Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.     

Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

SSeCKS is a Suppressor in Schwann Cell Differentiation and Myelination

Src-suppressed protein kinase C substrate (SSeCKS) plays an important role in the differentiation process. In regeneration of sciatic nerve injury, expression of SSeCKS decreases, mainly in Schwann cells. However, the function of SSeCKS in Schwann cells differentiation remains unclear. We observed that SSeCKS was decreased in differentiated Schwann cells. In long-term SSeCKS-reduced Schwann cells, cell morphology changed and myelin gene expression induced by cAMP was accelerated. Myelination was also enhanced in SSeCKS-suppressed Schwann cells co-culture with dorsal root ganglion (DRG). In addition, we found suppression of SSeCKS expression promoted Akt serine 473 phosphorylation in cAMP-treated Schwann cells. In summary, our data indicated that SSeCKS was a negative regulator of myelinating glia differentiation.     

Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.     

Lead and cadmium toxicity in established mammalian cell lines]

Lead and cadmium toxicity was evaluated in three mammalian cell lines (tumour : HeLa, transformed : XC and normal : NRK) by means of the modifications of the 3H-TdR incorporation rate in the nucleus of the treated cells. The three cell lines showed different degrees of sensitivity. Sensitivity depended on the line, metal, its concentration and duration of incubation. Cadmium was found to be at least five times more toxic than lead except at low concentration. The normal cell line was more sensitive to cadmium and less sensitive to lead than other lines.     

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

Production of selected baculoviruses in newly established lepidopteran cell lines

Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×10⁶TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×10⁶TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

Efficient DNA transfection in neuronal and astrocytic cell lines

We have studied different parameters for efficient DNA transfection in various cell types and with different size of the promoter. Here we report that the optimum condition for DNA transfection by electroporation is 350 V/960 microF for PC12, 450V/960 microF C6 cells, and 250 V/500 microF for COS-1 cells. For the human neuroblastoma (SK-N-SH) cells the optimum condition for DNA transfection is by the calcium phosphate method. In promoter mapping studies, a serial deletion approach is commonly used. To optimize transfection we have selected three DNA constructs that varied in size from 4.5 to 12.4 kilobases (kb). We measured the promoter activity of these constructs under conditions of 'equal amount', 'equimolar', and 'equimolar plus carrier DNA to make it equal amount'. We recommend that for comparative purpose, transfection should be carried out under 'equimolar condition' without a need to adjust the total amount of DNA by carrier DNA. Taken together, our results suggest that efficient methods for DNA transfection are important to study gene regulation by devising better ways to deliver DNA into the mammalian cells.     

Growth of three established cell lines on glass microcarriers

Three established cell lines were examined for growth on a newly developed microcarrier which consists of glass beads. The cells were simultaneously exmined for growth on commercially available microcarriers made from DEAE-dextran and from plastic. Cell yields on the glass microcarriers were comparble to the cell yields on the commercially available products. Cells grown on the glass microcarriers were easily separated from the substratum by trypsinization (as were the cells grown on the plastic substratum) while the cells grown on the DEAE-dextran particles were much more trypsin resistant. After removal of cells from the glass microcarriers, the cells reattached and spread out in plastic flasks as readily as cells harvested from monolayer. Scanning electron microscopy revealed dramatic differences in the appearence of the cell grown on the glass microcarriers and cells grown on the DEAE-dextran microcarriers. On the glass microcarriers, cells attached to the substratum through lond, slender filopodia while on the DEAE-dextran microcarriers, the entire edge of the cell appeared to be in contact with the substratum. This dissimilarity in attachment could underly the difference in sensitivity to trypsin-mediated detachment. Finally, the glass microcarriers were washed after being used once and retested for their ability to support cell growth a second time. Nearly identical results were obtained with the reprocessed beads as with previously unused ones.     

Growth and morphology of neuronal cell lines cultured in perfusion

Summary To optimize culture conditions and gain a more reliable culturing system for studies of metabolic properties of neuronal cells, a simplified perfusion chamber was developed. It consists of two parts: a perfusion block and a standard plastic culture dish. To confirm the suitability of this chamber for continuous culturing of anchorage-dependent cells, the growth and morphology of the four neuronal cell lines glioma C6 and glioma 138MG, neuroblastoma C1300, clones N1E115 and N18 were followed for 4 d using both traditional and perfusion techniques. A marked increase in growth and a decrease in the degree of morphological differentiation were obtained with the latter technique compared to the former. This work was supported by grants from the National Swedish Board for Technical Development (Grant 81-5009), the Swedish Work Environmental Foundation (Grant 76-53), and Ollie and Elof Ericssons Foundation for Scientific Research.     

Differential heat shock response of primary human cell cultures and established cell lines

The influence of hyperthermia on the cellular growth and protein synthesis pattern from primary human brain tumour cells and skin fibroblasts was compared with established and experimentally transformed tumour cell lines. Primary cell cultures did not show any visible morphological changes after 42 degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment.     

Characterization of pressure-induced calcium response in neuronal cell lines

BACKGROUND: Cation channels that respond to mechanical stress have been described in neuronal and nonneuronal cells. These nonselective cation ([C+(SA)]) channels are believed to regulate volume and osmolarity of cells in the central nervous system and are therefore believed to be involved in brain injury, resulting in intracellular calcium accumulation and cell death. METHODS: Activation of pressure-sensitive channels was monitored as an increase in [Ca2+](i) by flow cytometry using indo-1. Several neuronal cell lines including NH15-CA2 neuroblastoma x glioma cells were stimulated by rectangular pressure increase. RESULTS: Neuronal cell lines showed a pressure-sensitive increase in [Ca2+](i) but no pressure sensitivity was found in fibroblasts and embryonic P19 cells. [C+(SA)] channels in NH15-CA2 cells were not blocked by inhibitors of voltage-dependent calcium channels and G-proteins. Depletion of extracellular calcium and of internal Ca2+ stores inhibited pressure-induced [Ca2+](i) increase. Elevated [C+(SA)] channel activity was also observed in confluent NH15-CA2 thus accumulated in the G(0)/G(1)-phase of the cell cycle. P19 cells showed occurrence of [C+(SA)] channel activity only after neuronal differentiation. CONCLUSION: Pressure-sensitive channel activity is present in cells of neuronal origin. This activity depends on neuronal differentiation and might have a pivotal role in neuronal development and differentiation.     

Glycolipids and glycosyltransferases in permanent cell lines established from human medulloblastomas.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.     

C3 component of complement secreted by established cell lines

The hamster cell line NIL8 secretes the C3 component of complement as well as collagenous molecules and fibronectin (LETS protein). The C3 is found in the culture medium as a disulfidebonded complex of two polypeptides of 130,000 daltons (alpha) and 65,000 daltons (beta). The secreted C3 can be quantitatively cleaved to C3b and further cleaved by C3 inactivators. The activation of C3 to C3b is promoted by zymosan or by antibody-coated erythrocytes, demonstrating participation in both the classical and alternative complement pathways. The availability of this culture system has enabled us to show that C3 is synthesized as a 185,000 dalton precursor (proC3) which is biologically inactive and becomes cleaved to active C3. Some other established cell lines (NIL1 and BALB/c 3T3) also secrete C3, but some others do not.