Transferrin Receptor Antibody Research Articles

Abstract 2654: Transferrin receptor targeting PRINT® nanoparticles: Chemotherapeutic-free nanomedicine for B-cell lymphoma

Abstract Transferrin receptor (TfR, CD71) is an essential protein involved in cellular iron uptake and regulation of cell growth. This study is to convert PRINT® nanoparticles to targeted drug delivery system by conjugating TfR ligands to particle surface, taking advantage of TfR overexpression on many types of malignant tissues. 200 nm × 200 nm cylindrical PRINT nanoparticles with poly(ethylene glycol)-based composition were fabricated and labeled with transferrin receptor antibody (mouse anti-human TfR mAb, clone OKT-9) or human transferrin (hTf) via biotin-avidin binding or direct chemical conjugation strategies. Nanoparticles coated with OKT9 or hTf, but not isotype control mouse IgG1 or bovine transferrin (bTf), showed highly specific TfR-mediated uptake by all human tumor cell lines tested in this study, including solid tumors and disseminating B cell lymphomas. Non-cancerous HEK293 cell that expresses low level of surface TfR, in contrast, took up particles at much lower rate during short exposure to TfR-targeting particles. The targeting efficiency was dependent on particle concentration, ligand density, particle dosing time and cell surface receptor level. Interestingly, the PRINT nanoparticles coated with OKT9 or hTf showed little cytotoxicity on all solid tumor cell lines tested but were toxic to Ramos B-cell lymphoma. More than 70% of the Ramos cells were killed by OKT9 or hTf coated nanoparticles at a concentration of 100 µg/mL. In contrast, free OKT9 or hTf did not induce death of Ramos cells. There was a strong correlation between TfR ligand density on particle surface and cell viability. Elevated caspase 3 activity indicates the activation of apoptosis pathways upon particle treatment. Iron supplementation suppressed toxicity of NP-OKT9 but not NP-hTf, suggesting different mechanisms by which hTf and OKT9 exerts cytotoxicity on Ramos cells. Further study of cell death mechanism through DNA microarray analysis is under way. Overall, we have shown that human transferrin and transferrin receptor antibodies can be useful targeting ligands for nanoparticle delivery to tumors. More importantly, non-toxic transferrin and TfR antibodies can be turned into a potential chemotherapeutic-free medicine for B-cell lymphoma through multiplexing with PRINT nanoparticles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2654.

Abstract 3854: Newly designed nanobioconjugate for direct targeting and systemic treatment of HER2-positive breast cancer

Abstract Objective: We have developed an efficient nanobipolymer drug delivery system Polycefin based on natural-derived biodegradable, non-toxic and non-immunogenic polymalic acid (PMLA) platform. We have shown that Polycefin was able to block brain tumor growth and angiogenesis. Our goal is to engineer the new Polycefin variant for systemic (I.V.) treatment of HER2-positive breast tumors in order to enhance the efficacy of trastuzumab/Herceptin®. Materials and Methods: For in vitro and in vivo treatment, we used (1) Herceptin, (2) Antisense oligonucleotides (AON) to HER2/neu, (3) three versions of Polycefin carrying either (3a) Herceptin alone, (3b) anti-mouse (M) and anti-human (H) transferrin receptor antibodies and AON (P-AON-TfRM+H), (3c) combination of Herceptin, anti-mouse TfR, and AON (P-AON-Herceptin-TfRM), and (4) PBS as a control. Anti-mouse TfR antibody mediated polymer transcytosis through tumor vascular endothelial cells, and Herceptin (and anti-human TfR) ensured polymer binding and internalization by tumor cells. HER2 overexpressing (BT-474, SKBR-3) and HER2 low-expressing (MDA-MB-231, MDA-MB-435) breast cancer cells were used to evaluate cell viability in vitro after treatments. Western blot analysis of cell lysates upon various treatments was used to detect HER2, phosphorylated Akt, and cleaved PARP expression. For in vivo study, BT-474 cells were inoculated S.C. into nude mice. Drugs were injected I.V. and tumor size was measured. For Polycefin distribution, drugs labeled with Alexa Fluor 680 were I.V. injected and tumors and major organs were harvested in 24 hours. Drug accumulation was analyzed by Xenogen IVIS 200 imager and subsequent confocal microscopy. Results: Polycefin variant P-AON-Herceptin-TfRM (3c) inhibited tumor cell growth in vitro significantly more than Herceptin (P<0.01 for HER2 overexpressing cells and P<0.02 for HER2 low-expressing cells). It also displayed a stronger inhibition of HER2 expression and Akt phosphorylation leading to apoptosis (cleaved PARP increase by Western blot analysis) than Herceptin alone or any other Polycefin variant (3a or 3b). In vivo imaging revealed that drug accumulation was markedly enhanced in tumors treated with P-AON-Herceptin-TfRM (3c) compared to other treatments. The same Polycefin version P-AON-Herceptin-TfRM (3c) produced the strongest reduction of tumor size compared to PBS or Herceptin (P<0.001). Summary: The leading version of nanobioconjugate with covalently attached combination of TfRM, Herceptin (anti-HER2 antibody) and anti-HER2 AON showed significant anti-tumor effect on HER2-positive breast cancer by enhancing tumor targeting, inhibition of HER2 synthesis and signaling, as well as suppression of tumor growth as compared with Herceptin treatment alone. The new version of nanoconjugate Polycefin could be promising next generation therapy for HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3854.

