-dependent synaptic plasticity in the control of brain processes, like learning and memory and the known involvement of NRGs/ErbB signaling in the modulation of synaptic plasticity in brain’s pathological conditions. Current evidence points to a central role of NRGs/ErbB receptors in controlling glutamatergic LTP/LTD and GABAergic LTD at hippocampal CA3–CA1 synapses, as well as glutamatergic LTD in midbrain DA neurons, thus supporting that NRGs/ErbB signaling is essential for proper brain functions, cognitive processes, and complex behaviors. This suggests that dysregulated NRGs/ErbB-dependent synaptic plasticity might contribute to mechanisms underlying different neurological and psychiatric disorders.

A molecular dynamics-based algorithm for evaluating the glycosaminoglycan mimicking potential of synthetic, homogenous, sulfated small molecules.

Glycosaminoglycans (GAGs) are key natural biopolymers that exhibit a range of biological functions including growth and differentiation. Despite this multiplicity of function, natural GAG sequences have not yielded drugs because of problems of heterogeneity and synthesis. Recently, several homogenous non-saccharide glycosaminoglycan mimetics (NSGMs) have been reported as agents displaying major therapeutic promise. Yet, it remains unclear whether sulfated NSGMs structurally mimic sulfated GAGs. To address this, we developed a three-step molecular dynamics (MD)-based algorithm to compare sulfated NSGMs with GAGs. In the first step of this algorithm, parameters related to the range of conformations sampled by the two highly sulfated molecules as free entities in water were compared. The second step compared identity of binding site geometries and the final step evaluated comparable dynamics and interactions in the protein-bound state. Using a test case of interactions with fibroblast growth factor-related proteins, we show that this three-step algorithm effectively predicts the GAG structure mimicking property of NSGMs. Specifically, we show that two unique dimeric NSGMs mimic hexameric GAG sequences in the protein-bound state. In contrast, closely related monomeric and trimeric NSGMs do not mimic GAG in either the free or bound states. These results correspond well with the functional properties of NSGMs. The results show for the first time that appropriately designed sulfated NSGMs can be good structural mimetics of GAGs and the incorporation of a MD-based strategy at the NSGM library screening stage can identify promising mimetics of targeted GAG sequences.

Neuregulin-1 attenuates cognitive function impairments in a transgenic mouse model of Alzheimer's disease.

The neuregulin (NRG) family of epidermal growth factor-related proteins is composed of a wide variety of soluble and membrane-bound proteins that exert their effects via the tyrosine kinase receptors ErbB2-ErbB4. In the nervous system, the functions of NRG1 are essential for peripheral myelination, the establishment and maintenance of neuromuscular and sensorimotor systems and the plasticity of cortical neuronal circuits. In the present study, we report that an intracerebroventricular infusion of NRG1 attenuated cognitive impairments in 13-month-old Tg2576 mice, an animal model of Alzheimer's disease (AD). In addition, according to Golgi-Cox staining, NRG1 rescued the reduction in the number of dendritic spines detected in the brains of Tg2576 mice compared with vehicle (PBS)-infused mice. This result was also corroborated in vitro as NRG1 attenuated the oligomeric amyloid beta peptide1-42 (Aβ1-42)-induced decrease in dendritic spine density in rat primary hippocampal neuron cultures. NRG1 also alleviated the decrease in neural differentiation induced by oligomeric Aβ1-42 in mouse fetal neural stem cells. Collectively, these results suggest that NRG1 has a therapeutic potential for AD by alleviating the reductions in dendritic spine density and neurogenesis found in AD brains.

The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2(+) mammary cancer.

The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.

Repeated, intermittent exposures to diisopropylfluorophosphate in rats: protracted effects on cholinergic markers, nerve growth factor-related proteins, and cognitive function

Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in military and terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity (i.e. subthreshold levels) are poorly understood. Further, there is clinical evidence that such repeated exposures to OPs lead to prolonged deficits in cognition, although the mechanism for this effect is unknown. In this study, the behavioral and neurochemical effects of repeated, intermittent, and subthreshold exposures to the alkyl OP, diisopropylfluorophosphate (DFP) were investigated. Rats were injected with DFP s.c. (dose range, 0.25–1.0 mg/kg) every other day over the course of 30 days, and then given a 2 week, DFP-free washout period. In behavioral experiments conducted at various times during the washout period, dose dependent decrements in a water maze hidden platform task and a spontaneous novel object recognition (NOR) procedure were observed, while prepulse inhibition of the acoustic startle response was unaffected. There were modest decreases in open field locomotor activity and grip strength (particularly during the DFP exposure period); however, rotarod performance and water maze swim speeds were not affected. After washout, DFP concentrations were minimal in plasma and brain, however, cholinesterase inhibition was still detectable in the brain. Moreover, the 1.0 mg/kg dose of DFP was associated with (brain region-dependent) alterations in nerve growth factor-related proteins and cholinergic markers. The results of this prospective animal study thus provide evidence to support two novel hypotheses: (1) that intermittent, subthreshold exposures to alkyl OPs can lead to protracted deficits in specific domains of cognition and (2) that such cognitive deficits may be related to persistent functional changes in brain neurotrophin and cholinergic pathways.

