High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: Purification and production of blocking antibodies

Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 μg/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells. © 1993 Wiley-Liss, Inc.     

The platelet-derived growth factor-inducible KC gene encodes a secretory protein related to platelet alpha-granule proteins

A full length cDNA clone of the platelet-derived growth factor-inducible KC gene has been isolated, sequenced, and expressed in COS cells. Both sequence analysis and expression studies indicate that KC encodes a secretory protein. Sequence analysis shows that, furthermore, the protein encoded by KC belongs to a growing superfamily of inducible proteins with a common ancestral linkage to the platelet alpha-granule proteins, platelet factor 4, and connective tissue-activating peptide III. A computer-generated phylogenetic tree documents interrelationships between KC and six additional members of this peptide superfamily. The KC gene is, in all probability, the murine homologue of a human gene termed "gro." By extension, the KC protein is the murine counterpart of the protein encoded by the gro gene. The gro protein corresponds to a factor described as "melanoma growth-stimulating activity."     

Melamine causes apoptosis of rat kidney epithelial cell line (NRK-52e cells) via excessive intracellular ROS (reactive oxygen species) and the activation of p38 MAPK pathway

There was an outbreak of urinary stones associated with consumption of melamine-tainted milk products in 2008 in China, leading to serious illness of many infants and even death. We have recently demonstrated that melamine causes oxidative damage on the NRK (normal rat kidney)-52e cells. The objective of this study was to explore the cellular signalling pathway that mediates the cell apoptosis induced by melamine in the NRK-52e cells. Fluorescence microscope showed that melamine enhanced intracellular ROS (reactive oxygen species) levels of the NRK-52e cells. AO/EB (acridine orange/ethidium bromide) staining and flow cytometry revealed that melamine increased apoptotic and necrotic percentages of the NRK-52e cells in a dose-dependent manner. Notably, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assays and flow cytometry displayed that SB203580, an inhibitor for p38 MAPK (mitogen-activated protein kinase) pathway, increased the proliferation of the NRK-52e cells and reduced the apoptotic and necrotic percentages of the NRK-52e cells. Western blots further demonstrated that p38 phosphorylation was activated by melamine in the NRK-52e cells and inhibitor SB203580 blocked the increase of p38 phosphorylation induced by melamine. Together, these results suggested that melamine causes apoptosis of the NRK-52e cells via excessive intracellular ROS and the activation of p38 MAPK pathway. This study thus offers a novel insight into molecular mechanisms by which melamine has adverse cytotoxicity on renal tubular epithelial cells.     

Purification of a cytotoxic protein produced by the murine macrophage-like cell line J774.1 in response to Sarcophaga lectin

A tumor specific cytotoxic protein produced by the murine macrophage-like cell line J774.1 in response to stimulation with Sarcophaga lectin was purified to homogeneity in three steps from the culture medium. This cytotoxin, named tumor killing factor (TKF), was a protein with a molecular weight of 15,000, and aggregated forming an oligomer with a molecular weight of 48,000. Its amino acid composition was similar to that of human TNF. Purified TKF had a significant effect on transplanted murine ascites tumor sarcoma 180. The biological significance of TKF in terms of ontogeny is discussed from the view point of developmental biology.     

Purification of a lipoprotein lipase-inhibiting protein produced by a melanoma cell line associated with cancer cachexia

A human melanoma cell line, SEKI, induces severe cachexia in tumor-bearing nude mice. A factor with the ability to inhibit lipoprotein lipase (LPL) was isolated from the conditioned medium of this cell line. This factor was 40-K-dalton protein, and designated temporarily as melanoma-derived LPL inhibitor (MLPLI). Amino acid sequencing revealed that the amino-terminal portion consists of SPLPITPV-AT--IR-P. Unexpectedly, the sequence, as far as determined, was identical to those of leukemia inhibitory factor (LIF), suggesting that MLPLI is a protein closely related to LIF. The findings that MLPLI inhibits LPL activity and that MLPLI is produced by human cancer cells inducing cancer cachexia also suggest that this protein is a candidate for the factor responsible for cancer cachexia.     

The somatomedin-binding protein isolated from a human hepatoma cell line is identical to the human amniotic fluid somatomedin-binding protein

Serum-free medium conditioned by the human hepatoma cell line HEP G2 was shown to contain a somatomedin-binding protein with a relative molecular mass of about 35,000. This binding protein was purified to homogeneity by the use of immunoaffinity chromatography and subsequent size exclusion chromatography. Antibodies for the immunoaffinity step were raised in rabbits against a previously isolated human amniotic fluid somatomedin-binding protein. The total composition and N-terminal amino acid sequence showed the protein to be identical to the binding protein from human amniotic fluid. Both have the N-terminal structure Ala-Pro-Trp-Gln-. The HEP G2 cell line offers a useful model to study the regulation of the synthesis and secretion of human somatomedin-binding proteins.     