Aph-1 Associates Directly with Full-length and C-terminal Fragments of γ-Secretase Substrates

γ-Secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid β-peptide from the β-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the γ-secretase complex. Here, we provide evidence that both the β-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of γ-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an β-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical γ-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (β, αS, and αL), as they were all able to bind β-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for γ-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with γ-secretase substrates prior to their processing by the proteolytic complex.

Commentary [Research Highlights

Drug delivery to the central nervous system remains a vexing problem because of the presence of the blood-brain barrier (BBB), whose endothelial cell tight junctions limit the paracellular flux of hydrophilic molecules. One solution may be vector-mediated delivery to the brain. This employs chimeric peptide technology, wherein the drug is conjugated to a molecular carrier of protein nature (e.g. cationized albumin or a transferrin receptor antibody). Transferrin receptor antibodies selectively target BBB endothelium due to the high levels of receptor expressed by these cells, thereby triggering receptor-mediated transport across the BBB via transcytosis. Chitosan, a naturally occurring, abundant, and biocompatible polysaccharide has found widespread pharmaceutical application. In particular, the cationic nature of chitosan facilitates its interaction with negative charges on the brain endothelium, Building on their previous work on brain-deliverable chitosan-polyethylene glycol nanoparticles functionalized with monoclonal anti-transferrin antibody, Karatas and colleagues have now designed caspase-3 inhibitor-loaded chitosan nanospheres conjugated with an anti-mouse transferrin receptor monoclonal antibody that selectively recognizes the transferrin receptor type 1 on the cerebral vasculature. Caspase-3 has been implicated in neuronal cell death in cerebral ischemia, as well as in the pathophysiology of other neurological disorders. When given intravenously, the caspase-3-loaded nanospheres were rapidly transported across the BBB and dose-dependently decreased infarct volume, neurological deficit, and ischemiainduced caspase-3 activity in mice subjected to focal cerebral ischemia/reperfusion. Because significant opening of the BBB starts only 6 h after ischemia/reperfusion, the rapid transfer of nanospheres to the brain was not likely caused by an increased permeability of the BBB. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17, after closure of the BBB. These observations convincingly demonstrate that transferrin monoclonal antibody-conjugated chitosan nanospheres are an efficient delivery system for bringing neuroprotective, small peptide drugs into the brain. This opens intriguing new avenues for the treatment of central nervous system disorders in which the administration of therapeutic agents is handicapped by insufficient diffusion across the BBB. Further studies are eagerly awaited to see if this technology will be applicable to polypeptide factors, for example, brain-derived neurotrophic factor and ciliary neurotrophic factor.

Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders

Brain-targeted Tempol-loaded poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) conjugated with a transferrin antibody (OX 26) were developed using the nanoprecipitation method. These NPs may have utility in treating neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Central to these diseases is an increased production of reactive oxygen and nitrogen species which may take part in the development of these conditions. As proof of principle, the NPs were loaded with Tempol, a free radical scavenger that has been shown to be protective against oxidative insults. To enhance the delivery of NPs to the central nervous system (CNS), we conjugated the transferrin receptor antibody covalently to PLGA NPs using the NHS-PEG3500-Maleimide crosslinker. The NPs showed a particle size suitable for blood brain barrier (BBB) permeation (particle size 80–110 nm) and demonstrated a sustained drug release behavior. A high cellular uptake of antibody-conjugated NPs was demonstrated in RG2 rat glioma cells. The ability of the Tempol-loaded NPs to prevent cell death by resveratrol in RG2 cells was determined using the MTT assay. The conjugated NPs containing Tempol were more effective in preventing cell viability by resveratrol when compared with unconjugated NPs or free Tempol in solution. Our findings suggest that transferrin-conjugated NPs containing antioxidants may be useful in the treatment of neurodegenerative diseases.

Differential effects of transferrin receptor antibodies on growth and receptor expression of human lymphocytic and myelocytic cell lines.

J64, a monoclonal antibody against the human transferrin receptor, has been shown to induce interleukin-2 production by HUT78 cells. It also causes growth inhibition of several cell lines and stimulated lymphocytes. These effects were also present using transferrin-free culture conditions. In this paper, we dissect cell membrane and intracellular events after binding of J64 and other transferrin receptor antibodies. Incubation of HUT78 and several other cell lines with J64 resulted in an increased number of receptor molecules expressed on the cell surface in contrast to a downmodulation seen with other monoclonal antibodies to the transferrin receptor. This upregulation after treatment with J64 was not due to an increased concentration of transferrin receptor mRNA in these cells or a higher protein synthesis rate. We therefore suggest that J64 causes a redistribution of transferrin receptor molecules from intracellular pools to the cell surface. Additional experiments investigating signal transduction mechanisms revealed no influence of J64 on intracellular Ca2+ concentrations or translocation of protein kinase C. However, an increase of transferrin receptor phosphorylation was seen in HL60 cells after treatment with phorbolester or J64. This phosphorylation of the transferrin receptor might be a signal transduction pathway involved in activation and growth control.

Morphological definition of CD71 positive reticulocytes by various staining techniques and electron microscopy compared to reticulocytes detected by an automated hematology analyzer

Background The enumeration of peripheral blood reticulocytes plays an important part in clinical hematology. Although reticulocyte enumeration is currently performed with visible dyes such as New Methylene Blue (NMB), fluorescent dyes, or anti-CD71 (transferrin receptor) antibody, it has not been demonstrated whether the reticulocytes detected in each method are the same or not. Methods We prepared the reticulocyte rich fraction with density gradient centrifugation, stained with both anti-CD71 and Sysmex's fluorescent stain RET SEARCH (II)™, and detected the cells by both confocal laser scanning microscopy and flow cytometry. We also stained the reticulocyte rich fraction and the CD71 + reticulocytes with NMB and compared them by microscopy. We also observed the CD71 + reticulocytes by electron microscopy. Results Almost all CD71 + reticulocytes were intensely stained with both NMB and RET SEARCH (II)™. These cells were therefore classified as highly immature reticulocytes. During the stages of reticulocyte maturation, the expression of CD71 antigen decreased prior to the reduction of the reticular structures. The electron microscopic observation showed that CD71 + reticulocytes had some typical morphological characteristics found in highly immature reticulocytes. Conclusions The CD71 + reticulocytes consisted of highly immature reticulocytes and were not equal to the reticulocytes defined with NMB or RET SEARCH (II)™.

Direct tumor targeting using nanobioconjugate with a combination of monoclonal antibodies for breast cancer treatment.

