Incubation In Serum-containing Medium Research Articles

Kinetics and isotherm of fibronectin adsorption to three-dimensional porous chitosan scaffolds explored by125I-radiolabelling

In this study, 125I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using 125I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.

Cadmium-Induced Apoptosis in Rat Kidney Epithelial Cells Involves Decrease in Nuclear Factor-Kappa B Activity

Renal epithelial cells undergo apoptosis upon exposure to cadmium (Cd). Transcription factors, such as nuclear factor-kappa B (NF-kappaB), mediate the expression of a number of genes involved in apoptosis. The present study was designed to examine the involvement of this transcription factor in Cd-induced apoptosis. Rat kidney proximal tubular epithelial cells, NRK-52E, were incubated with up to 20 microM CdCl2 in serum-free medium for 5 h followed by incubation in serum-containing medium (without Cd) for an additional 12 h. The cells accumulated 582 +/- 19 ng Cd/mg protein after 5-h exposure to 20 microM Cd. As a result of Cd exposure, the DNA-binding activity of the p65 subunit of NF-kappaB was decreased in a concentration- and time-dependent manner. The activity of tumor necrosis factor-alpha-induced inhibitor of kappa B (IkappaB) kinase alpha was also inhibited by Cd. In addition, the phosphorylation of IkappaB-alpha and NF-kappaB p65, as well as the levels of NF-kappaB target gene products, cIAP-1 and cIAP-2, were reduced. Pretreatment of the cells with the antioxidant U83836E or butylated hydroxytoluene preserved the DNA-binding activity and blocked the Cd-induced decease in IkappaB-alpha phosphorylation. Cd exposure caused the activation of caspase-3, -7, and -9 and DNA fragmentation. By flow cytometry, 14.6 and 30.5% apoptosis was detected at 6 and 12 h after stopping the Cd exposure. Overexpression of NF-kappaB p65 by transient transfection protected the cells from the Cd-induced apoptosis. Conversely, attenuation of NF-kappaB activity by pretreatment with SN50, an NF-kappaB nuclear translocation inhibitor, potentiated apoptosis. These results suggest that Cd-induced apoptosis involves suppression of NF-kappaB activity which may be mediated by oxidative stress.

Inhibin, activin, and follistatin in human fetal pituitary and gonadal physiology.

Activin has been previously demonstrated to directly stimulate the synthesis of gonadotropin-releasing hormone (GnRH) receptors and to increase follicle-stimulating hormone (FSH) secretion in nonhuman pituitary cell cultures (PCCs). Currently, knowledge of the physiological role of these peptides in primates is still far from complete. Moreover, several results in macaque monkeys failed to support an unequivocal role for inhibin in FSH suppression. Whereas the bioactivity of inhibin and activin has been demonstrated in rat PCCs, no data exist on human pituitary response to these peptides either in vivo or in vitro. We studied the secretion of FSH and luteinizing hormone (LH) by dispersed human fetal pituitary cells from midtrimester abortions in response to recombinant human (rh-) activin-A, inhibin-A, and other secretagogues. After mechanical and enzymatic dispersion, the human fetal pituitary cells were cultured on an extracellular matrixlike-material-coated 24-well plate. After 3 days' incubation in serum-containing medium, the PCCs were washed and preincubated for 90 min in serum-free medium and incubated with activin-A, inhibin-A, TGF-beta, follistatin, sex steroids, and GnRH, in quadruplicate. Activin-A was a potent secretagogue for FSH secretion. GnRH (20 ng/ml) was more potent than rh-activin-A for LH secretion. Nevertheless, a significant increase in LH secretion into the medium was brought about by rh-activin-A. Inhibin decreased FSH and LH secretion, but the LH response to inhibin was less prominent than that of FSH. GnRH opposed the inhibitory effect of inhibin on LH secretion. In dynamic, short-term, repetitive exposure of fetal pituitary fragments to rh-activin-A (superfusion), we could not receive a similar increase in LH and FSH as in static incubations, as opposed to a short GnRH exposure. In addition to their endocrine, paracrine, and autocrine effects, and in addition to their role as possible markers, the TGF-beta superfamily members may affect embryogenesis and possibly immunomodulation of the embryo and fetus. The role of activin and inhibin as intragonadal regulators is hypothesized. The pro-alphaC inhibin precursor may act as an FSH receptor antagonist. Human fetal PCCs express the previously reported physiologic responses to activin and inhibin generated in nonhuman experiments on gonadotropin secretion in vitro and may serve as a physiologic model for studying human gonadotrope responses to the TGF-beta family of peptides.

DNA repair and survival in human lens epithelial cells with extended lifespan

Purpose. Ultraviolet-B radiation (290–320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. Methods. Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. Results. HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6–4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6–4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. Conclusions. These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca 2+-containing medium after exposure to Ca 2+-free medium caused an increase in intracellular Ca 2+ ([Ca 2+ i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40–44°C for 10–20 min in fetal calf serum-free medium, incubated at 37°C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca 2+. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca 2+] i nor the Na +-Ca 2+ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca 2+] i.

