Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Animals ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Transferrin ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; metabolism

Mitochondrial ferritin (MtF), a new player in iron metabolism, first identified in 2001, is highly homologous to ferritin both structurally and functionally. Preliminary studies have suggested that MtF might play very important roles in the regulation of mitochondrial iron homeostasis. Leukemic cells, just like other malignant cells, demand more iron for their greater proliferation potential. However, little is known about what roles MtF might play in leukemic cell iron metabolism and cell proliferation. The aim of this study was to investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during TPA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules. K562 cells cultured with or without TPA (16 nmol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward monocyte lineage was confirmed by microscopic study (Wright's staining) and flow cytometry. Semiquantitative RT-PCR was performed to determine mRNA expression, with house-keeping gene beta-actin as control reference. This study revealed that over 95% of K562 cells showed morphological features of monocyte/macrophage, and the expression of CD64 on cell surface increased significantly at day 5 with TPA treatment. K562 cells could express a certain level of MtF before TPA-induced differentiation. With increase of TPA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 50.3% and 68.2% of that before TPA treatment. While Fn mRNA expression increased to 1.97 folds of that before TPA treatment. It is concluded that MtF expression is downregulated during TPA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.
Antigens, CD ; metabolism ; Cell Proliferation ; Cell Transformation, Neoplastic ; drug effects ; Ferritins ; biosynthesis ; genetics ; Humans ; Iron-Regulatory Proteins ; metabolism ; K562 Cells ; Mitochondria ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo explore the effect of T(3) on the expression of transferrin receptor (TfR) and ferritin (Fn) in K562 cells and its possible mechanism.

METHODSFlow cytometry was used for the detection of TfR expression, radioimmunoassay for Fn expression, RNA/protein band shift assay for the binding activity of iron regulatory protein (IRP) and iron responsive elements (IRE), and RT-PCR for TfR and Fn mRNA levels.

RESULTSDifferent concentration of T(3) significantly increased Fn expression of K562 cells, especially at 100 nmol/L and 200 nmol/L (p < 0.05). However, T(3) had no effect on TfR expression. T(3) decreased the binding activity between IRP and IRE, particularly at concentration of 50 nmol/L. Different concentration of T(3) increased Fn-H mRNA level at different time point while it had no effect on TfR mRNA level.

CONCLUSIONT(3) increased Fn expression of K562 cells through the possible mechanisms of either the post-transcriptional regulation or transcriptional modulation.

Ferritins ; biosynthesis ; drug effects ; genetics ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; Radioimmunoassay ; Receptors, Transferrin ; biosynthesis ; drug effects ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Triiodothyronine ; pharmacology

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics

Transferrin receptor (TR) performs the major function of binding and internalizing its specific iron-loaded ligand, transferrin, and its expression is closely linked to the proliferation status of the cell. This study was undertaken to elucidate TR expression in the hyperplastic lesion of hepatocyte in chemically induced hepatic carcinogenesis. The resistant hepatocyte model was chosen for a rat model of carcinogenesis and Sprague-Dawley rats were divided into the following groups: the control groups of normal diet and iron-rich diet with or without hydroxyquinoline and the groups of carcinogen alone and carcinogen plus iron-rich diet with or without administration of hydroxyquinoline. Microscopic changes in the liver, expression of transferrin receptor and glucose-6-phosphatase were studied. The hepatocyte of the control group showed both cytoplasmic and membranous expression of TR. The liver of rats fed on high iron diet accumulated iron and the expression of TR was down regulated by intrahepatic iron accumulation. In the carcinogen administered group the resistant hepatocyte of hyperplastic lesion revealed strong membranous expression of TR and failed to accumulate iron in spite of high iron diet but in contrast the surrounding non-resistant hepatocyte expressed TR in both the membrane and cytoplasm and stored iron when fed on high iron diet. The strong membranous expression of TR is one of the characteristics of the resistant hepatocyte of hyperplastic lesion and it seems to be related to the inability to accumulate iron in spite of a high iron diet.
Animal ; Glucose-6-Phosphatase/metabolism ; Immunohistochemistry ; Iron/analysis/pharmacology ; Liver/chemistry/enzymology/pathology ; Liver Neoplasms, Experimental/enzymology/*ultrastructure ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Transferrin/*biosynthesis ; Support, Non-U.S. Gov't

OBJECTIVETo explore impaired erythropoiesis and relative inadequacy of erythropoietin production in the anemic cancer patients and the correlation of tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) with inadequate erythropoietin (EPO) response and impaired erythropoiesis in cancer patients with anemia.