An IL-17F/A heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4(+) T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.     

The expression of glial fibrillary acidic protein in a rat cerebellar cell line

A rat cerebellar cell line, WC5, derived by transformation with Rous sarcoma virus, which is temperature-sensitive for transformation (ts-RSV), can be induced to express glial fibrillary acidic protein (GFAP). Immunofluorescence, radioimmune assay, and electron microscopy studies show that GFAP is expressed in WC5 cells grown at the nonpermissive temperature (NPT), but not at the permissive temperature (PT) for transformation. GFAP is first detectable about 3 days after incubating cells at the NPT, and reaches an apparent plateau by the seventh or eighth day. The expression of GFAP is reversible; shifting cells from the NPT to the PT causes a dramatic decrease in GFAP after 96 hr. In order to determine if the expression of GFAP is linked to the temperature-sensitive transforming activity of the viral src gene product, phenotype revertants of WC5 were established. By the criteria of morphology and growth in agar, the revertant lines, in contrast to the parent cell line WC5, were shown to exhibit a transformed phenotype at both the NPT and PT. Immunofluorescence studies on several of the revertant cell lines show that they do not express GFAP at either the PT or NPT. These findings suggest that the expression of GFAP in WC5 is linked to the expression of the src gene product. The advantage of using ts-RSV to derive neural cell lines which exhibit differentiated properties is discussed.     

Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.     

A comparison of 60, 70, and 90 kDa stress protein expression in normal rat NRK-52 and human HK-2 kidney cell lines following in vitro exposure to arsenite and cadmium alone or in combination

Arsenite and cadmium are two potent nephrotoxicants and common Superfund site elements. These elements are included among the stress protein inducers, but information regarding relationships between toxicity produced by combinations of these agents to the stress protein response is lacking. In this study, the immortalized cell lines normal rat kidney NRK-52E and human kidney HK-2 were exposed in vitro to arsenite (As(3+)), cadmium (Cd(2+)), or to equimolar As(3+) plus Cd(2+) mixture combinations for 3 and 5 h over a concentration range of 0.1-100 microM. After a 12-h recovery period, cultured cells were then evaluated for expression of the 60, 70, and 90 kDa major stress protein families. Results indicated that expression of stress proteins varied depending on the species of kidney cells exposed, the exposure concentrations, and the length of exposure to each element on an individual basis and for combined mixtures. For the HK-2 kidney cell line, increased levels of the 70 kDa stress protein was observed for single and combined element exposures whereas there was no change or a decrease of stress proteins 60 and 90 kDa. Increased 70 kDa expression was observed for 10-microM doses of single elements and for a lower dose of 1 microM of the As plus Cd mixture at 3- and 5-h exposures. NRK-52 kidney cells exposed to equivalent doses of As(3+) and Cd(2+) alone or in combination showed increased levels of all stress proteins 60, 70, and 90 kDa. This increase was seen for 10 microM of the As plus Cd mixture at 3 h whereas for single element exposures, increased stress protein levels were generally observed for the 100-microM doses. At 5 h- exposure, 60 and 90 kDa levels increased for 10 microM of Cd(2+) and 60 kDa levels increased for 1 microM of As(3+). However, exposures to 10 microM of the As plus Cd mixture decreased 60 kDa protein expression to control levels at 5 h. For both kidney cell lines, there was a decrease in the stress protein expression levels for all three stress protein families for 100-microM doses of the mixture combination for 3- and 5-h exposures. These data indicate a dose- and combination-related correlation between depression of the stress protein response and the onset of overt cellular toxicity and/or cell death. The threshold for these changes was cell line specific.     

New rat cell line that is highly susceptible to transformation by several oncogenes.

We describe here a new cell line, EL2, which spontaneously arose from primary rat embryo fibroblasts and has the distinctive property of being highly susceptible to a number of different transforming genes. The high susceptibility is expressed not only in high transformation frequencies but, most importantly, in an unusually high rate of growth of EL2 transformants under selective conditions, i.e., in soft agar or as foci. The biological characteristics of EL2 cells greatly accelerate the isolation of transformants from known oncogenes and could be useful to detect new transforming genes.     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

Stimulated release of histamine by a rat mast cell line is inhibited during mitosis

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine. Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca2+ that preceded secretion of histamine and a similar Ca2+ influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca2+ to the fusion of secretory granules with the plasma membrane.     

The expression of interleukin-6 by a rat macrophage-derived cell line

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rate macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.