Hepatoma‐derived growth factor‐related protein‐3: A new neurotrophic and neurite outgrowth‐promoting factor for cortical neurons

Hepatoma-derived growth factor-related proteins (HRPs) make up a family of six members. Hepatoma-derived growth factor-related protein-3 (HRP-3) is the only family member whose expression is almost restricted to nervous tissue. Here we show that soluble HRP-3 acts as a novel neurotrophic factor for cultured primary cortical neurons. Antibody-mediated neutralization of HRP-3 function results in neuronal degeneration. In contrast, HRP-3 as the only addition to a culture medium not supporting neuronal survival rescues neurons to an extent comparable to the addition of FCS. Besides this neuroprotective capability, the protein exerts a neurite outgrowth-promoting effect when it is presented as a coated substrate but not as a soluble factor. This study points to an important role of HRP-3 during the development of the nervous system.

Immunohistochemical Array for Clear Cell Type Mucoepidermoid Carcinoma

Background : The protein expression profile of clear cell type mucoepidermoid carcinoma (MEC) is not well known. Methods : We examined a case of clear cell type MEC by immunohistochemical (IHC) array using 59 antibodies against oncoproteins, proliferation-related proteins, apoptosis-related proteins, growth factor-related proteins, angiogenesis-related proteins, and matrix proteins. Results : MEC tumor cells showed 40 to 60% more expression of BCL-2 and cyclin-dependent kinase 4 than normal gingival tissue, and 20-40% more expression of BCL-2-associated agonist of cell death, deleted in malignant brain tumors 1, E-cadherin, eIF5A, hypoxia-inducible factor, vimentin, and Wnt-1. Expression of other proteins, including p53, epidermal growth factor receptor, proliferating cell nuclear antigen, survivin, carcinoembryonic antigen, b-catenin, poly-ADP ribose-polymerase, etc. were relatively weak in MEC tumor cells. Conclusions : The IHC array for our MEC contained strong oncogenic signals involving Wnt-1/ adenomatous polyposis coli, tumor necrosis factor a/signal transducer and activator of transcription 3/BCL-2, and pAKT pathways, signals that could result in the prolonged survival of clear tumor cells.

Hepatoma-derived Growth Factor-related Protein-3 Interacts with Microtubules and Promotes Neurite Outgrowth in Mouse Cortical Neurons

Hepatoma-derived growth factor-related proteins (HRP) comprise a family of 6 members, which the biological functions are still largely unclear. Here we show that during embryogenesis HRP-3 is strongly expressed in the developing nervous system. At early stages of development HRP-3 is located in the cytoplasm and neurites of cortical neurons. Upon maturation HRP-3 relocalizes continuously to the nuclei and in the majority of neurons of adult mice it is located exclusively in the nucleus. This redistribution from neurites to nuclei is also found in embryonic cortical neurons maturing in cell culture. We show that HRP-3 is necessary for proper neurite outgrowth in primary cortical neurons. To identify possible mechanisms of how HRP-3 modulate neuritogenesis we isolated HRP-3 interaction partners and demonstrate that it binds tubulin through the N-terminal so called HATH region, which is strongly conserved among members of the HRP family. It promotes tubulin polymerization, stabilizes and bundles microtubules. This activity depends on the extranuclear localization of HRP-3. HRP-3 thus could play an important role during neuronal development by its modulation of the neuronal cytoskeleton.

Regulation of Cripto-1 Signaling and Biological Activity by Caveolin-1 in Mammary Epithelial Cells

Human and mouse Cripto-1 (CR-1/Cr-1) proteins play an important role in mammary gland development and tumorigenesis. In this study, we examined the relationship between Cripto-1 and caveolin-1 (Cav-1), a membrane protein that acts as a tumor suppressor in the mammary gland. Cripto-1 was found to interact with Cav-1 in COS7 cells and mammary epithelial cells. Using EpH4 mouse mammary epithelial cells expressing Cr-1 (EpH4 Cr-1) or Cr-1 and Cav-1 (EpH4 Cr-1/Cav-1), we demonstrate that Cav-1 expression markedly reduced the ability of Cr-1 to enhance migration, invasion, and formation of branching structures in EpH4 Cr-1/Cav-1 cells as compared to EpH4 Cr-1 cells. Furthermore, coexpression of Cav-1 together with Cr-1 in EpH4 Cr-1/Cav-1 cells inhibited Cr-1-mediated activation of c-src and mitogen-activated protein kinase signaling pathways. Conversely, primary mammary epithelial cells isolated from Cav-1 null(-/-)/mouse mammary tumor virus-CR-1 transgenic animals showed enhanced motility and activation of mitogen-activated protein kinase and c-src as compared to Cav-1(+/-)/CR-1 mammary cells. Finally, mammary tumors derived from mouse mammary tumor virus-CR-1 mice showed a dramatic reduction of Cav-1 expression as compared to mammary tissue from normal FVB/N mice, suggesting that in vivo Cav-1 is down-regulated during the process of CR-1-mediated mammary tumorigenesis.

