Development of a generalized model for kidney depth estimation in the Chinese population: A multi-center study.

PURPOSE: To establish an accurate and reliable equation for kidney depth estimation in adult patients from different Chinese geographical regions. METHOD: This multicenter study enrolled Eastern Asian Chinese patients with abdominal PET/CT scans at 26 imaging centers from six macro-regions across China in 3 years. Age, gender, height, weight, primary disease and its extent on PET scans of the participants were collected as potential predictive factors. Kidney depth on CT, defined as the average of the vertical distances from the posterior skin to the farthest anterior and closest posterior surfaces of each kidney, was measured as the standard reference. The new kidney depth model was constructed using a multiple regression model, and its performance was compared to those of three established models by computing the absolute value of estimation errors in comparison with CT-measured kidney depth. RESULTS: A total of 2502 patients were enrolled and classified into training (n=1653) and testing (n = 849) subsets. In the training subset, two kidney depth models were constructed: Left (cm): 0.013×age+0.117×gender-0.044×height+0.087×weight+7.951, Right (cm): 0.005×age+0.013×gender-0.035×height+0.082×weight+7.266 (weight: kg, height: cm, gender = 0 if female, 1 if male). In the testing subset, one-way analysis of variance showed that the estimation errors of the new models did not significantly differ among the 6 regions. Bland-Altman analysis determined that new equations had lower estimated biases (left: 0.039 cm, right: 0.018 cm) compared with other existing models. CONCLUSION: The new equations were highly accurate for kidney depth estimation in adults from all over China, with lower estimation errors compared to other established models.

NIS and epithelial-mesenchymal transition marker expression of circulating tumor cells for predicting and monitoring the radioactive iodine-131 therapy effect in differentiated thyroid cancers.

Current methods, such as serum thyroglobulin measurement and medical imaging, have limitations in the routine monitoring of the disease status and treatment response of patients with differentiated thyroid cancers (DTCs), and additional methods remain to be explored. The aim of this study was to investigate the clinical value of the sodium/iodide symporter (NIS) expression and epithelial-mesenchymal transition (EMT) phenotypes of circulating tumor cells (CTCs) in monitoring the disease status and treatment response of DTC. Blood samples were obtained from DTC patients before (1 to 3 months after total thyroidectomy) and 4 to 6 months after radioactive iodine-131 (RAI) therapy for the CTC assessments. The number, NIS expression, and EMT phenotypes of CTCs were enumerated and characterized with CanPatrol™ CTC enrichment and mRNA in situ hybridization. Postoperative NIS high expression was independently correlated with a better response to first RAI therapy and good treatment efficacy. Postoperative NIS-/epithelial-/mesenchymal+ CTCs presence was independently correlated with a worse response to first RAI therapy. The numbers of total NIS+ CTCs and NIS+/epithelial+/mesenchymal+ CTCs after first RAI therapy were negatively correlated with a better response to RAI therapy only in univariate analyses. Univariate and multivariate analyses showed that a decreased or unchanged number of total NIS+ CTCs after RAI therapy may denote good efficacy and effective RAI therapy. These preliminary data suggest that assessment of the NIS expression and EMT phenotypes of CTCs may serve as potential adjuncts for predicting and monitoring the curative effect of RAI therapy in DTC patients and avoid ineffective treatment. Further validation is warranted.

Novel small peptides derived from VEGF125-136: potential drugs for radioactive diagnosis and therapy in A549 tumor-bearing nude mice.

Vascular endothelial growth factor receptor (VEGFR) is a critical factor in tumor angiogenesis and has been considered a potential target for receptor-mediated radionuclide imaging and therapy. In this study, we identified two peptides (QKRKRKKSRKKH and RKRKRKKSRYIVLS) derived from VEGF125-136 that displayed high binding affinities to VEGFR and strong inhibition of A549 cell growth. 99mTc- and 188Re-labeled peptides displayed high labeling efficiency and favorable stability in saline and human plasma. At the cellular level, the radiolabeled peptides could bind with A549 cells and be internalized via the VEGFR-1-mediated pathway. 99mTc/188Re-labeled peptide was significantly accumulated at xenograft tumors, as observed with single-photon emission computed tomography (SPECT) planar imaging. Moreover, 188Re-labeled peptides significantly inhibited tumor growth, prolonged the survival time of the tumor-bearing nude mice and resulted in much more necrotic regions and apoptotic cells in the A549 xenograft tumors. These results demonstrated that these two peptides as candidate drugs for radionuclide imaging and tumor therapy.

