Receptor-mediated Endocytic Process Research Articles

Anti-CD123 antibody-modified niosomes for targeted delivery of daunorubicin against acute myeloid leukemia

A novel niosomal delivery system was designed and investigated for the targeted delivery of daunorubicin (DNR) against acute myeloid leukemia (AML). Anti-CD123 antibodies conjugated to Mal-PEG2000-DSPE were incorporated into normal niosomes (NS) via a post insertion method to afford antibody-modified niosomes (CD123-NS). Next, NS was modified with varying densities of antibody (0.5 or 2%, antibody/Span 80, molar ratio), thus providing L-CD123-NS and H-CD123-NS. We studied the effect of antibody density on the uptake efficiency of niosomes in NB4 and THP-1 cells, on which CD123 express differently. Our results demonstrate CD123-NS showed significantly higher uptake efficiency than NS in AML cells, and the uptake efficiency of CD123-NS has been ligand density-dependent. Also, AML cells preincubated with anti-CD123 antibody showed significantly reduced cellular uptake of CD123-NS compared to control. Further study on the uptake mechanism confirmed a receptor-mediated endocytic process. Daunorubicin (DNR)-loaded H-CD123-NS demonstrated a 2.45- and 3.22-fold higher cytotoxicity, compared to DNR-loaded NS in NB4 and THP-1 cells, respectively. Prolonged survival time were observed in leukemic mice treated with DNR-H-CD123-NS. Collectively, these findings support that the CD123-NS represent a promising delivery system for the treatment of AML.

Examination of the Relative Rates of Rituximab-Mediated Loss of CD20 From B Cells Via Trogocytosis Versus Internalization. Implications for Rituximab Therapy of CLL

AbstractAbstract 2404The use of the anti-CD20 monoclonal antibody (mAb) rituximab (RTX) in the immunotherapy of cancer has led to substantial advances in the treatment of B cell malignancies. However, many patients are refractory to RTX therapy and a substantial fraction of responding patients suffer relapses. In some patients the loss or lack of efficacy can be explained because levels of CD20 are substantially reduced on target B cells as a consequence of RTX therapy. This phenomenon may be due to growth and proliferation, over a period of at least several months, of a CD20-negative B cell clone. In contrast, we have reported that levels of CD20 expressed on circulating CLL cells can be reduced > 90% in less than one hour following RTX infusion. These clinical observations, as well as our results seen in vitro and in mouse models, suggest that after the body's effector mechanisms that promote mAb-mediated cell killing are exhausted or saturated, an alternative reaction predominates. RTX-CD20 complexes on targeted B cells are rapidly removed from the B cells by monocytes and macrophages through an Fc receptor-mediated endocytic process known as trogocytosis, or the shaving reaction. Beers et al. recently reported (Blood, 2010) that human CD20 transgenic mouse B cells opsonized with Rit m2a, a mouse IgG2a mAb with RTX activity, internalized both bound mAb and up to half of cell-surface CD20 after incubation for 2 hrs at 37C. They also reported that on average 40% of cell-surface CD20 was internalized when CLL cells opsonized with RTX were similarly treated. It would thus appear that as a consequence of RTX therapy, both trogocytosis by effector cells and direct internalization by B cells can promote loss of CD20 from B cells targeted by RTX. Both of these reactions will reduce the efficacy of RTX therapy, and a key question must focus on the relative rates of trogocytosis and internalization in promoting CD20 loss in vivo.To address these questions we examined human CD20 transgenic mouse 38C13 B cells (a kind gift from Dr. Josee Golay) for RTX-mediated CD20 internalization versus shaving. To test for internalization of cell-bound RTX, CD20+ 38C13 cells were reacted with Al488- or biotinylated RTX for 10 min to 2 hours at 37C, then washed and secondarily probed with the following reagents: 1, Al647 mAb HB43, specific for the Fc region of human IgG; 2, Al488-streptavidin to detect surface-bound biotinylated RTX; 3, Anti-Al488 antibodies that induce quenching of surface-bound Al488 RTX; or 4, The cells were subjected to an acid wash, re-equilibrated at neutral pH, and re-tested for Al488 RTX binding. Our flow cytometry results reveal rapid and stable binding of Al488-labeled or biotinylated RTX to the CD20+ 38C13 cells. After two hours at 37C, the amount of cell-bound RTX that had been internalized, defined by the 4 different assays, was much less than that reported by Beers, and ranged between 5 and 25%, suggesting that the degree of internalization may be model-dependent. Moreover, reaction of acceptor THP-1 cells (a monocytic line) with the RTX-opsonized cells leads to rapid shaving and removal of ~ 90% of bound RTX and CD20 from the 38C13 cells. After just 45 min at 37C in the presence of adhered THP-1 monocytes, there is a 10-fold reduction in fluorescence intensity signal (molecules of equivalent soluble fluorochrome) on the 38C13 cells attributable to either bound Al488 RTX or due to secondary development with Al647 anti-human IgG mAb HB43. Alternatively, Al488 RTX-opsonized CD20+ 38C13 cells were reacted in solution with THP-1 monocytes at varying THP-1/38C13 cell ratios. Within 45 min at 37C at a 3/1 ratio more than half of cell bound Al488 RTX and CD20 were removed from the 38C13 cells, and large amounts of Al488 RTX were found on the acceptor THP-1 cells. Our results indicate that the shaving reaction is considerably more rapid and indeed leads to the removal of much more bound anti-CD20 mAb RTX (and CD20) than the internalization reaction, suggesting that the shaving reaction is most likely primarily responsible for the rapid down-modulation of CD20 that is observed when CLL patients are treated with RTX. Disclosures:No relevant conflicts of interest to declare.

