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1.
The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density. 相似文献
2.
Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%. 相似文献
3.
Exogenous hyaluronic acid (HA) added to a confluent monolayer of 3T3 BALB cells facilitates cell detachment which can be enhanced by gently pipetting. When HA is added to a cell culture with the cell inoculum, the cells are able to grow and form a confluent monolayer, but the cellular density is lower than in the control cultures, in a concentration-dependent way. This difference seems due to the ease of detachment promoted by HA on the cells near confluency. In fact only near confluency is the amount of the detached cells greater in the culture plates containing HA than in controls. Culture dishes containing substrate-attached material (SAM) left behind by the confluent 3T3 BALB cells have been prepared by removing the cells with different detaching agents. The SAM-containing dishes have been incubated in the presence of HA for 24 h and, after washing, were used for cell cultures. The cells grown on such HA-treated dishes show a very low density at confluency and in some cases are prevented from forming a confluent monolayer. When the SAM-containing dishes are treated with Streptomyces hyaluronidase, the effect of HA is abolished and the cells are able to grow normally. Among the chondroitin sulphates, only chondroitin sulphate C shows the same effects as HA, whereas A and B are ineffective. 相似文献
4.
Potassium influx has been investigated in XTH-2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Density-related changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were 1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; 2) the relative amount of 86Rb taken up via the Na+ -K+ -2Cl- cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide-sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; 3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either "masking" or "unmasking" of the enzyme; 4) no alterations in K+ uptake occur that would be characteristic of the "stationary growth phase." The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+-ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity. 相似文献
5.
6.
The growth of chick heart cells in culture declines when the cells reach confluency. The decline in growth rate is associated with both a decrease in the pH of the bicarbonate-CO2 buffered medium and a reduced capacity for glucose oxidation by the pentose phosphate pathway. The pH of proliferating cultures supplemented with either 14 mM NaHCO3 or with a mixture of organic buffers (pK 7.4) was increased by 0.3 pH unit over that of the controls. The rate of glucose oxidation by the pentose phosphate pathway in confluent cultures supplemented with NaHCO3 or organic buffer increased by 60% 24 h after pH correction. This was associated with an increase in glucose uptake from the medium. We conclude that pH elevation in confluent heart cell cultures stimulates both growth and the capacity for glucose oxidation by the pentose phosphate pathway. The data also provide further evidence for a relationship between activity of the pentose phosphate pathway and cell growth. 相似文献
7.
Michael D. Aleo Mary L. Taub Peter A. Nickerson Paul J. Kostyniak 《In vitro cellular & developmental biology. Plant》1989,25(9):776-783
Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This
study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over
a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures
within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after
confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity
of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers
into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular
protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent
cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal
tubule cell culture system for future studies in in vitro toxicology.
This paper was resented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society
of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988.
This work was supported by grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid
of Research Award. 相似文献
8.
Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor 总被引:13,自引:2,他引:11
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We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent. 相似文献
9.
The mechanisms by which hepatocytes regulate their cell numbers in culture have been examined. We found that when murine hepatocytes were cultured at an overconfluent stage, the number of viable cells were reduced to that of the confluent stage 48 h later by cell death. Cell death was accompanied by LDH release, and it was observed only in primary cultured hepatocytes but not in hepatoma cells. Genomic DNA analysis using electrophoresis showed that DNA fragmentation, a biochemical hallmark of apoptosis, was induced in superconfluent cultures of hepatocytes in a cell-density-dependent fashion, but not in pre-confluent cells. DNA fragmentation was rapidly induced 2 h after the beginning of the in vitro culture and continued up to 24 h later. Flow cytometry analysis demonstrated that the nuclei from the hepatocytes in a high density culture were condensed and that the DNA content was reduced. These data suggest that the mechanism of cell death is apoptosis. The DNA fragmentation seen in the high density hepatocyte culture was not observed in hepatoma cell lines. Moreover, apoptosis was induced in hepatocytes of MRL/lpr mice, suggesting that the Fas antigen was not involved in the apoptotic process. Apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, and by a calmodulin antagonist, W-7. Taken together, the results indicate that high density culture of murine hepatocytes though not hepatoma cells regulate their cell numbers by an apoptotic mechanism. The apoptosis is dependent on de novo protein synthesis and intracellular calcium metabolism. 相似文献
10.
