Production of recombinant human erythropoietin in Bowes melanoma cells in suspension culture

Summary Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO₂ accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000 1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.Offprint requests to: L. Keay     

Relationship of an unstable argG gene to a 5.7-kilobase amplifiable DNA sequence in Streptomyces lividans 66.

The relationship between an unstable argG gene and a 5.7-kilobase (kb) amplifiable DNA sequence in Streptomyces lividans 66 was investigated. Spontaneous, high-frequency Arg mutants deleted for this gene typically contain 200 to 300 copies of the tandemly reiterated sequence. A library of S. lividans 66 (strain 1326) wild-type genomic DNA was prepared in the vector lambda Charon 35. Chromosome walking over 44 kb established that argG is located 25 kb distant from a duplicated amplifiable DNA structure. A sequence was characterized, located farther distal from the amplifiable structure, containing strong homology with an internal sequence of the amplifiable DNA, which may have a role in the deletion of argG. Genetic mapping showed that argG and the 5.7-kb amplifiable sequence are linked to another unstable gene, determining chloramphenicol resistance (Camr) and that together these genes may be located in a silent chromosomal arc.     

The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells.

A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock.     

Endogenous melanin-concentrating hormone receptor SLC-1 in human melanoma SK-MEL-37 cells.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that regulates several physiological functions. The orphan G protein-coupled receptors SLC-1 and MCHR2 were recently found to bind MCH with high affinity. We show here that the human melanoma cell line SK-MEL-37 expresses SLC-1 mRNA but not MCHR2 by RT-PCR analysis and immunofluorescence studies. Using Chinese hamster ovary cells and 293 cells overexpressing SLC-1 by cDNA transfection, it was shown that SLC-1 coupled to both G alpha(i)/G alpha(o) and G alpha(q) proteins. In SK-MEL-37 cells, MCH inhibited forskolin-stimulated cyclic AMP accumulation and induced mitogen-activated protein kinase (MAPK) in a pertussis toxin-(PTX)-sensitive manner. The MAPK activity leads to the production of phosphorylated forms of p42/p44 MAPK. However, an increase in the intracellular free Ca(2+) concentration was not elicited by MCH in SK-MEL-37 cells. These results show that SLC-1 is coupled only to PTX-sensitive G alpha(i)/G alpha(o) in SK-MEL-37 cells. This study provides for the first time a skin-derived cellular model to analyze the molecular mechanism of the MCH signaling pathway.     

Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells

Bowes melanoma cells synthesize more tissue plasminogen activator (tPA) in monolayer cultures than in multicell spheroids. Cellular production of tPA in these cells was measured during a cultivation period of 800 h. Without changing the cell culture assay, we were able to obtain monolayers, multilayers, and multicell spheroids (cell aggregates) by stirring microcarrier beads in 500-mL spinner flasks operated at 50 rpm. Thus, the medium conditions in the liquid were similar for cells in monolayers and in multicell spheroids. Probes for measurements of intracellular and extracellular parameters were taken from the same culture at distinct times; therefore, their variations during cultivation can directly be compared. Because cells were cultured in an unregulated (with regard to pH, glucose, etc.) spinner flask, their concentration was kept below 10(6) cells/mL, thus avoiding too fast and too severe depletion of oxygen and other medium factors. Nevertheless, the tPA productivity decreased from 8 ng/h/10(6) cells (monolayer) to 4 ng/h/10(6) cells (multicell spheroids with microcarrier nucleus, 800 mum diameter), matching the decrease of total cellular protein. Due to medium depletion, the cell cycle distribution changed from 45% to 68% G(1) cells in a characteristic way during growth of multicell spheroids. This is accompanied by changes in amino acids, glucose, lactate, and pH, which may account for the reduction of tPA productivity. (c) 1996 John Wiley & Sons, Inc.     

组织型纤溶酶原激活剂cDNA表达质粒的构建及其在CHO细胞中的表达

人组织型纤溶酶原激活剂(t-PA)cDNA克隆片段。采用两种方式构建成真核表达质粒。第一:切除t-PA3＇端非编码区序列后插入SR启动子和SV_(40)晚期Poly(A)终止信号之间,形成pMGZ6001质粒;第二:将3＇端部分切除并带有Poly(A)加尾信号的t-PA片段插入由金属硫蛋白MT启动子调控的载体中,分别组建成含大T抗原与不含大T抗原的两个表达质粒pMGZ6002和pMGZ6003。这三种质粒用磷酸钙共沉淀法和电穿孔法转染CHO-dhfr细胞,阳性克隆细胞均能合成并分泌rt-PA。其分子量约68kD,并能与t-PA单克隆抗体特异结合,溶解纤维蛋白。阳性克隆经MTX选择培养扩增基因,培液中rt-PA表达水平可达3000IU/(10~6细胞·48h)。     

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.     

Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators

Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed.     

A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator.

We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo

The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, "cross-talk" between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissue in vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.     

Beta and gamma interferons act synergistically to produce an antiviral state in cells resistant to both interferons individually.

We showed previously that the mouse fibroblastoid cell line Ltk-aprt- is resistant to the antiviral effects of beta interferon. This lack of response reflects a partial sensitivity to the interferon that is accompanied by a failure to activate expression of several interferon-regulated genes, although certain other genes respond in a normal manner. We show here that Ltk-aprt- cells were also unable to establish an antiviral state and to activate expression of 2,5-oligo(A) synthetase when treated with gamma interferon. Strikingly, however, treatment with a combination of beta interferon and gamma interferon provided complete protection against viral replication. Although the cells were completely insensitive to up to 250 U of the interferons per ml added singly, essentially complete protection from viral cytopathic effects was achieved when as little as 10 U of each of the interferons per ml were combined. Expression of 2,5-oligo(A) synthetase was also sensitive to this synergistic effect. Activation of an antiviral state could also be achieved by sequential treatment, first with gamma interferon and then with beta interferon. Partial protection against viral replication could be achieved by pretreatment with gamma interferon for as little as 1 h before incubation with beta interferon and could be blocked by the addition of specific antibodies or by cycloheximide, indicating that gamma interferon induces the synthesis of a protein which can act synergistically with a signal produced by the beta-interferon receptor. We suggest that Ltk-aprt- cells suffer from defects in one or more components of the gene activation pathways for both type I and type II interferons. Nonetheless, gamma interferon is able to activate the expression of a gene encoding a protein required for signal transduction. This protein acts synergistically with a transient signal produced in response to beta interferon, thereby activating the expression of a further group of genes.