Modulation, bioinformatic screening, and assessment of small molecular peptides targeting the vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1-Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF125-136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of (125)I-VEGF to VEGFR in a dose-dependent manner. The IC50 values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF125-136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF125-136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.

Identification of phosphocaveolin-1 as a novel protein tyrosine phosphatase 1B substrate.

Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B-catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B.

Identification of granulocyte-macrophage colony-stimulating factor and lipopolysaccharide-induced signal transduction pathways that synergize to stimulate HIV type 1 production by monocytes from HIV type 1 transgenic mice.

HIV-1-infected monocyte/macrophages located in lymph nodes and tissues are highly productive sources of HIV-1 and may function as a persistent reservoir contributing to the rebound viremia observed after highly active antiretroviral therapy is stopped. Mechanisms activating latently infected, primary monocyte/macrophages to produce HIV-1 were investigated using monocytes isolated from a transgenic mouse line carrying a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF), regulated by the endogenous long terminal repeat (LTR) (JR-CSF mice). Granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) induced infectious HIV-1 production by JR-CSF mouse monocytes over 10-fold and 100-fold higher than that stimulated by GM-CSF or LPS alone, respectively. We examined mechanisms of GM-CSF synergy with LPS and demonstrated that GM-CSF up-regulated the LPS receptor, TLR-4, and also synergized with LPS to activate mitogen-activated protein (MAP) kinase/ERK kinase and the Sp1 transcription factor. Inhibitors of either MAP kinase/ERK kinase or p38 kinase but not PI 3-kinase potently suppressed GM-CSF and LPS-induced HIV-1 production by JR-CSF mouse monocytes. Because Sp1 is activated by both the MAP kinase/ERK kinase and p38 kinase pathways, we postulate that synergistic activation of these pathways by GM-CSF and LPS induced sufficient levels of Sp1 to activate the HIV-1 LTR in a Tat-independent manner and induced HIV-1 production by JR-CSF mouse monocytes. Thus, our study delineated the pathway of HIV-1 LTR activation by GM-CSF and LPS and indicated that JR-CSF transgenic mice may provide a new in vitro and in vivo system for investigating the mechanism by which inflammatory and infectious stimuli activate HIV-1 production from latently infected monocytes.

Cellular effects of small molecule PTP1B inhibitors on insulin signaling.

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type 2 diabetes and other associated metabolic syndromes. To further define the role of PTP1B in insulin signaling and to test the hypothesis that blocking the activity of PTP1B would augment the action of insulin, we prepared several cell permeable, potent and selective, small molecule PTP1B inhibitors, and evaluated their biological effects in several insulin sensitive cell lines. Our data indicate that PTP1B inhibitors bind to and colocalize with PTP1B on the surface of the endoplasmic reticulum and PTP1B exerts its negative effect on insulin signaling upstream of phosphatidylinositol 3-kinase and MEK1. Treatment of cells with PTP1B inhibitors, both in the presence and in the absence of insulin, markedly enhances IRbeta and IRS-1 phosphorylation, Akt and ERK1/2 activation, Glut4 translocation, glucose uptake, and Elk1 transcriptional activation and cell proliferation. These results indicate that small molecule inhibitors targeted to PTP1B can act as both insulin mimetics and insulin sensitizers. Taken together, our findings combined with results from PTP1B knockout, antisense, and biochemical studies provide strong evidence that PTP1B negatively regulates insulin signaling and that small molecule PTP1B inhibitors have the ability to potentiate and augment the action of insulin.

SDF-1alpha production is negatively regulated by mouse estrogen enhanced transcript in a mouse thymus epithelial cell line.

SDF-1/CXCR4 plays an important role in promoting survival, expansion, and differentiation of T cell progenitors. The present study investigates the mechanism by which estrogen inhibits SDF-1alpha expression in mouse thymus. Mouse estrogen enhanced transcript (mEET) is endogenously expressed in a mouse thymus epithelial cell line 1 (MTEC1). In MTEC1 cells that express the transfected sense mEET, the SDF-1alpha transcription and its chemotactic activity were profoundly inhibited. Conversely, in MTEC1 that express the transfected anti-sense mEET, the SDF-1alpha transcription and its chemotactic activity were substantially augmented. Moreover, we disclosed that mEET inhibited the production of SDF-1alpha by its suppression of NF-kappaB translocation into nucleus. Using a combinatorial induction of doxycycline (Dox) and 17beta-estradiol (E2) on the sense and anti-sense mEET transfectants, it was demonstrated that an increase of mEET expression enhanced E2-induced inhibition of SDF-1alpha production, while a blockade of mEET expression alleviated E2-induced inhibition of SDF-1alpha production. In conclusion, the E2-imposed suppression of SDF-1alpha production is partly mediated by mEET involved signaling pathway.

