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1.
Protein Expr Purif ; 137: 26-33, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28651975

ABSTRACT

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 µg and 202.6 µg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins.


Subject(s)
Factor VII , Gene Expression , Cell Line , Factor VII/biosynthesis , Factor VII/genetics , Factor VII/isolation & purification , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
2.
Bioengineered ; 7(3): 189-97, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27116572

ABSTRACT

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.


Subject(s)
Cloning, Molecular/methods , Factor VII/genetics , Immunoglobulins/genetics , Protein Sorting Signals/genetics , Transcription, Genetic , Animals , Batch Cell Culture Techniques , CHO Cells , Cell Count , Cell Cycle/genetics , Cell Survival , Cricetulus , Culture Media/chemistry , Factor VII/biosynthesis , Gene Expression Regulation , Immunoglobulins/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Replication Origin
3.
Cell Transplant ; 24(12): 2541-55, 2015.
Article in English | MEDLINE | ID: mdl-25622096

ABSTRACT

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Hepatocytes/transplantation , Liver Diseases/therapy , Organ Preservation Solutions/pharmacology , Adult , Aged , Albumins/biosynthesis , Animals , Cell Proliferation , Cell Survival , Factor VII/biosynthesis , Female , Ganciclovir/adverse effects , Hepatocytes/cytology , Humans , Liver/cytology , Male , Mice , Mice, Inbred BALB C , Middle Aged , Serum Albumin/analysis , Spleen/cytology , Transplantation, Heterologous , Urea/metabolism
4.
Thromb Res ; 131(5): 444-9, 2013 May.
Article in English | MEDLINE | ID: mdl-23566532

ABSTRACT

INTRODUCTION: Factor VIII (FVIII) treatment for hemophilia A has difficulties in correcting bleeding diathesis in the presence of inhibitors. MATERIALS AND METHODS: An adeno-associated virus type 8 (AAV8) vector containing the factor VII (FVII) gene or the activated factor VII (FVIIa) gene was used to investigate the therapeutic effect of FVII or FVIIa overexpression in FVIII-deficient mice with inhibitors. RESULTS: Following repeated human FVIII injection, FVIII-deficient mice developed anti-human FVIII antibodies that cross-reacted with mouse FVIII. High transgene expression of murine FVII or murine FVIIa was achieved using the AAV8 vector and resulted in increased blood FVII activity greater than 800% of normal murine FVII levels in vector-injected FVIII-deficient mice. Thromboelastography analysis showed significant improvements in clotting time, clot formation time, α angle, and mean clot firmness in AAV8 vector-injected FVIII-deficient mice with inhibitors. Overexpression of FVIIa ameliorated the bleeding phenotype of FVIII-deficient mice with inhibitors and significantly increased the survival rate after tail clipping. In addition, overexpression of FVII increased the survival rate of FVIII-deficient mice with inhibitors after tail clipping though it was not as efficient as FVIIa overexpression. CONCLUSIONS: These data suggest that FVII overexpression is an alternative strategy for the treatment of hemophilia A with inhibitors.


Subject(s)
Factor VII/biosynthesis , Hemorrhage/therapy , Animals , Dependovirus/genetics , Factor VII/genetics , Factor VIIa/biosynthesis , Factor VIIa/genetics , Genetic Therapy , Hemophilia A/drug therapy , Hemophilia A/genetics , Hemophilia A/immunology , Hemophilia A/therapy , Hemorrhage/drug therapy , Hemorrhage/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Survival Rate , Transfection
5.
Biochem J ; 449(1): 231-9, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23050902

ABSTRACT

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.


Subject(s)
Antimetabolites/pharmacology , Factor VII/genetics , Gene Expression Regulation , Guanosine Triphosphate/deficiency , Intracellular Fluid/metabolism , Ribavirin/pharmacology , Transcription Elongation, Genetic/physiology , Factor VII/biosynthesis , Gene Expression Regulation/drug effects , Guanosine Triphosphate/antagonists & inhibitors , Hep G2 Cells , Humans , Transcription Elongation, Genetic/drug effects
6.
Genet Mol Res ; 12(4): 6813-24, 2013 Dec 16.
Article in English | MEDLINE | ID: mdl-24391029

ABSTRACT

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.