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

Dynamin 2 Regulates Riboflavin Endocytosis in Human Placental Trophoblasts

Riboflavin is thoroughly established to be indispensable in a multitude of cellular oxidation-reduction reactions through its conversion to coenzyme forms flavin mononucleotide and flavin adenine dinucleotide. Despite its physiological importance, little is known about specific mechanisms or proteins involved in regulating its cellular entry in humans. Studies involving biochemical modulators and immunological inhibition assays have indirectly revealed that riboflavin internalization and trafficking occurs at least in part through a clathrin-dependent receptor-mediated endocytic process. Here, using a two-tiered strategy involving RNA interference and the overexpression of dominant-negative constructs, we directly show the involvement of this endocytic mechanism through the requirement of the pluripotent endocytic vesicle scission enzyme, dynamin 2 GTPase, in human placental trophoblasts. Similar to the endocytic control ligand, transferrin, riboflavin is shown to exhibit 50% dependence on the functional expression of dynamin 2 for its active cellular entry. Furthermore, this reduced vitamin uptake correlates with >2-fold higher riboflavin association at the cell surface. In addition, fluorescent ligand endocytosis assays showing colocalization between rhodamine-riboflavin and the immunostained caveolar coat protein, caveolin 1, suggest that the active absorption of this important nutrient involves multiple and distinct endocytosis pathways.

CAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process.

Riboflavin (RF, vitamin B(2)), an essential micronutrient central to cellular metabolism through formation of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors, is internalized, at least in part, via a proposed receptor-mediated endocytic (RME) process. The purpose of this study was to delineate the cellular RF distribution using human placental trophoblasts and evaluate the regulatory role of cAMP in this process. Subcellular fractionation and three-dimensional confocal microscopy analyses were carried out to define the RF accumulation profile. Biochemical assays evaluating the cAMP dependence of this pathway were also performed. This study records an intracellular RF distribution pattern that shows dynamic accumulation of the ligand predominantly in the endosomal and lysosomal compartments and to a lesser extent in the Golgi and mitochondria. In contrast, transferrin (TF) colocalizes rapidly within endosomes with minimal accumulation in the other organelles. The temporal and spatial distribution of RF and TF colocalized with unique markers of the endocytic machinery provides added morphological evidence in support of the RME process with ultimate translocation to the mitochondrial domain. Colocalized staining with the Golgi also suggests a possible recycling or exocytic mechanism for this ligand. Furthermore, this study demonstrates cAMP regulation of the putative ligand-bound RF receptor and its association into endocytic vesicles. Delineating the dynamics of the process governing cellular RF homeostasis presents an untapped resource that can be further exploited in improving our current understanding of nutritional biology and fetal growth and development, and perhaps in targeting the endogenous system for developing novel therapeutic approaches.