Bello RI Gómez-Díaz C Navarro F Alcaín FJ Villalba JM 《The Journal of biological chemistry》2001,276(48):44379-44384
The aim of this work was to study the role of H(2)O(2) in the regulation of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC ) with relation to cell density of HeLa cells cultures and the function played by NQO1 in these cells. Levels of NQO1 activity were much higher (40-fold) in confluent HeLa cells than in sparse cells, the former cells being much more resistant to H(2)O(2). Addition of sublethal concentrations of H(2)O(2) (up to 24 microm) produced a significant increase of NQO1 (up to 16-fold at 12 microm) in sparse cells but had no effect in confluent cells. When cells reached confluency in the presence of pyruvate, a H(2)O(2) scavenger, NQO1 activity was decreased compared with cultures grown to confluency without pyruvate. Inhibition of quinone reductases by dicumarol substantially decreased viability of confluent cells in serum-free medium. This is the first demonstration that regulation of NQO1 expression by H(2)O(2) is dependent on the cell density in HeLa cells and that endogenous generation of H(2)O(2) participates in the increase of NQO1 activity as cell density is higher. This enzyme is required to promote survival of confluent cells. 相似文献
11.
A human renal carcinoma from a patient with an erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency the cultured cells formed multicellular hemicysts (domes) which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase in Ep levels in the culture medium after the cultures reached confluency, in parallel with an increase in dome formation. The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a significant dose-related inhibitory effect on Ep production and dome formation in the renal carcinoma cell cultures, suggesting an important role of protein kinase C, the only known receptor for TPA, in inhibiting the expression of differentiated phenotypes in the renal carcinoma cells. TPA also suppressed Ep secretion over a period of 96 h, indicating a time course of suppression of this differentiated function of the renal carcinoma cells in culture. This hypothesis was further supported by the observation that diacylglycerol, the endogenous activator of protein kinase C, likewise inhibited Ep production and dome formation in the renal carcinoma cell cultures. These studies suggest a role of the inositol-lipid second messenger path and protein kinase C in the regulation of Ep production. 相似文献
12.
Ju-Hye Lee Yeong-Seon Won Ki-Hun Park Mi-Kyung Lee Hirofumi Tachibana Koji Yamada Kwon-Il Seo 《Apoptosis : an international journal on programmed cell death》2012,17(12):1275-1286
Celastrol has been reported to possess anticancer effects in various cancers; however, the precise mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by celastrol treatment in melanoma cells remains unknown. We showed that celastrol effectively induced apoptotic cell death and inhibited tumor growth using tissue culture and in vivo models of B16 melanoma. In addition to apoptotic cell death in B16 cells, several apoptotic events such as PARP cleavage and activation of caspase were confirmed. Pretreatment with caspase inhibitor modestly attenuated the celastrol-induced increase in PARP cleavage and sub-G1 cell population, implying that caspases play a partial role in celastrol-induced apoptosis. Moreover, ROS generation was detected following celastrol treatment. Blocking of ROS accumulation with ROS scavengers resulted in inhibition of celastrol-induced Bcl-2 family-mediated apoptosis, indicating that celastrol-induced apoptosis involves ROS generation as well as an increase in the Bax/Bcl-2 ratio leading to release of cytochrome c and AIF. Importantly, silencing of AIF by transfection of siAIF into cells remarkably attenuated celastrol-induced apoptotic cell death. Moreover, celastrol inhibited the activation of PI3K/AKT/mTOR signaling cascade in B16 cells. Our data reveal that celastrol inhibits growth and induces apoptosis in melanoma cells via the activation of ROS-mediated caspase-dependent and -independent pathways and the suppression of PI3K/AKT signaling. 相似文献
13.
Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures. 相似文献
14.
Gröbner S Autenrieth SE Soldanova I Gunst DS Schaller M Bohn E Müller S Leverkus M Wesselborg S Autenrieth IB Borgmann S 《Apoptosis : an international journal on programmed cell death》2006,11(11):1959-1968
Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of κB kinase (IKK)-β
resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells
(DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating
simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and
measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk
prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential ΔΨm and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial
death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent
apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs
independently from apoptotic events or as an apoptotic epiphenomenon. 相似文献
15.
The Cytotoxic Effects of Camptothecin and Mastoparan on the Unicellular Green Alga Chlamydomonas reinhardtii
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Jürgen Voigt Markus Morawski Johannes Wöstemeyer 《The Journal of eukaryotic microbiology》2017,64(6):806-819
We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well‐known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag‐phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occurred during the cell‐division phase. Essentially no differences between wall‐deficient and wild‐type cells were observed with respect to dose‐response and time‐course of camptothecin and mastoparan. In cultures of the wall‐deficient strain, cell death was accompanied by swelling and subsequent disruption of the cells, established markers of necrosis. In case of the wild‐type strain, camptothecin and mastoparan caused accumulation of apparently intact, but dead cells instead of cell debris due to the presence of the wall. Both in cultures of the wall‐deficient and the wild‐type strains, cell death was accompanied by an increase of the protein concentration in the culture medium indicating a lytic process like necrosis. Taking together, we have severe doubts on the existence of an apoptotic program in case of C. reinhardtii. 相似文献
16.
Taxol caused apoptotic cell death of Taxus cuspidata in suspension cultures. Typical morphological and biochemical changes of apoptosis were observed by microscopy and total DNA agarose gel electrophoresis. Taxus cuspidata responded to the added Taxol by increasing the biosynthesis of Taxol. The percentage of apoptotic cells in total cells increased with the concentration of added Taxol. With Taxol added at 10 mg l–1, the maximum concentration of Taxol produced was 23 mg l–1, 3 times higher than that of the control culture. 相似文献
17.
The expression of transgenes in eukaryotic cells is a powerful approach in cell biology. In most cases, it is based on the activity of strong and constitutive viral cis-acting elements in eukaryotic expression vectors. Here we show that a widely used such element derived from an early gene of human cytomegalovirus is strongly and differentially regulated in mouse cell lines. We analyzed cytomegalovirus promoter-driven expression of stably transfected transgenes in growing, confluent, and differentiating mouse 3T3 fibroblasts, C2C12 myoblasts, and P19 teratocarcinoma cells. In the fibroblasts, transgene expression was strongly downregulated in confluent cultures and was upregulated in growing or confluent cultures by phorbol ester. In contrast, no downregulation by confluency, nor upregulation by phorbol ester, was detected in C2C12 cells. In addition, while marked upregulation was detected in differentiating myotubes, transgene expression was downregulated when differentiating teratocarcinoma cells assumed a neuronal phenotype. These results demonstrate the existence of drastic differences in the regulation of transgene expression in different types of cell lines, indicating that when studying transgene function in cells that are not growing exponentially, viral promoter-driven expression should not be taken for granted. 相似文献
18.
Andre P. Boulet Michel A. Fortier 《In vitro cellular & developmental biology. Plant》1987,23(2):93-99
Summary Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits.
Isolated cells were cultured in PRMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC)
at a plating density of 0.5×106 cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded
myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent
cultures. The addition of increasing doses of 17β-estradiol (E2) (0.1 nM to 1 μM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was
due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein
was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell
pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.
This research was supported by Natural Sciences and Engineering Research Council of Canada, Ottawa, Ontario, grant no. U-0389. 相似文献
19.
A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line. 相似文献
20.
Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells 总被引:8,自引:1,他引:7
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Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics. 相似文献