Subject(s)
Cloning, Molecular , Factor VII/genetics , Recombinant Proteins/genetics , Animals , Base Sequence , Blood Coagulation/genetics , Blood Coagulation/physiology , CHO Cells , Cell Line , Cricetinae , Cricetulus , Factor VII/biosynthesis , Hep G2 Cells , Humans , Real-Time Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Sequence Analysis, DNA
7.
PLoS One ; 7(7): e40994, 2012.
Article in English | MEDLINE | ID: mdl-22848420

ABSTRACT

BACKGROUND: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. METHODOLOGY/PRINCIPAL FINDINGS: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. CONCLUSIONS/SIGNIFICANCE: Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.


Subject(s)
Activating Transcription Factor 4/metabolism , Factor VII/biosynthesis , Glucose/metabolism , Response Elements/physiology , Transcription, Genetic/physiology , Up-Regulation/physiology , Activating Transcription Factor 4/genetics , Glucose/pharmacology , Hep G2 Cells , Humans , RNA, Small Interfering , Sweetening Agents/metabolism , Sweetening Agents/pharmacology , Transcription, Genetic/drug effects , Up-Regulation/drug effects
8.
Blood ; 119(4): 957-66, 2012 Jan 26.
Article in English | MEDLINE | ID: mdl-22134170

ABSTRACT

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.


Subject(s)
Dependovirus , Factor VII Deficiency/therapy , Factor VII/therapeutic use , Genetic Therapy/methods , Genetic Vectors , Hemorrhage/prevention & control , Perinatal Care , Animals , Animals, Newborn , Codon , Dependovirus/genetics , Factor VII/analysis , Factor VII/biosynthesis , Factor VII/genetics , Factor VII Deficiency/blood , Factor VII Deficiency/genetics , Factor VII Deficiency/physiopathology , Female , Fetal Therapies/adverse effects , Gene Expression , Genetic Therapy/adverse effects , Genetic Vectors/administration & dosage , Genetic Vectors/adverse effects , Hemorrhage/etiology , Hep G2 Cells , Humans , Injections, Intravenous , Macaca mulatta , Male , Mice , Pregnancy , Sex Characteristics , Survival Analysis
9.
J Biomed Biotechnol ; 2011: 873874, 2011.
Article in English | MEDLINE | ID: mdl-21912483

ABSTRACT

The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.


Subject(s)
Factor VII/biosynthesis , Leishmania/metabolism , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Animals , Blood Coagulation Tests , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Factor VII/chemistry , Factor VII/genetics , Hep G2 Cells , Humans , Leishmania/cytology , Leishmania/genetics , Lizards/parasitology , Models, Molecular , Plasmids/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
10.
Biotechnol Lett ; 32(6): 803-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20213530

ABSTRACT

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.


Subject(s)
Baculoviridae/genetics , Factor VII/biosynthesis , Gene Expression , Genetic Vectors , Animals , Carbon-Carbon Ligases/biosynthesis , Carbon-Carbon Ligases/genetics , Cell Line , Factor VII/genetics , Humans , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence Analysis, DNA , Spodoptera
11.
Chin Med J (Engl) ; 123(24): 3559-65, 2010 Dec.
Article in English | MEDLINE | ID: mdl-22166631

ABSTRACT

BACKGROUND: Blood coagulation factor VII (FVII) is physiologically synthesized in the liver and released into the blood. Binding of FVII to tissue factor (TF) is related to the metastatic potential of tumor cells, also a significant risk factor in the development of hepatic metastasis in patients with colorectal cancer (CRC). It has been found that some cancer cells can produce FVII extrahepatically. However, little is known about FVII and CRC. We therefore hypothesized that CRC cells may synthese FVII, leading to tumor invasion and metastasis. METHODS: We detected the expression of FVII protein in 55 CRC specimens by immunohistochemical staining. The FVII mRNA in 45 of 55 CRC cases, 6 colon cancer cell lines and one hepatoma cell line was measured by real-time reverse transcription-PCR (RT-PCR). Transwell invasion assays were performed to evaluate the changes of cell migration and invasion of LoVo cancer cells in vitro. We further observed the likely effectors regulated by the TF/FVIIa complex Western blotting assay. RESULTS: Extrahepatic synthesis of FVII was detected in the cytoplasm of 32 (58.2%) CRC specimens by immunohistochemistry, but not in normal mucosa. Liver metastasis (P = 0.003) and TNM staging (P = 0.005) were significantly correlated with FVII antigen expression. The positive ratios in stages I, II, III and IV were 33.3%, 40.0%, 52.4% and 87.5%, respectively. The expression of FVII mRNA in CRC with hepatic metastasis was significantly higher than CRC without hepatic metastasis (5.33 ± 2.88 vs. 1.47 ± 0.51, P = 0.03). Ectopic FVIIa induced a slight increase (1.34-fold) in the number of migrating cells, which was inhibited by the specific TF antibody. The formation of TF/FVIIa complex resulted in a marked increase in the expression of matrix metalloproteinases (MMP)-2 (3.5-fold) and MMP-9 (4.7-fold) in a time-dependent and dose-dependent manner. CONCLUSIONS: Extrahepatic synthesis of FVII by CRC cells may promote tumor invasion and metastasis. MMPs, as downstream effectors of TF/FVIIa signaling, facilitate the development of metastasis in colon cancer.