Abstract Abstract #2124 Objective: Nanobioconjugate engineered based on polymalic acid (PLMA), which is derived from slime mold that is non-toxic, non-immunogenic, and biodegradable. Using combination of targeting antibodies, the nanopolymer candidates for drug delivery can efficiently reach breast cancer without affecting normal tissue in vivo. Controllable, unique drug releases tumor inhibiting agents specifically into cancer cells without affecting normal surrounding cells. Active drug blocks several cancer-specific tumor markers at the same time (Ljubimova JY, Nanomedicine 2008). We evaluated whether PMLA with covalently attached antibodies to transferrin receptor and nucleosome 2C5 reaches tumor site effectively and provides anti-tumor effect.
 Materials and Methods: Human breast cancer MDA-MB-435 and MDA-MB-231 cell lines were inoculated subcutaneously in the right thighs of nude mice. When tumors grew to 5 mm, each drug variant was injected intravenously. The nanoconjugates had different antibodies attached: non-immune IgG as a negative control (P-IgG), anti-human transferrin receptor (TfR) antibody (P-H), anti-human and anti-mouse TfR antibodies (P-H/M), and anti-tumor 2C5 antibodies (P-2C5). All drug variants were labeled with Alexa Fluor 680 tracking dye. For assessment of drug distribution at different time points and localization in various organs after intravenous injection, Xenogen IVIS 200 imager was used. The tumor and major organs were harvested after 24 hours to analyze drug distribution. Confocal microscopy of frozen sections was used for histological confirmation of drug accumulation in tissues.
 Results: Anti-mouse TfR antibody delivered nanoconjugate across the mouse endothelial system and the drug reached the tumor site specifically through anti-human TfR or anti-tumor 2C5 antibodies. The targeting efficacy of Polycefin variants was quantitatively compared against P-IgG. Weak P-IgG signal was detected in tumor probably based on non-specific enhanced permeability and retention effect. Drugs P-H and P-M/2C5 with antibodies for active targeting showed high accumulation in human tumor (p=0.01 vs. P-IgG). The best breast tumor targeting was obtained through P-H/M variant bearing a combination of anti-mouse and anti-human TfR antibodies (p=0.0001 vs. P-IgG) The drug candidate is selected to block several molecular targets specific for breast tumors treatment Her-2, EGFR or tumor endothelial marker laminin 411.
 Summary: Nanobioconjugate with a combination of anti-TfR and 2C5 antibodies showed strong tumor targeting effect in vivo. Targeting effect of antibody was more effective when the different targeting antibodies were combined than when the single antibody was used. Anti-nucleosome antibody 2C5 and TfR antibody along with polymalic acid are promising targeting tools for drug delivery. Currently, we are conducting in vivo study attaching antisense oligos/siRNA to universal nanoplatform, PLMA, to block the molecular marker such as HER-2, EGFR and laminin 411 to suppress the breast tumor growth. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2124.

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II α in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 ( Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19, 1415-1426 ). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIalpha inclusion into AP-3 complexes. BLOC-1, PI4KIIalpha, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIalpha, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIalpha with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIalpha along the endocytic route.

Intravenous siRNA of Brain Cancer with Receptor Targeting and Avidin–Biotin Technology

The effective delivery of short interfering RNA (siRNA) to brain following intravenous administration requires the development of a delivery system for transport of the siRNA across the brain capillary endothelial wall, which forms the blood-brain barrier in vivo. siRNA was delivered to brain in vivo with the combined use of a receptor-specific monoclonal antibody delivery system, and avidin-biotin technology. The siRNA was mono-biotinylated on either terminus of the sense strand, in parallel with the production of a conjugate of the targeting MAb and streptavidin. Rat glial cells (C6 or RG-2) were permanently transfected with the luciferase gene, and implanted in the brain of adult rats. Following the formation of intra-cranial tumors, the rats were treated with a single intravenous injection of 270 microg/kg of biotinylated siRNA attached to a transferrin receptor antibody via a biotin-streptavidin linker. The intravenous administration of the siRNA caused a 69-81% decrease in luciferase gene expression in the intracranial brain cancer in vivo. Brain delivery of siRNA following intravenous administration is possible with siRNAs that are targeted to brain with the combined use of receptor specific antibody delivery systems and avidin-biotin technology.

Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(β- l-malic acid)

Tumor-specific targeting using achievements of nanotechnology is a mainstay of increasing efficacy of anti-tumor drugs. To improve drug targeting we covalently conjugated for the first time two different monoclonal antibodies, an anti-mouse transferrin receptor antibody and a mouse autoimmune anti-nucleosome antibody 2C5, onto the drug delivery nanoplatform, poly(β- l-malic acid). The active anti-tumor drug components attached to the same carrier molecule were antisense oligonucleotides to vascular protein laminin-8. The resulting drug, a new Polycefin variant, was administered intravenously into glioma-bearing xenogeneic animals. The drug delivery system was targeted across mouse endothelial system by the anti-mouse transferring receptor antibody and to the tumor cell surface by the anti-nucleosome antibody 2C5. The targeting efficacies of the Polycefin variants bearing either two antibodies or each single antibody were compared in vitro and in vivo. ELISA confirmed the co-existence of two antibodies on the same nanoplatform molecule and their functional activities. Fluorescence imaging analysis after 24 h of intravenous injection demonstrated significantly higher tumor accumulation of Polycefin variants with the tandem configuration of antibodies than with single antibodies. The results suggest improved efficacy for tandem configuration of antibodies than for single configurations carried by a drug delivery vehicle.

Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis

We have previously shown that laminin-8, a vascular basement membrane component, was overexpressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 alpha4 and beta1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.

Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.

Expression of a KCC1 Splice Variant Is Suppressed by NF-ΚB in Erythroid Cells.

The K+Cl- cotransporter (KCC) plays a significant role in the maintenance of red cell volume. Activity of the cotransporter is higher in sickle (SS) compared to normal (AA) reticulocytes, and contributes to SS dehydration. Thus, KCC is considered a potential modifier gene for sickle cell disease (SCD). We have demonstrated the presence of transcripts for KCC1, KCC3, and KCC4 in human reticulocytes (Exp.Hem. 2005; 33:624–31) and shown that one splice variant of KCC1 (KCC1ex1b), which codes for a protein with a small (7aa) alternative N-terminal exon, is detected in AA, but not SS reticulocytes. Studies with murine KCC1 have demonstrated that proteins produced by N-terminal truncation are inactive for K+Cl- cotransport, and function as dominant negative regulators of full-length KCC1 and KCC3 proteins. Since high level expression of this variant in AA cells compared to SS cells might explain the relatively low KCC activity in AA reticulocytes, we have identified the promoter for the KCC1ex1b transcript and investigated the regulatory elements that control its expression. Here we report the involvement of TNFα and NF-ΚB in the transcriptional regulation of the KCC1ex1b variant. Although KCC1ex1b is not expressed in reticulocytes isolated from sickle cell patients, we found that SS erythroid precursor cells cultured in vitro express this variant. SS and AA peripheral blood mononuclear cells were cultured in semi-liquid media with stem cell factor and erythropoietin, and collected after 5, 10, and 14 days in culture. Cells harvested at 14 days and isolated by binding to micromagnetic beads coated with transferrin receptor antibody were 95–98% positive for glycophorin A. RNA was extracted and analyzed by semi-quantitative RT-PCR, using primers for KCC1ex1 and KCC1ex1b. In both AA and SS cells, the transcript level for KCC1ex1b rose over the time in culture, while the KCC1ex1 transcript was constant. This difference between the in vitro and in vivo expression patterns for the KCC1ex1b variant could be explained by regulation via an external factor, such as a cytokine present in the blood of sickle cell patients, but absent in the in vitro culture system. The levels of numerous cytokines, including TNF, VEGF, and various interleukins, are elevated in SCD. We therefore assayed the effect of TNFα on endogenous KCC1ex1b expression in K562 cells by RT-PCR analysis at 24 and 48 hours after the addition of TNFα to the tissue culture medium. The steady-state mRNA levels of the KCC1ex1b variant decreased approximately 40% in response to TNF treatment. The transcription factor NF-ΚB is activated by TNF signaling, and an NF-ΚB consensus site is present in the KCC1ex1b promoter region. We assayed the effect of co-expressing NF-ΚB and our KCC1ex1b promoter constructs in K562 cells. NF-ΚB expression produced an 8-fold decrease in luciferase activity from these promoter constructs indicating NF-ΚB transcriptionally represses this promoter, either directly or indirectly. Our current model proposes that induction or modulation of the expression of the KCC1ex1bvariant could be an important factor in the control of red cell hydration.