The Phylogenetically Conserved Molluscan Chitinase-like Protein 1 (Cg-Clp1), Homologue of Human HC-gp39, Stimulates Proliferation and Regulates Synthesis of Extracellular Matrix Components of Mammalian Chondrocytes

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Identification of genes expressed differentially in an in vitro human lung carcinogenesis model

Lung cancer is the deadliest among all cancers in the US for both men and women. Early detection of premalignant lesions or tumors appears to be a promising approach to reducing the morbidity and mortality from lung cancer because the survival of early stage lung cancer patients is better than that of patients with advanced cancers. We approached the identification of early biomarkers of human lung carcinogenesis, by combining the use of cDNA array and an in vitro human lung carcinogenesis model that consists of normal (NHBE), immortalized (BEAS-2B and 1799), transformed (1198) and tumorigenic (1170-I) human bronchial epithelial (HBE) cells. We hypothesized that certain genes that are expressed differentially among these cells can serve as both biomarkers for early detection and targets for intervention. Nineteen genes were down-regulated and six were up-regulated in tumorigenic 1170-I HBE cells compared to normal HBE cells (NHBE) using cDNA array. Down- regulated genes encode cell-to-cell and cell-to-matrix adhesion-related proteins, calcium-binding proteins and some enzymes or growth factor-related proteins. The functions of up-regulated gene were more variable. Similar expression of selected genes was found in different non-small cell lung cancer cell lines analyzed by Northern blotting. Furthermore, the differential expression of some genes was also observed by in silico analysis of database of human lung tumors. The differentially expressed genes encode calcium-binding proteins and cell-cell and cell-matrix adhesion proteins. Furthermore, Northern blotting analysis of RNA from different cell lines comprising an in vitro carcinogenesis model including normal, immortalized, transformed, and tumorigenic cells indicated that the expression of subsets of the identified genes changed at different stages of carcinogenesis. These data show that the in vitro carcinogenesis cell system could be useful for discovering potential biomarkers of early and late stages of lung carcinogenesis and targets for chemoprevention.

Potential role of HER-2; in primary breast tumor with bone metastasis

HER-2 is an important prognostic factor in breast cancer, and its overexpression is observed in 20-60% of cases with micrometastases in the bone marrow. The aim of this study was to explore the potential functional role of HER-2 in primary breast tumors with bone metastases. Forty-eight primary breast tumors with simultaneous or non-simultaneous bone metastases were studied. The expression of hormone receptors, and metastasis and growth factor-related proteins were assessed by immunohistochemical staining. The correlation in statistical significance was assessed by the Chi-square test. Of the 48 breast tumors, 11 (22.9%) were HER-2-positive and 37 were HER-2-negative. There was no significant difference in HER-2 status and clinicopathologic factors between the two groups. Of the 11 HER-2-positive tumors, there were 2 and 3 cases that showed positive nuclear expression for estrogen and progesterone receptors, respectively. No extranuclear expression of HRs was detected in these tumors. For metastasis-related proteins such as c-Met, VEGF, and MTA-1, which are activated by HER-2, only some insignificant focal expression of these proteins was observed. An increased level of pAkt was observed in 9 (81.8%) of 11 tumors, and an increased expression of CXCR4 was observed in 6 (54.5%) of 11 tumors. The frequency of increased levels of pAkt and CXCR4 was not significant between HER-2-positive and -negative tumors. The increased levels of pAkt and CXCR4 are induced by factors that are both dependent and independent of HER-2, and the activation of HER-2/CXCR4/ Akt signaling pathway in primary breast tumors may contribute to the formation of bone metastases in breast cancer.

ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain

The central cholinergic system has a fundamental role in normal cognitive function, and in diseases that exhibit cognitive dysfunction. The purpose of this study was to design ELISA methods to measure proteins that have essential functions in the central cholinergic system. We were particularly interested in quantifying proteins that respond directly or indirectly to nerve growth factor (NGF). ELISAs offer advantages over Western blot analyses and other methods, such as increased sensitivity, decreased assay variability, increased efficiency, and decreased cost. We developed indirect ELISA methods for: choline acetyltransferase (ChAT); the vesicular acetylcholine transporter (VAChT); the high affinity choline transporter (HACT/CHT); TrkA, the high affinity NGF receptor; the p75 neurotrophin receptor (p75 NTR). A sandwich ELISA was developed to measure tyrosine-phosphorylated TrkA in brain lysates. We used these ELISAs to compare levels of the above proteins in important memory-related brain regions – basal forebrain, hippocampus, cortex, and prefrontal cortex – from old and young rats. We identified age-related differences in the levels of the aforementioned proteins (e.g., VAChT and HACT/CHT in hippocampus). Thus, these ELISA methods should be particularly useful for comparing the effects of age, disease, drugs, and toxicants on brain levels of key cholinergic and growth factor-related proteins.