Subject(s)
Colorectal Neoplasms/pathology , Factor VII/biosynthesis , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Cell Movement , Colorectal Neoplasms/metabolism , Factor VII/analysis , Factor VII/genetics , Female , Humans , Immunohistochemistry , Liver Neoplasms/secondary , Male , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Middle Aged , Neoplasm Invasiveness , Neoplasm Staging , RNA, Messenger/analysis , Thromboplastin/physiology
12.
Mol Cancer Res ; 7(12): 1928-36, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19996301

ABSTRACT

Tissue factor/coagulation factor VII (fVII) complex formation on the surface of cancer cells plays important roles in cancer biology, such as cell migration and invasion, angiogenesis, and antiapoptotic effects. We recently found that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion. Here, we characterized mechanisms of hepatic and ectopic fVII (FVII) gene expression to identify molecular targets enabling selective inhibition of the ectopic expression. Unlike hepatic expression, hepatocyte nuclear factor-4 binding to the promoter is not required for ectopic FVII expression, although Sp1 binding is essential. Furthermore, we found novel nuclear targets of basal hepatocytic and ectopic FVII expression. Notably, histone acetyltransferases p300 and cyclic AMP-responsive element binding protein-binding protein (CBP) are exclusively recruited to the promoter region of the FVII gene specifically in breast cancer cells. We further show that curcumin, a dietary compound, can selectively inhibit ectopic fVII expression by targeting p300/CBP activity. These results suggest a strategy to inhibit ectopic fVII-induced tumor progression without impairment of the physiologic hemostatic process.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Factor VII/biosynthesis , Histone Acetyltransferases/antagonists & inhibitors , Acetylation/drug effects , Animals , Binding Sites , Breast Neoplasms/genetics , Cell Line, Tumor , Chromatin/drug effects , Chromatin/metabolism , Curcumin/pharmacology , E1A-Associated p300 Protein/antagonists & inhibitors , Factor VII/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Hepatocyte Nuclear Factor 4/metabolism , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Liver/drug effects , Liver/enzymology , Mice , Neoplasm Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/drug effects
13.
Blood Transfus ; 7(4): 305-12, 2009 Oct.
Article in English | MEDLINE | ID: mdl-20011642

ABSTRACT

BACKGROUND: Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology. METHODS: To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma. RESULTS: The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII. CONCLUSION: As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.


Subject(s)
Factor VII/biosynthesis , Factor VII/isolation & purification , Gene Expression , Histidine/biosynthesis , Histidine/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Animals , CHO Cells , Cricetinae , Cricetulus , Factor VII/genetics , Genetic Vectors/genetics , Hep G2 Cells , Histidine/genetics , Humans , Recombinant Fusion Proteins/genetics
14.
Blood ; 113(25): 6461-4, 2009 Jun 18.
Article in English | MEDLINE | ID: mdl-19387004

ABSTRACT

Our previous studies with genomic minigenes have demonstrated that an engineered small nuclear RNA-U1 (U1+5a) partially rescued coagulation factor VII (FVII) mRNA processing impaired by the 9726+5G>A mutation. Here, to evaluate the U1+5a effects on FVII function, we devised a full-length FVII splicing-competent construct (pSCFVII-wt). This construct drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 +/- 4 ng/mL), which were virtually undetectable upon introduction of the 9726+5G>A mutation (pSCFVII-9726+5a). Cotransfection of pSCFVII-9726+5a with pU1+5a resulted in a partial rescue of FVII splicing and protein biosynthesis. The level increase in medium was dose dependent and, with a molar excess (1.5x) of pU1+5a, reached 9.5% plus or minus 3.2% (5.0 +/- 2.8 ng/mL) of FVII-wt coagulant activity. These data provide the first insights into the U1-snRNA-mediated rescue of donor splice sites at protein level, thus further highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels.