The SH3 Domain of Alpha Spectrin Binds to Galectin-1 during Erythroid Differentiation.

A major function of the spectrin skeleton in erythrocytes is to provide mechanical support for the membrane bilayer and allow survival of these cells in the circulation. An interesting feature of spectrin is that the alpha subunit contains a highly conserved Src homology 3 domain (SH3) of unknown function. The SH3 domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. A possible direct role of spectrin in signal transduction, recruiting or activating signaling molecules through the SH3 domain, has been recently explored. It seems that spectrin and its SH3 domain promote Rac activation in the specialized integrin cluster which initiate cell adhesion and spreading. In the present study, we used the yeast two-hybrid system to screen a human bone marrow cDNA library, looking for a protein that interacts with a 62 aa sequence containing the SH3 domain of spectrin, which comprises Ala977 to Glu1038 of alpha-spectrin protein (SPTA1). We screened 5.9 x 105 clones from a human bone marrow cDNA library. Thirteen positive clones were obtained, of which two contained the region K51-K123 of Niemann Pick C2 protein (NP-C2), one contained the region A2-M121 of the galectin 1 (LGALS1) and one contained the region P4-F128 of interleukin 2 receptor gamma (IL2RG). We considered the possible interaction between spectrin and galectin-1 (GAL1), a beta-galactoside-binding lectin involved in cell cycle progression. Galectin-1 over expression is associated with neoplastic transformation and loss of differentiation. In order to verify the physical association between galectin-1 and erythroid spectrin we differentiated erythroid progenitor cells obtained from peripheral blood mononuclear cells to erythroblasts stages, subsequent to the proerythroblast stage, using a two phase liquid culture system. Cells were collected at day zero, 7 and 13 of the second phase. FACS analysis using glicophorin A and transferrin receptor antibodies confirmed the erythroid differentiation at days 7 and 13. Cellular extracts were immunoprecipitated with a goat polyclonal anti-galectin-1 antibody (Santa Cruz) and the proteins were submitted to 6% SDS-PAGE and transferred to a nitrocellulose membrane which was immunobloted with a rabbit polyclonal anti-spectrin alpha 1 antibody (Santa Cruz). At the 13th day of differentiation, the immunoblotting showed a 240 kDa band, compatible to the alpha spectrin molecular weight, suggesting the physical association between erythroid alpha spectrin and galectin-1. Corroborating our data, Marion et al (Mol Biochem Parasitol. 2004, 135(1):31–8) recently described a protein from the spectrin family co-localizing with the Gal-GalNAc lectin in Entamoeba histolytica and confirmed this interaction by in vitro assay. The acquisition of an erythroid phenotype is associated with externalization of galectin-1. Spectrin may have a role in initiating the externalization of galectin-1, an event related to late stages of erythroid differentiation and this association might involve conformational changes mediated by SH3 domain of erythrocyte alpha spectrin. The autocrine binding of galectin-1 to cell surface ligands of nonadherent cells such as K562 suggest that galectin-1 is implicated in signal transduction rather than in cell-cell or cell-matrix interaction. In conclusion, this is the first report identifying the interaction between galectin-1 and the SH3 domain of alfa-spectrin in erythroid cell.