METHODSFifty adult anemic and 15 non-anemic tumor patients were studied. Serum EPO levels were measured by radioimmunoassay (RIA) and serum soluble transferrin receptor (sTfR). TNF-alpha and IFN-gamma levels by enzyme-linked immunosorbent assay (ELISA). Log transformed EPO and sTfR values were used in statistical analysis. The R correlation analyses were performed.

RESULTSThe mean serum immunoreactive erythropoietin level in anemic cancer patients [(23.11 +/- 10.00) IU/L] was not significantly higher than in healthy people (P = 0.053), but significantly lower than in IDA patients with similar degree of anemia [(43.00 +/- 22.00) IU/L, P < 0.01]. Both O/P EPO [0.88 (0.54-1.10)] and O/P sTfR [0.89 (0.57-1.22)] were significantly lower in anemic cancer patients than in controls and in non-anemic cancer patients. There was no significant difference between the latter two groups. Furthermore, the expected inverse linear relation between serum EPO and hemoglobin levels was absent in the anemic cancer patients, and so did the relation between serum sTfR and hemoglobin levels. There was no correlation between O/P EPO and O/P sTfR. The serum levels of both TNF-alpha and IFN-gamma in anemic cancer patients [(25.75 +/- 26.71) ng/L, (50.49 +/- 42.12) ng/L, respectively] were significantly higher than those in healthy controls (both P < 0.01) or in nonanemic cancer patients (both 0.01 < P < 0.05), and so did between non-anemic cancer patients and controls. The serum levels of TNF-alpha were inversely correlated with hemoglobin levels (r = - 0.40, P = 0.004), O/P EPO (r = -0.32, P = 0.025) or O/P sTfR (r = -0.36, P = 0.01); while serum levels of IFN-gamma were inversely correlated with hemoglobin levels (r = -0.36, P = 0.01) or O/P sTfR (r = 0.39, P = 0.006), but not with O/P EPO. Conclusions Anemia of cancer is due to impaired erythropoiesis and relative inadequacy of EPO production. TNF-alpha might inhibit EPO production and erythropoiesis, while IFN-gamma maybe directly inhibit erythropoiesis and be independent of EPO response inadequacy.

Adolescent ; Adult ; Aged ; Anemia ; blood ; etiology ; physiopathology ; Erythropoiesis ; physiology ; Erythropoietin ; biosynthesis ; blood ; Female ; Humans ; Interferon-gamma ; blood ; Male ; Middle Aged ; Neoplasms ; complications ; Receptors, Transferrin ; blood ; Tumor Necrosis Factor-alpha ; metabolism

As an essential metal for sustaining life, iron is involved in a number of metabolic processes, including DNA synthesis, electron transport, oxygen delivery, and so on. Iron metabolism involves the absorption, transport, and use of iron and is strictly regulated. Numerous studies have found a positive correlation between iron storage and the risk of tumors, such as colorectal carcinoma, hepatic cancer, renal carcinoma, lung cancer, and gastric cancer. In tumor cells, iron metabolism changes by several mechanisms, such as regulating the growth of tumor cells by transferrin, accelerating the uptake of iron by the overexpressions of transferrin receptors 1 and 2 (TfR1 and TfR2), synthesizing or secreting ferritin by some malignant tumor cells, and upregulating the level of hepcidin in patients with cancer. Some advances on diagnosis and treatment based on iron metabolism have been achieved, such as increasing the transfection and target efficiency of transferrin-polyethylenimine (PEI), inducing cell apoptosis by beta-guttiferin through interacting with TfR1.
Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigens, CD ; genetics ; metabolism ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Apoptosis ; Cell Proliferation ; Doxorubicin ; pharmacology ; Ferritins ; metabolism ; physiology ; Hepcidins ; Humans ; Interleukin-18 ; pharmacology ; Iron ; metabolism ; physiology ; Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptors, Transferrin ; genetics ; metabolism ; Transferrin ; metabolism ; physiology ; Tumor Suppressor Protein p53 ; pharmacology

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors