Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.     

降低细胞内tTG活性可使细胞获得对TNF-α的抗性

 用 Northern印迹证实在 He La细胞中有 t TG的表达 ,且这种表达受 TNF-α的上调 .构建t TG反义真核表达质粒并转染 He La细胞 ,用 G41 8抗性筛选稳定表达的转染细胞 ,并用 Northern印迹和 t TG活性测定进一步分析反义 t TG的转录 .用结晶紫及 MTT法证实阳性细胞克隆获得了对 TNF- α的抗性 ,而转染表达载体 pc DNA3的细胞仍对 TNF- α敏感 .结果表明降低 t TG活性能使细胞对 TNF- α诱发的细胞凋亡敏感性降低     

Insulin-like growth factor-I (IGF-I) attenuation of growth hormone is enhanced by overexpression of pituitary IGF-I receptors.

Insulin-like growth factor-I (IGF-I) attenuates GH gene expression by a receptor-mediated mechanism in pituitary cells. We, therefore, isolated neomycin-resistant stable GC cell transfectants over-expressing human IGF-I receptor cDNA (IGFIR-cDNA) cloned in an Rous sarcoma virus-directed expression vector. A transfection control contained the IGFIR-cDNA cloned in the reverse orientation. Southern analysis confirmed incorporation of human IGFIR-cDNA sequences into rat genomic DNA. Immunoprecipitation of metabolically labeled [35S]methionine stably transfected cells revealed a 200-kDa human IGF-I receptor precursor protein. Growth rate and basal GH secretion were not altered in transfected cells. Although transfected and control cells had a similar Kd for IGF-I binding (0.43 and 0.40 nM, respectively), IGF-I-binding sites were induced 17-fold (384,000 vs. 22,000 sites/cell). Treatment of cells with IGF-I (6.5 nM) maximally attenuated GH secretion by 80% compared to 40% attenuation in control cells (P less than 0.0001). Maximal suppression of GH in transfectants occurred within 15 h of treatment, and GH secretion by control cells was only maximally suppressed after 42 h. The ED50 of IGF-I suppression of GH secretion in transfectants after 15 h was 0.5 nM. These results demonstrate that transfectants overexpressing human IGF-I receptor are hyperresponsive to exogenous IGF-I. These data indicate that IGF-I receptor number plays an important role in mediating the signal transduction of IGF-I to the GH gene.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Receptor density and cAMP accumulation: analysis in CHO cells exhibiting stable expression of a cDNA that encodes the beta 2-adrenergic receptor

The relationship between hormone receptor number and hormone-stimulated cAMP accumulation was probed in CHO cells that were transfected with the cDNA encoding the beta-adrenergic receptor under the control of the SV40 early promoter (expression vector pSV2BAR). CHO cells were cotransfected with pSV2BAR and expression vector pHOMER that directs the expression of a neomycin-resistance gene, and stable transfectants were selected. Clones expressing receptor at levels from 30 (wild-type) to 6000 fmol/mg membrane protein were isolated and further characterized for receptor mRNA content (measured by solution hybridization with a single-stranded cDNA probe), steady-state expression of receptor (measured by immunoblotting and indirect immunofluorescence), and their ability to accumulate intracellular cAMP in response to a beta-adrenergic agonist. Receptor mRNA content and the steady-state level of receptor protein and its expression at the cell surface were found to increase with receptor density as measured by radioligand binding. Over a 200-fold range of receptor expression, CHO transfectants displayed increasing efficacy of agonist-stimulated cAMP accumulation and increasing maximal cAMP accumulation in response to agonist. These data provide for the first time an analysis of the relationship between the density of a G-protein-linked receptor and a receptor-mediated response under conditions where the levels of G-proteins and adenylate cyclase are unaltered.     

Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.

Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

Bcl—2基因加强表达对SK细胞编程死亡的效应

TNF和OA诱发人神经母细胞瘤SK细胞编程死亡(PCD)。将编码Bcl-2完整蛋白质的cDNA植入pXJ 41neo载体中,由HCMV病毒启动子控制其表达。形成的正向(pBcl-2-S)及反向(pBcl-2-AS)表达质粒经转染导入SK细胞中获得稳定转染子。Western印迹表明正向转染子表达较大量的26kd Bcl-2蛋白,而反向转染子则不表达。增强表达的Bcl-2基因产物能抑制由TNF引发的PCD,但不影响由OA引发的PCD,从而证明Bcl-2基因产物抗细胞死亡效应的特异性。     

Growth hormone (GH) stimulates protein synthesis in cells transfected with GH receptor complementary DNA

An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nM) specific binding of GH. Cross-linking of [125I]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response.     