The v-SNARE Vti1a Regulates Insulin-stimulated Glucose Transport and Acrp30 Secretion in 3T3-L1 Adipocytes

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Targeting Anti—Transferrin Receptor Antibody (OX26) and OX26-Conjugated Liposomes to Brain Capillary Endothelial Cells Using In Situ Perfusion

Brain capillary endothelial cells (BCECs) express transferrin receptors. The uptake of a potential drug vector (OX26, or anti-transferrin receptor antibody IgG2a) conjugated to polyethyleneglycol-coated liposomes by BCECs was studied using in situ perfusion in 18-day-old rats in which the uptake of OX26 is almost twice as high as in the adult rat. Using radio-labeling, the uptake of OX26 by BCECs after 15-minute perfusion was approximately 16 times higher than that of nonimmune IgG2a (Ni-IgG2a). OX26 and OX26-conjugated liposomes selectively distributed to BCECs, leaving choroid plexus epithelium, neurons, and glia unlabeled. Ni-IgG2a and unconjugated liposomes did not reveal any labeling of BCECs. The labeling of BCECs by OX26 was profoundly higher than that of transferrin. Perfusion with albumin for 15 minutes did not reveal any labeling of neurons or glia, thus confirming the integrity of the blood-brain barrier. The failure to label neurons and glia shows that OX26 and OX26-conjugated liposomes did not pass through BCECs. The expression of transferrin receptors by endothelial cells selective to the brain qualifies OX26 as a candidate for blood-to-endothelium transport. A specifically designed formulation of liposomes may allow for their degradation within BCECs, leading to subsequent transport of liposomal cargo further into the brain.

α-Synuclein Up-regulation and Aggregation during MPP+-induced Apoptosis in Neuroblastoma Cells: INTERMEDIACY OF TRANSFERRIN RECEPTOR IRON AND HYDROGEN PEROXIDE

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP(+)-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of alpha-synuclein (alpha-syn) in MPP(+)-induced oxidative stress and apoptosis. Results show that MPP(+) inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H(2)O(2) generation, TfR expression and alpha-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, alpha-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing alpha-syn exacerbated MPP(+) toxicity, whereas antisense alpha-syn treatment totally abrogated MPP(+)-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of alpha-syn in MPP(+)-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP(+)-induced iron signaling is responsible for intracellular oxidant generation, alpha-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed.

1-Methyl-4-phenylpyridinium-induced Apoptosis in Cerebellar Granule Neurons Is Mediated by Transferrin Receptor Iron-dependent Depletion of Tetrahydrobiopterin and Neuronal Nitric-oxide Synthase-derived Superoxide

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP(+) toxicity. Results show that MPP(+) treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP(+) caused a time-dependent depletion of tetrahydrobiopterin (BH(4)) that was mediated by H(2)O(2) and transferrin iron. Depletion of BH(4) decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP(+)-mediated "uncoupling" of nNOS decreased *NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH(4) biosynthesis or cell-permeable iron chelator and TfR antibody to prevent iron-catalyzed BH(4) decomposition inhibited MPP(+) cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP(+)-induced iron uptake, BH(4) depletion, proteasomal inactivation, and apoptosis. We conclude that MPP(+)-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH(4), leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed.

Identification of CD4 and transferrin receptor antibodies by CXCR4 antibody-guided Pathfinder selection.

To generate human antibodies against CXCR4, a seven-transmembrane chemokine receptor and a principal coreceptor for HIV-1, several rounds of Pathfinder and Step-back selection from a large phage display antibody library were performed on Jurkat cells. A mAb against CXCR4 or biotinyated phage antibodies were used as guide molecules. Over 100 pan-Jurkat-cell-positive antibodies were characterized, but none were CXCR4 specific. However, several antibodies against CD4 and the transferrin receptor were identified. Our results indicate that, although Pathfinder and Step-back selection can be used to select phage antibodies on whole cells, the successful selection of certain targets is still complex and limited. The reason is probably, in part, due to the inaccessibility of the targeted extracellular structures and the range of the horseradish peroxidase-labeled guide molecule. Refinements of these techniques are required to improve target specificity and selectivity.