Subject(s)
Factor VII/genetics , RNA Splice Sites/genetics , RNA, Small Nuclear/physiology , Animals , COS Cells/metabolism , Chlorocebus aethiops , Factor VII/biosynthesis , Factor VII/metabolism , Genes, Synthetic , Genetic Engineering , Humans , Point Mutation , RNA/genetics , RNA/physiology , RNA Splicing , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection
15.
Vox Sang ; 96(4): 309-15, 2009 May.
Article in English | MEDLINE | ID: mdl-19175565

ABSTRACT

BACKGROUND: Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. It is converted to factor VIIa that plays an important role in the coagulation cascade. The aim of this study was isolating and cloning the genes of human factor VII and hepsin and subsequent co-transfection of the constructs to Chinese hamster ovary (CHO) cell line to obtain rFVIIa. METHODS: Factor VII and hepsin cDNAs were isolated from HepG2 cell line and cloned into pcDNA3.1 (+) vector. The constructs were co-transfected to CHO cell line. A cell line that permanently expressed recombinant factor VII (rFVII) and hepsin was established. The expression of rFVII was confirmed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot analysis. Biological activity of rFVII was evaluated by prothrombin time assay. RESULTS: The results showed that the genes of FVII and hepsin were successfully cloned and expressed. Stable CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin expressed FVII and hepsin mRNA, but there was no expression in the CHO cells transfected with insert free pcDNA3.1. FVIIa protein was secreted to medium of CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin. The expected band of rFVII was detected in Western blot analysis. A three- to fourfold decrease in clotting time was observed when human FVII-depleted plasma was used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII. CONCLUSION: As we are aware, this is the first report of establishing a cell line expressing FVIIa using genetic engineering methods.


Subject(s)
Factor VII/genetics , Factor VIIa/metabolism , Genetic Engineering/methods , Animals , Blotting, Western , CHO Cells , Cell Line, Tumor , Cloning, Molecular , Cricetinae , Cricetulus , DNA, Complementary/genetics , Enzyme-Linked Immunosorbent Assay , Factor VII/biosynthesis , Factor VII/metabolism , Humans , Prothrombin Time , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transfection/methods , Tumor Cells, Cultured
16.
J Pediatr Surg ; 42(10): 1768-71, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17923213

ABSTRACT

PURPOSE: A 3-year-old girl developed extrahepatic portal vein obstruction (EHPVO) after a liver transplant. She had sequelae of portal hypertension that required another transplantation. The circumstances allowed for comparison of liver-dependent coagulation factor production between the second donor liver and the explanted liver with EHPVO. METHODS: Liver samples from the explanted first graft and the second transplant were obtained. Fresh tissue was used to perform reverse transcription-polymerase chain reaction with primers against factors V, VII, as well as VIII, protein C, and paraffin-embedded sections for hepatocyte proliferation using Ki-67 antibody as well as for apoptosis using TUNEL assay. RESULTS: The transcription of factor VII and that of protein C were decreased in the explant as compared with the newly transplanted liver (factor VII, 77% of the donor; protein C, 88% of the donor). The transcription of factor V and that of factor VIII were unchanged. The explant had a greater percentage of proliferating hepatocytes than the new organ (0.85% +/- 0.75% vs 0.11% +/- 0.21%). The percentage of apoptotic cells was similar between the 2 livers (0.09% +/- 0.13% vs 0.09% +/- 0.13%). CONCLUSIONS: Idiopathic EHPVO is associated with a reduction in liver-dependent coagulation factor transcription and an increase in hepatocyte proliferation. Portal blood flow deprivation alters hepatic homeostasis and initiates mechanisms that attempt to restore liver-dependent coagulation factors.