Anopheles gambiae lipophorin: Characterization and role in lipid transport to developing oocyte

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here, we describe purification and characterization of the lipid–apolipoprotein complex, lipophorin (Lp), in the malaria vector mosquito Anopheles gambiae. We also describe the Lp-mediated lipid transfer to developing eggs and the distribution of the imported lipid in developing embryos. The density of the Lp complex was 1.135 g/ml with an apparent molecular weight of 630 kDa. It is composed of two major polypeptides, apoLp I (260 kDa) and apoLp II (74 kDa) and composed of 50% protein, 48% lipid and 2% carbohydrate (w/w). Hydrocarbon, cholesterol, phosphatidyl choline, phosphatidyl ethanolamine, cholesteryl ester and diacylglyceride were the major Lp-associated lipids. Using fluorescently tagged lipids, we observed patterns that suggest that in live developing oocytes, the Lp was taken up by a receptor-mediated endocytic process. Such process was blocked at low temperature and in the presence of excess unlabeled Lp, but not by bovine serum albumin. Imported Lp was segregated in the spherical yolk bodies (mean size 1.8 μm) and distributed evenly in the cortex of the oocyte. In embryonic larvae, before hatching, a portion of the fatty acid in vesicles was found evenly distributed along the body, whereas portion of phospholipids was accumulated in the intestine.

Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity

Azurin is a member of a group of copper-containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature-dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28-amino-acid fragment of azurin fused to glutathione S-transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37 degrees C, but not at 4 degrees C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor-mediated endocytic process. The 28-amino-acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST-GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.

Accumulation of smooth tubular membrane aggregates in the vitellogenic oocytes of an insect, Dysdercus cingulatus

The accumulation of vitellogenins in the oocytes of insects is a receptor-mediated endocytic process, which leads to the formation of yolk granules. We have been studying the hormonal regulation of vitellogenin accumulation and yolk granule formation in the red cotton stainer, Dysdercus cingulatus (Insecta, Hemiptera). During our investigation we observed the appearance of extensive aggregates of smooth tubular membranes in the vitellogenic oocytes. In this report we describe the ultrastructural features of these new cellular organelles.In D. cingulatus (as in other insects) the onset of vitellogenesis (i.e. yolk accumulation) is marked by the appearance of coated pits between the microvilli (Fig. 1). The coated pits transform into electron dense coated vesicles upon invagination into the cortical ooplasm and their subsequent fusion leads to the formation of yolk granules. At a later stage the fusion and continuous growth of yolk granules gives rise to large yolk platelets. During the early phase of yolk accumulation the cortical ooplasm also contains non-coated vesicles of different size in a large number (Fig. 1).

Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: possible function in insulin processing in the blood-brain barrier.

The binding of 125I-insulin to primary cultures of bovine brain microvessel endothelial cells was examined. Insulin binding was both time and temperature dependent and inhibited by excess unlabeled insulin. Furthermore, the specific binding of insulin was polarized to the apical side of the cell monolayers. Upon binding, the labeled insulin was internalized, with approximately 70% resistant to acid wash over a 90-min period. The inhibition of insulin internalization observed with cell monolayers exposed to either phenylarsine oxide or unlabeled insulin suggests a receptor-mediated endocytic process. Furthermore, the ability of chloroquine to reduce the metabolism of insulin indicates a significant portion of the peptide is processed through a lysosomal pathway. In contrast to the fluid-phase endocytosis marker, Lucifer yellow, as much as 65% of internalized insulin undergoes apical to basolateral trancytosis in brain microvessel endothelial cells. While most of the effluxed insulin was degraded, as assessed by trichloroacetic acid precipitation, the results of the present study suggest insulin receptors within the brain microvasculature may be involved in the processing and transport of blood-borne insulin.

Erythrocyte membrane abnormalities in sickle cell disease.