Measles virus-substance P receptor interactions: Jurkat lymphocytes transfected with substance P receptor cDNA enhance measles virus fusion and replication

1. We have demonstrated previously (Harrowe et al., 1990), using a lymphoblastoid cell line that constitutively expresses the substance P receptor (SPR) (Payan et al., 1984, 1986), that this receptor may facilitate measles virus (MV) fusion with these cells. In order to test this hypothesis further, a stable cell line transfected with SPR cDNA has been established, and various stages of MV infection in SPR positive and negative cells compared. 2. Jurkat cells, a human T-lymphoblastoid cell line, were transfected with a cDNA clone encoding the SPR. Cells transfected with only the plasmid were used as controls. Jurkat cells and Jurkat vector control cells (J-vo) failed to demonstrate any detectable 125I-SP binding, whereas a clonally selected population of cells transfected with SPR cDNA (J-SPR) expressed about 50,000 receptors/cell (Sudduth-Klinger et al., 1992). 3. Using the J-vo- and J-SPR-transfected cell lines, the following experiments were conducted to investigate the effect of SPR expression on MV infection. To determine if MV would preferentially attach to J-SPR as compared to J-vo, we absorbed virus to cells at 37 degrees C for various times and measured bound MV using a fluorescence activated cell sorter (FACS). Using this approach, we found that MV bound to a greater degree to J-SPR compared with J-vo. In addition to equilibrium being reached faster for J-SPR, the total amount of bound MV was higher on J-SPR. The effect was greater at lower MOIs, suggesting that there existed multiple binding sites for MV on these cells and that the affinity is higher for those cells expressing the SPR. 4. Since binding does not necessitate a successful viral infection, we needed to know if this difference in binding reflected a difference in infection. This was demonstrated by showing an approximate twofold increase in infected cells after a 2-hr binding period with J-SPR as compared to J-vo at an MOI of 1 in an infectious cell-center assay. Moreover, when both cells types were subjected to continuous infection in culture, J-SPR-infected cells produced a seven- to ninefold increase in measles viral titer in 24 hr as compared with J-vo. The observed increase in viral titer may have resulted in more of the J-SPR cells binding virus, as indicated by our binding and infectious cell-center data, or alternatively, the virus might have entered the J-SPR cells faster and begun replication before the J-vo-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells

We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.     

Truncated activin type II receptor inhibits erythroid differentiation in K562 cells

Two receptor serine/threonine kinases (types I and II) have been identified as signaling transducing activin receptors. We studied the possibility of inhibiting activin A-dependent differentiation in K562 cells, using a dominant negative mutant of type II receptor. A vector was constructed expressing activin type II truncated receptor (ActRIIa) that lacks the cytoplasmic kinase domain. Since activin type I and II receptors form heteromeric complexes for signaling, the mutant receptors compete for binding to endogenous receptors, hence acting in a dominant negative fashion. K562 cells were stably transfected with ActRIIa, and independent clones were expanded. The truncated cDNA was integrated into the genome of the transfectants, as shown by polymerase chain reaction; and the surface expression of truncated receptors was shown by affinity cross-linking with (125)I-activin A. In wild-type K562 cells, activin A induced erythroid differentiation and cells started to express hemoglobins. In transfected cells expressing ActRIIa, the induction of erythroid differentiation was abrogated and less than 10% of cells were hemoglobin-containing cells after culture with activin A. Further transfection with wild-type type II receptors rescued the mutant phenotype of these transfectants, indicating that the effect of ActRIIa is dominant negative. In addition, phosphorylation of the cytoplasmic kinase domain of the type II receptor in vitro confirms the autophosphorylation of this portion of the receptor. Therefore, induction of erythroid differentiation in vitro is mediated through the cell surface activin receptor, and interference with this receptor signaling inhibits this process of differentiation in K562 cells.     

Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.

A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.     

Expression and rescuing of a cloned human tumour necrosis factor gene using an EBV-based shuttle cosmid vector.

A cosmid vector carrying the Epstein-Barr virus origin of replication, the EBNA-1 gene, the hygromycin phosphotransferase (hph) gene and pBR322 sequences has been constructed. This cosmid can replicate autonomously in the nucleus of human tissue culture cells, even when it carries a 35-kb long insert. The cosmid can be rescued from the transfected cells by cloning it directly into ampicillin-sensitive Escherichia coli. A gene for human tumour necrosis factor (TNF) cloned into this cosmid vector was introduced in tissue culture cells, where it was transcribed into mature mRNA.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.     

Use of a sensitive receptor binding assay to discriminate between full-length and truncated human recombinant tumor necrosis factor proteins

A radioreceptor assay (RRA) capable of detecting picomolar concentrations of human recombinant tumor necrosis factor (TNF) was used to compare the relative binding affinities of genetically engineered full-length and truncated TNF proteins. The specific cell-surface receptors for TNF present on the human cervical carcinoma cell line ME-180 were characterized as having a Kd of 0.2 nM and a density of 2700 sites/cell. Conditions were then defined for an RRA that maximized the specific binding of 125I-TNF to this adherent cell line. Incubation of ME-180 cells with 125I-TNF at 37 degrees C in the presence of 0.02% sodium azide resulted in a 4-fold increase in assay sensitivity and a doubling of specific counts bound, as compared to binding done at 4 degrees C with or without sodium azide. Inhibition of receptor-ligand internalization under these conditions was a likely reason for the increases. This system was utilized to compare low concentrations of the full-length TNF protein and a genetically altered TNF protein (mutein) which lacks the 10 N-terminal amino acids and contains an N-terminal methionine. Previous studies showing the truncated TNF to be 2- to 3-fold lower in cytotoxic activity on a variety of tumor cell lines were corroborated by our findings that the mutein was also three and one-half times lower in relative affinity for the TNF receptor on ME-180 cells. These results suggest a possible role for these residues in receptor binding and illustrate the use of a highly sensitive RRA for the evaluation of TNF molecules altered by recombinant DNA technology.     

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.     

Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades.

The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.