Characterization and applicability of a cytotoxicity assay determining growth inhibition after a 1-hour treatment with xenobiotics in human cell culture

A cytotoxicity assay to determine growth inhibition after a 1-hr treatment with xenobiotics in serum-free medium and subsequent re-incubation in serum-containing medium without test chemicals was characterized and optimized for cytotoxicity screening. Our results indicated that after treatment of human fibroblasts with the test chemicals a 44-hr re-incubation period is most suitable for measuring the growth inhibitory effects of a broad spectrum of chemicals. Applying the assay, the activity to inhibit growth by 10% (log IC 10) correlated with the lipophilicity (specified as octanol-water partition coefficient = log K ow) of 19 xenobiotics. The linear regression coefficient was r = 0.91. This close correlation between toxicity and lipophilicity shows that the test, which avoids long-term incubation in serum-containing medium, is suitable for cytotoxicity screening and structure-activity analysis.

Placental lactogen-binding sites in isolated fetal fibroblasts: characterization, processing, and regulation.

Placental lactogen (PL) stimulates amino acid transport, DNA synthesis, and insulin-like growth factor production in isolated fetal fibroblasts and myoblasts. To clarify the mechanisms by which PL exerts its effects in fetal tissues, we have examined the binding and processing of PL and its receptor in cultured ovine fetal skin fibroblasts. Fetal sheep fibroblasts bound ovine PL (oPL) with high affinity (Kd, 0.2 nM) and ovine (o) GH (Kd, 1.6 nM) and oPRL (Kd, > 200 nM) with lower affinities, as recently reported. Maximal specific binding of oPL was noted after a 24-h incubation at 4 C. When fibroblasts were incubated with [125I]oPL at 4 C and then warmed to 37 C, the radiolabeled hormone was internalized within 2-4 min. Most of the internalized hormone, however, was recycled rapidly to the cell surface by 6-10 min and released intact by retroendocytosis into the incubation medium. Of the small amount of remaining internalized radioligand, approximately 50% appeared to be degraded, as assessed by solubility in trichloracetic acid. To directly examine the cellular processing of receptor proteins, we used the membrane-impermeant cross-linking reagent bis-(sulfosuccinimidyl)suberate (BS3) to detect oPL-binding sites confined to the cell surface. These studies detected a specific oPL-receptor complex with a mol wt of 130 kilodaltons, suggesting that the mol wt of the membrane receptor is 108 kilodaltons. At 37 C, oPL-receptor complexes were internalized rapidly, reducing cell surface binding activity to 25% of the initial values within 4 min. oPL receptor activity reappeared on the cell surface at 6 min, suggesting receptor recycling, but declined precipitously during the ensuing 30 min, reflecting receptor processing and turnover. After proteolysis of surface receptors with trypsin, binding activity was recovered fully during a 12- to 24-h incubation in serum-containing medium. Recovery of surface receptors was blocked by cyclohexamide (2 microM), suggesting that repopulation of receptors depends upon new protein synthesis. Specific binding of PL was 1.5- to 5-fold higher in cells preincubated in medium containing dexamethasone (0.1 microM), suggesting that glucocorticoids may regulate expression of the fetal PL receptor. These studies provide insight into the cellular mechanisms by which the fetus may regulate its sensitivity and response to PL.

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominately diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.

Role of pronase-resistant peptide segments of the antitumor protein antibiotics, auromomycin and macromomycin, in stabilizing cytocidal activity of the chromophore moieties to carcinoma cells.

The free chromophores isolated from the antitumor protein antibiotics, auromomycin (AUR) and macromomycin (MCR), were rapidly inactivated by incubation in serum-containing medium at 37 degrees C in the dark with respect to cytocidal activity to human lung carcinoma A549 cells. Under the same conditions, the intact antibiotics, their pronase-hydrolysates and reconstituents from the chromophores and apo-proteins were stable. Intact and reconstituted AUR and MCR were more resistant to pronase digestion than the apo-proteins. The analyses of the pronase-hydrolysates of AUR and MCR by SDS-polyacrylamide gel electrophoresis and ultrafiltration showed that the antibiotics (13 kilodaltons (kDa] were degraded to produce peptide fragments (1-3 kDa) in which most cytotoxicity of the pronase-hydrolysates resided. The pronase-hydrolysates exhibited a differential cytocidal activity to normal diploid fibroblasts (WI38), their SV40-transformants (VA13) and carcinoma cells (A549) of human lung origin as was observed for the intact antibiotics. These results indicate that specific interaction between the chromophores and the pronase-resistant peptide segments (1-3 kDa) of the protein moiety stabilizes the cytocidal activity of the chromophores and also protects the peptide segments from pronase digestion.

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl 2 (0.3 mM) and NaF (10 mM) and moderately by l-tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl 2, c-GMP and l-tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.

Decrease and recovery in cell surface charge induced by anesthetics.

We applied cell electrophoresis to detect the effect of anesthetics on membrane, and clarified the concentration - effect relationship and the term of reversibility. The cell electrophoretic mobility of rat erythrocytes decreased with time of incubation with intravenous anesthetics (thiopental or droperidol) at clinical concentrations and reached a minimum value of 25% reduction in 30 min. The effect changed gradually in the concentration range of 0.5-1000 microgram/ml of thiopental, and changed markedly with 0.3-1 microgram/ml of droperidol. Washing out of the anesthetics and subsequent incubation in serum-containing medium resulted in the complete recovery in the mobility when 5 microgram/ml thiopental or 1 microgram/ml droperidol was used. With high concentration of thiopental (100 microgram/ml) or droperidol (10 microgram/ml, however, unrecovered fraction of cells increased with time of treatment. The critical time for retaining the reversibility was about 2 hr.