A large fraction of circulating sickle red cells contain one or more large vesicles which are not found in normal erythrocytes. These vesicles contain very high levels of Ca2+, and probably account for the long-known elevation of cellular Ca2+ in sickle cells. These vesicles contain the plasma membrane CaATPase and leak Ca2+ by a nitrendipine-sensitive pathway. The question of whether an abnormal endocytic process occurs in sickle cells which could give rise to these vesicles was examined using the nonspecific endocytic marker Lucifer yellow (LY). The kinetics of formation of LY-labeled endocytic vesicles in sickle cells includes a slow component which is not found in normal (or sickle) reticulocytes. In addition, the number of sickle cells in which endocytosis can be demonstrated with this nonspecific marker consistently exceeds the number which can be labeled with markers of the receptor-mediated endocytic process. The results suggest that a slow, abnormal endocytosis takes place in sickle cells which may be the source of the Ca2(+)-containing vesicles, and therefore of the elevated Ca2+ levels characteristic of the circulating cells in this disease.

Drug targeting to the liver with lactosylated albumins: Does the glycoprotein target the drug or is the drug targeting the glycoprotein?

The isolated perfused rat liver preparation was employed to study hepatic disposition of the model drug-carrier conjugate fluorescein-lactosylated albumin (F-LnHSA) with special reference to the influence of the organic anion fluorescein on liver cell specificity of the endocytosed neoglycoprotein. Hepatic clearance of fluoresceinated neoglycoproteins was significantly faster than clearance of radioiodinated neoglycoproteins. Perfusate clearance of F-L7HSA and F-L25HSA could not completely be inhibited by a dose of 10 mg asialoorosomucoid that saturates the hepatocyte receptor-mediated endocytic process. From these data, we inferred an additional hepatic uptake mechanism, competing with the Ashwell-receptor-mediated internalization of galactose-terminated glycoproteins. Clearance experiments with fluoresceinated 125I-human serum albumin in the presence of the polyanionic probe dextran sulfate revealed a nearly complete (approximately 90%) inhibition of hepatic uptake, while also a pronounced effect was obtained with colloidal carbon. These data point to nonparenchymal cell uptake of fluoresceinated protein via interaction with scavenger receptors. In wash-out studies, it was shown that about 25% of ligand sequestrated by sinusoidal liver cells escaped degradation and recycled to the perfusion medium. Our results show that care should be taken in the use of neoglycoproteins as drug carriers to hepatocytes, since a load of only 2 to 3 moles fluorescein per mole neoglycoprotein considerably affects intrahepatic distribution. The relative contribution of nonparenchymal cell uptake by coupling of acidic drugs to the neoglycoproteins is very probably inversely related to the number of exposing galactose groups per molecule neoglycoprotein. This phenomenon of "inversed targeting" could therapeutically both be useful or detrimental, dependent on the spectrum of cell types that should be reached by the drug.

A specific subunit of vitellogenin that mediates receptor binding.

Vitellogenin, an estrogen-induced serum protein synthesized in the liver, is composed of two Mr 250K polypeptides. It is specifically transported by a receptor-mediated endocytic process into the developing oocytes of virtually all oviparious animals. Following endocytosis, in the chicken, vitellogenin is specifically processed to yield several smaller products including the phosvitins (PV) and the lipovitellins (LV). These products are then stored within the oocyte until they are degraded during embryogenesis to provide nutrients for the developing embryo. Direct binding studies using iodinated vitellogenin demonstrate that vitellogenin binds to isolated oocyte membranes with a KD of 2.5 microM. Competition studies indicate that PV is a competitive inhibitor of vitellogenin binding. This leads us to propose that the PV portion of the circulating vitellogenin molecule mediates binding and uptake. Direct binding studies using iodinated PV show that PV binds to isolated oocyte membranes with a KD of 2.4 microM. Competition studies also demonstrate that 3.1 microM vitellogenin inhibits 50% of control 125I-PV binding, but IgG and bovine serum albumin at concentrations up to 10 microM have no effect on 125I-PV binding. Another series of competition experiments using a constant amount of vitellogenin and increasing amounts of 125I-PV indicate that vitellogenin acts as a competitive inhibitor of PV binding and has a KI of 2-3 microM. These results support our hypothesis that the receptor which mediates vitellogenin binding and uptake recognizes determinants on the PV portion of the native vitellogenin molecule.