Subject(s)
Factor VII Deficiency/etiology , Hypertension, Portal/etiology , Liver Transplantation , Portal Vein/pathology , Postoperative Complications/pathology , Protein C Deficiency/etiology , Apoptosis , Biliary Atresia/surgery , Cell Division , Child, Preschool , Factor VII/biosynthesis , Factor VII/genetics , Female , Gastrointestinal Hemorrhage/etiology , Hepatocytes/pathology , Humans , Hypertension, Portal/surgery , Liver/metabolism , Liver/pathology , Liver Circulation , Postoperative Complications/etiology , Protein C/biosynthesis , Protein C/genetics , Reoperation , Transcription, Genetic
17.
J Biol Chem ; 282(43): 31156-65, 2007 Oct 26.
Article in English | MEDLINE | ID: mdl-17675296

ABSTRACT

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.


Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Factor VII/genetics , Gene Expression Regulation/genetics , Insulin/metabolism , 5' Untranslated Regions , Animals , Base Sequence , CCAAT-Enhancer-Binding Protein-beta/genetics , CHO Cells , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cricetinae , Cricetulus , DNA/genetics , DNA/isolation & purification , Factor VII/biosynthesis , Factor VII/metabolism , Genes, Reporter , Glucose/genetics , Glucose/metabolism , Humans , Insulin/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription, Genetic , Transfection
18.
Cancer Res ; 66(19): 9453-60, 2006 Oct 01.
Article in English | MEDLINE | ID: mdl-17018600

ABSTRACT

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.


Subject(s)
Factor VII/biosynthesis , Neoplasm Invasiveness/physiopathology , Neoplasm Proteins/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/physiology , Blood Coagulation , Carbon-Carbon Ligases/biosynthesis , Carbon-Carbon Ligases/genetics , Cell Hypoxia/genetics , Cell Line, Tumor/metabolism , Cell Movement/physiology , Factor VII/genetics , Factor VII/physiology , Factor Xa/physiology , Female , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neoplasms/blood , Neoplasms/pathology , Ovarian Neoplasms/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Receptor, PAR-1/physiology , Recombinant Fusion Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Thromboplastin/biosynthesis , Thromboplastin/genetics , Transfection
19.
J Am Coll Cardiol ; 46(4): 707-13, 2005 Aug 16.
Article in English | MEDLINE | ID: mdl-16098440

ABSTRACT

OBJECTIVES: The purpose of this study was to test the hypothesis that activated monocytes with soluble plasma tissue factor (pTF) activate factors VII and X to generate thrombin. BACKGROUND: Despite heparin, thrombin is progressively generated during cardiac surgery with cardiopulmonary bypass (CPB), produces intravascular fibrin and fibrinolysis, and causes serious thromboembolic and nonsurgical bleeding complications. Thrombin is primarily produced in the surgical wound, but mechanisms are unclear. METHODS: In 13 patients, interactions of mononuclear cells, platelets, pTF, and pTF fractions to activate factors VII and X were evaluated in pre-bypass, perfusate, and pericardial wound blood before and during CPB. RESULTS: Monocytes are activated in wound, but not in pre-bypass or perfusate plasma (monocyte chemotactic protein-1 = 29.5 +/- 2.1 pmoles/l vs. 2.8 +/- 1.2 pmoles/l and 3.3 +/-1.4 pmoles/l, respectively). Wound pTF is substantially elevated compared to other locations (3.64 +/- 0.45 pmoles/l vs. 0.71 +/- 0.65 pmoles/l and 1.31 +/- 1.4 pmoles/l). Supernatant wound pTF contains 81.7% of TF antigen; wound microparticle pTF contains 18.3%. Wound monocytes and all C5a-stimulated monocytes (but not activated platelets) completely convert factor VII to factor VIIa with wound pTF. Activated monocytes more efficiently activate factor X with wound supernatant TF/factor VII(VIIa) complex than with wound microparticle TF/factor VII(fVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wound pTF concentration is 0.944 (p = 0.0004). CONCLUSIONS: During cardiac surgery with CPB, wound monocytes plus wound pTF or wound microparticle-free supernatant pTF preferentially accelerate activation of factor VII and factor X. This system represents a novel mechanism for thrombin generation via the TF coagulation pathway.


Subject(s)
Blood Coagulation/physiology , Cardiopulmonary Bypass/adverse effects , Factor VII/biosynthesis , Factor X/biosynthesis , Monocytes/physiology , Plasma/chemistry , Postoperative Hemorrhage/physiopathology , Thrombin/biosynthesis , Thromboplastin/analysis , Aged , Aged, 80 and over , Blood Coagulation Factors/physiology , Blotting, Western , Factor VII/analysis , Factor X/analysis , Female , Humans , Male , Middle Aged , Postoperative Care , Wound Healing
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