Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased ³²P phosphate incorporation into the Cx45 protein in transfected cells.The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and –26 were coexpressed in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.     

Intracellular localization of the HLA class II gene products in transfected mouse L cells.

To investigate the impaired cell surface expression of human major histocompatibility antigen (HLA) in transfected L cells, we examined their intracellular localization by immunocytochemistry. HLA class II molecules produced in transfected L cells were mainly detected in the intracellular vesicles and in the nuclear envelope as granular precipitates. The results suggest that the intracellular transport of the newly synthesized molecules in transfected L cells is impaired at some point along the pathway from the rough endoplasmic reticulum (RER) to the medial-, trans-Golgi apparatus.     

Nodamura virus nonstructural protein B2 can enhance viral RNA accumulation in both mammalian and insect cells

During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.     

Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells

We describe a female patient with developmental delay, dysmorphic features and multiple congenital anomalies who presented a normal G-banded karyotype at the 550-band resolution. Array and multiplex-ligation probe amplification (MLPA) techniques identified an unexpected large unbalanced genomic aberration: a 17.6Mb deletion of 9p associated to a 14.8 Mb duplication of 20p. The deleted 9p genes, especially CER1 and FREM1, seem to be more relevant to the phenotype than the duplicated 20p genes. This study also shows the relevance of using molecular techniques to make an accurate diagnosis in patients with dysmorphic features and multiple anomalies suggestive of chromosome aberration, even if on G-banding their karyotype appears to be normal. Fluorescence in situ hybridization (FISH) was necessary to identify a masked balanced translocation in the patient's mother, indicating the importance of associating cytogenetic and molecular techniques in clinical genetics, given the implications for patient management and genetic counseling.     

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.     

Gene amplification-associated cytogenetic aberrations and protein changes in vincristine-resistant Chinese hamster, mouse, and human cells

We carried out cytogenetic studies of four Chinese hamster, mouse, and human cell lines selected for high levels of resistance (500- to 4,000-fold) to vincristine (VCR) by a multistep selection procedure. All cells examined contained gene amplification-associated metaphase chromosome abnormalities, either homogeneously staining regions (HSRs), abnormally banding regions (ABRs), or double-minute chromosomes (DMs); control actinomycin D- and daunorubicin-resistant hamster lines did not exhibit this type of chromosomal abnormality. VCR-resistant Chinese hamster sublines exhibited both increased synthesis of the protein V19 (Mr 19,000; pl = 5.7) and increased concentrations of V19 polysomal mRNA. When VCR-resistant cells were grown in drug-free medium, level of resistance, synthesis of V19, and amount of V19 mRNA declined in parallel with mean length of the HSR or mean number of DMs per cell. Cross-resistance studies indicate that VCR-resistant cells have increased resistance both to antimitotic agents and to a wide variety of agents unrelated to VCR in chemical structure and/or mechanism of action. Our studies of tubulin synthesis in Chinese hamster cells indicate no overproduction of tubulin or presence of a mutant tubulin species. Comparison with antifolate-resistant Chinese hamster cells known to contain amplified dihydrofolate reductase genes localized to HSRs or ABRs strongly suggests that the HSRs, ABRs, or DMs of the Vinca alkaloid-resistant sublines likewise represent cytological manifestations of specifically amplified genes, possibly encoding V19, involved in development of resistance to VCR.     

Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

By using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or CW3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogeneous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.     

Sensitivity of influenza a viruses to human interferons in human diploid cells

Abstract The sensitivity of influenza virus to the action of natural human interferon (IFN)-α+β and -γ, and to the action of highly purified recombinant HuIFN-αB, -αD, and -αF, has been investigated. A plaque assay for the fowl-plague strain of influenza A virus has been established using human embryonic foreskin (HEF) cells. The sensitivity of influenza virus to all IFNs tested in this assay was comparable to that shown by vesicular stomatitis virus (VSV) which was taken as the reference standard. The high sensitivity to IFN action found for the fowl-plague strain was confirmed for the WSN strain of human origin in a yield reduction assay.     

Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene.

Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells. dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1. SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E. coli (about 18,800) suggests that the former was glycosylated.     

Kinetics of folding and assembly of the human chorionic gonadotropin beta subunit in transfected Chinese hamster ovary cells.

We have employed Chinese hamster ovary (CHO) cell lines transfected with either the wild type human chorionic gonadotropin beta (hCG-beta) gene alone (CHO beta cells) or in conjunction with the gene expressing the alpha subunit (CHO alpha,beta cells) to study the folding pathway of the hCG-beta subunit. In both CHO beta and CHO alpha,beta cells, the earliest detectable hCG-beta precursor, p beta 1, which had two of six potential disulfide bonds (34-88 and 38-57) formed, was converted to p beta 2, a form that, following the formation of disulfide bonds between cysteines 9-90 and 23-72, migrated more slowly than p beta 1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The t1/2 for the conversion of p beta 1 to p beta 2 in CHO alpha,beta and CHO beta cells was 5 min, demonstrating that the alpha subunit had no effect on the rate of this conversion. Furthermore, the tryptic-releasable peptides generated from nonreduced p beta 1 or p beta 2 were the same in both CHO alpha,beta and CHO beta cells. Thus, both the rate and order of disulfide bond formation during the conversion of the folding intermediate p beta 1 into p beta 2 were the same whether or not the alpha subunit was present. A comparison between cell types expressing different alpha/beta subunit ratios revealed that the higher the glycoprotein hormone alpha subunit to beta subunit ratio, the greater the rate and extent of hCG heterodimer assembly.     

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.     

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.     

RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity

RNA interference (RNAi) has been recently applied to improve the yield and quality of recombinant proteins produced in Chinese hamster ovary (CHO) cells, the most commonly used mammalian cell line for production of complex biopharmaceuticals. Proteomic profiling of CHO cells undergoing gene amplification identified cofilin, a key regulatory protein of actin cytoskeletal dynamics, as a cellular target for genetic engineering studies. Transient reduction of cofilin by small interfering RNA (siRNA) enhanced specific productivity in recombinant CHO cells by up to 80%. CHO cell lines expressing cofilin-specific short hairpin RNA (shRNA) vectors showed up to a 65% increase in specific productivity. These results suggest that modulation of cofilin, and its regulatory pathways, may be a new approach to enhance recombinant protein productivity in CHO cells.     

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.     

Mouse RanBPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.