Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells

We evaluated the toxicity of CdCl2, HgCl2, and MeHgCl on the C6/36 cell line of Aedes albopictus. This cell line proved to be a suitable tool for studying heavy-metal toxicity in insect cells. Since data on heavy-metal toxicity in invertebrate cell cultures are almost nonexistent, our results are discussed in relation to in vivo invertebrate and in vitro vertebrate studies. Viability and proliferation were assessed by dye exclusion and DNA quantification, respectively. Viability tests were carried out with and without 5% fetal calf serum in the medium. The three metal species decreased viability to different extents (MeHgCl>HgCl2>CdCl2), and fetal calf serum had a protective effect. In serum-deprived cultures, LD50 values were 140.20, 2.51, and 2.08 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. For cultures with fetal calf serum, LD50 values were 149.71, 12.01, and 5.47 µmol/L, respectively. The viability curve for CdCl2 under serum-free conditions suggests the induction of a cell defense system. The three metal species also inhibited cell proliferation (MeHgCl> CdCl2> HgCl2). The IC50 values were 1.75, 18.36, and 0.96 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. In summary, low MeHgCl concentrations caused both cell death and inhibition of cell proliferation; HgCl2 primarily disrupted the plasma membrane, whereas CdCl2 primarily inhibited cell proliferation.     

Influence of long-term limitation of cell proliferation on the cell cycle duration

The hypothesis was tested that constant, long-term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L-929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 10⁴ cells. No changes in the duration of the cell cycle were observed when, following long-term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence.     

Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.     

Non‐Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification

The fields of regenerative medicine and tissue engineering require large‐scale manufacturing of stem cells for both therapy and recombinant protein production, which is often achieved by culturing cells in stirred suspension bioreactors. The rheology of cell suspensions cultured in stirred suspension bioreactors is critical to cell growth and protein production, as elevated exposure to shear stress has been linked to changes in growth kinetics and genetic expression for many common cell types. Currently, little is understood on the rheology of cell suspensions cultured in stirred suspension bioreactors. In this study, we present the impact of three common cell culture parameters, serum content, cell presence, and culture age, on the rheology of a model cell line cultured in stirred suspension bioreactors. The results reveal that cultures containing cells, serum, or combinations thereof are highly shear thinning, whereas conditioned and unconditioned culture medium without serum are both Newtonian. Non‐Newtonian viscosity was modeled using a Sisko model, which provided insight on structural mechanisms driving the rheological behavior of these cell suspensions. A comparison of shear stress estimated by using Newtonian and Sisko relationships demonstrated that assuming Newtonian viscosity underpredicts both mean and maximum shear stress in stirred suspension bioreactors. Non‐Newtonian viscosity models reported maximum shear stresses exceeding those required to induce changes in genetic expression in common cell types, whereas Newtonian models did not. These findings indicate that traditional shear stress quantification of cell or serum suspensions is inadequate and that shear stress quantification methods based on non‐Newtonian viscosity must be developed to accurately quantify shear stress.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Evaluation of cell proliferation kinetics by the differential staining of the sister chromatids at 2 points of cell fixation

Lymphocyte proliferation in blood cultures from 23 normal individuals was studied. A simple method of evaluation of cell proliferative kinetics with sister chromatid differential staining using two different points of cell harvesting was suggested. It was shown that different composition of tissue culture medium plays a significant role during the cell exit from G0 stage, the mean doubling generation time of cellular population remaining constant.     

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.     

厄贝沙坦对人脐静脉内皮细胞株EA.hy926增殖、凋亡、VEGF mRNA表达的影响

目的:应用不同浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy 926的增殖、凋亡生物学效应及血管发生主要基因VEGFmRNA的表达进行体外研究,探讨厄贝沙坦对内皮细胞的血管生成效应。方法:各种浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy926共同孵育24 h。细胞增殖采用CCK8法分析,Annexin V/PI双染法检测细胞凋亡。RT-PCR验证VEGFmRNA的表达。结果:厄贝沙坦各浓度干预组细胞形态无明显变化,CCK8结果提示厄贝沙坦各干预组相比对照组细胞增殖活力增高(P<0.05),呈浓度非依赖性。流式细胞仪分析厄贝沙坦各浓度干预组细胞无明显凋亡。RT-PCR发现厄贝沙坦1×10-4,1×10-5,1×10-6mol/L浓度组VEGFmRNA表达增高(P<0.05)。结论:厄贝沙坦促进EA.hy926细胞株细胞增殖,上调VEGFmRNA的表达。这提示除了降压效应,血管紧张素受体拮抗剂在缺血性心脏病如慢性心力衰竭治疗中具有一定作用。     

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 predominantly 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.     

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular 'mechanism' behind a rule-in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand-receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.     

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.     

Influence of supramammary lymph node extract on in vitro cell proliferation

Abstract.   Objectives: Experiments were conducted to evaluate whether or not bovine supramammary lymph node extract (LNE) could support cell proliferation when it was substituted for bovine growth serum (BGS) in cell culture media. Materials and Method s: Two different preparations of LNE were tested. The first yielded protein concentration of 3 mg/mL and the second contained 27 mg/mL protein. Three cell lines (MDA-MB-435, MAC-T and 1C6) were used in serum starvation assays to evaluate LNE. Cell proliferation assays were used to determine growth stimulation in the presence of LNE, and short-term or rapid adaptation cultures were evaluated for LNE effects on cell survival. Results : Heat-inactivated preparation 1 supported cell proliferation as well as or better (12–39%) than BGS following 2 days of serum starvation in culture. The second lymph node preparation provided a stimulatory effect (263–702% greater than BGS across all cell lines) following serum starvation at 2.7 and 5.4 mg/mL protein supplementation. A gradual adaptation process with lymph node supplementation into media maintained cell population growth on a short-term basis. However, once cells were trypsinized or scraped and re-seeded into 2.7 mg/mL LNE protein containing media, cells were unable to re-adhere, leaving them detached, and eventually appearing to be dead. Conclusion s: Substitution of BGS with LNE protein dramatically stimulated cells to proliferate, but did not allow for rapid cell population growth adaptation in vitro .     

Lower total cell numbers in mouse preimplantation embryos cultured in human assisted reproductive technique (ART) media are not induced by apoptosis

A common feature of assisted reproductive techniques such as IVF or intracytoplasmic sperm injection is the IVC of oocytes or preimplantation embryos in artificial culture media. The IVC conditions are selected to mimic the environment of the female genital tract. We have shown that murine preimplantation embryos respond to different culture media with changes in developmental rates, cellular lineage composition, and gene expression patterns. In this study, we explored whether apoptosis is responsible for cell loss in mouse preimplantation embryos after exposure to different human culture media. We examined total embryonic cell count as well as the pattern of protein expression for caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and the executioner caspase-3 via immunohistochemical staining. Total cell counts decline in embryos cultured either in innovative sequential medium 1 and Blast Assist (Origio) or human tubal fluid and MultiBlast (Irvine Scientific) when compared to KSOM(aa). Few cells were caspase-9 and -3 positive in all experimental groups. Staining for caspase-8 was almost undetectable. We conclude that embryonic cell loss is not associated with higher rates of intrinsic apoptotic cell loss. Our results suggest that the culture medium–dependent decline in total cell count and the developmental restriction in embryos cultured in innovative sequential medium 1/Blast Assist and human tubal fluid/MultiBlast are related to processes affecting cell proliferation.     

Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model

Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p?<?0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p?>?0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells.     

Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation

Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.     

Properties of the H-4-II-E tumor cell system. I. Growth and cell proliferation kinetics of an experimental hepatoma.

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo--in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is rapidly growing tumor with a mean doubling time of 49-2 hr. The cell cyle time is 39-1 hr with a cell loss factor of 0-32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

Inhibition of human endothelial cell proliferation by heparin and steroids

Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.     

Different growth patterns of a cancer cell population as a function of its starting growth characteristics: analysis by mathematical modelling

The growth kinetics of a cancer cell population as a function of the total number of cells and the proportion of proliferating and resting cells at the beginning of the growth has been analysed by a mathematical model. The model takes into account the processes of cell division, death and transition from proliferation to rest and backwards. It is shown that a single cell population growing under the same environmental conditions has an extremely broad spectrum of growth patterns. The whole multiplicity of possible growth patterns has been determined by the inherent cellular growth characteristics of the population, while the growth pattern actually realized of the variety of growth curves depends on the total number of cells and the proportion of proliferating and resting cells at the initial moment of growth. The model is shown to provide a good prediction of experimentally measured kinetics of regrowth of tumour cells subcultured after various times of the growth in unfed cultures, and the kinetics of tumour cell growth after severe hypoxia. The role of cell transitions between proliferating and resting stages in the problem of growth control is discussed.     

Antagonistic effect of selenite on tumor promoter induced cell proliferation in cultures of rat tongue epithelium

In cultures of rat tongue epithelial cells, cell proliferation following incubation with different doses of the potent tumor promoter TPA has been studied by using a stathmokinetic method counting colchicine arrested metaphases. It was demonstrated that 24 h incubation with concentrations higher than 5 ng TPA/mL medium caused inhibition, whereas below 5 ng TPA/mL medium caused stimulation of the mitotic activity reaching a maximum around 30 h from the start of the incubation period. Based on the evidence of the anticarcinogenic effect of selenium in several animal models, experiments have been performed elucidating the influence of an atoxic dose (1/1.000.000M) of selenite on the observed TPA-induced cell proliferation. Our results indicate that addition to the culture medium of an atoxic dose of selenite, not affecting the mitotic activity of control cultures, inhibits the TPA-induced stimulation of cell proliferation.     

Usefulness of PKHs for studying cell proliferation

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies, (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.