Negative regulation of monocyte chemoattractant protein-1 gene expression by a mouse estrogen-enhanced transcript.

A novel gene containing two typical estrogen responsive elements (ERE) was cloned from MTEC1 cells, a mouse thymus epithelial cell line that produces constitutively many chemokines. This gene is a homologue of the rat estrogen-enhanced transcript (EET) gene, and is called the mEET gene. mEET protein is expressed in cytoplasm. Addition of 17 beta-estradiol (E2) to the MTEC1 cell cultures enhanced mEET mRNA expression and, meanwhile, significantly inhibited monocyte chemoattractant protein-1 (MCP-1) production. To analyze the functional links between the expression of mEET and of MCP-1, we transfected MTEC1 cells with ERE-deleted antisense- or sense-mEET complementary (c)DNA construct and activated the transfected mEET cDNA in stable MTEC1 transfectants with doxycycline (Dox). Dox-induced activation of the mEET gene profoundly inhibited MCP-1 expression at both mRNA and protein levels and alleviated its chemotactic activity. Conversely, inactivation of the mEET gene substantially augmented MCP-1 expression. Activation of the mEET gene markedly attenuated activity of nuclear NF-kappaB. In summary, we have first demonstrated that estrogen-imposed inhibition of MCP-1 expression occurs through the activation of the mEET gene, its product suppresses nuclear NF-kappaB and negatively regulates MCP-1 gene activation.

Modulation of protein kinase signaling by protein phosphatases and inhibitors.

Protein tyrosine phosphatases (PTPs) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTP activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases. In addition to controlling the phosphorylation states of protein kinase substrates, PTPs can also directly modulate protein kinase activity. Evidence suggests that PTPs can exert both positive and negative effects on a signaling pathway. Thus, further understanding of the fundamental role of protein tyrosine phosphorylation in complex and critical signal transduction pathways requires detailed studies of both the kinases and the phosphatases. In this review, we first summarize our current understanding of PTP structure and function. We then discuss the molecular basis of PTP substrate specificity, focusing primarily on mitogen-activated protein (MAP) kinase phosphatase 3. We demonstrate that the MAP kinase phosphatases display exquisite substrate specificity requiring extensive protein-protein interactions for precise down-regulation of MAP kinase activity. We also highlight our recent progress in developing small molecule PTP1B inhibitors. Using a novel combinatorial approach that is designed to target both the active site and a unique peripheral site in PTP1B, we have obtained a PTP1B inhibitor with 2.4 nM affinity and orders of magnitude selectivity against a panel of PTPs. Currently, some of the compounds are being evaluated in both cell and animal models to further define the role of PTP1B in insulin signaling.

Design and characterization of an improved protein tyrosine phosphatase substrate-trapping mutant.

Although members of the protein tyrosine phosphatase (PTPase) family share a common mechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involved can be both highly specialized and fundamentally important. Identification of cellular PTPase substrates will help elucidate the biological functions of individual PTPases. Two types of substrate-trapping mutants are being used to isolate PTPase substrates. In the first, the active site Cys residue is replaced by a Ser (e.g., PTP1B/C215S) while in the second, the general acid Asp residue is substituted by an Ala (e.g., PTP1B/D181A). Unfortunately, only a limited number of PTPase substrates have been identified with these two mutants, which are usually relatively abundant cellular proteins. Based on mechanistic considerations, we seek to create novel PTPase mutants with improved substrate-trapping properties. Kinetic and thermodynamic characterization of the newly designed PTP1B mutants indicates that PTP1B/D181A/Q262A displays lower catalytic activity than that of D181A. In addition, D181A/Q262A also possesses 6- and 28-fold higher substrate-binding affinity than those of D181A and C215S, respectively. In vivo substrate-trapping experiments indicate that D181A/Q262A exhibits much higher affinity than both D181A and C215S for a bona fide PTP1B substrate, the epidermal growth factor receptor. Moreover, D181A/Q262A can also identify novel, less abundant substrates, that are missed by D181A. Thus, this newly developed and improved substrate-trapping mutant can serve as a powerful affinity reagent to isolate and purify both high- and low-abundant protein substrates. Given that both Asp181 and Gln262 are invariant among the PTPase family, it is predicted that this improved substrate-trapping mutant would be applicable to all members of PTPases for substrate identification.