Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.     

Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1

Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-alpha/beta) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-alpha4, IFN-beta, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-alpha4, IFN-beta, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-alpha4, IFN-beta, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.     

Inhibition of human placental aromatase in a perfusion model. Comparison with kinetic, cell-free experiments

In vitro perfusion of human placenta was evaluated for characterization of aromatase inhibitors. The results were compared with those in kinetic experiments in cell-free system. Inhibition constants (Ki) were determined by measuring the release of tritiated water during coincubation of human placenta microsomes with varying amounts of [1 beta,2 beta 3H]androstenedione and inhibitor in the presence of NADPH-generating system. Irreversible inactivation constants (Kinact) were determined in a similar manner following preincubation of the microsomes with different amounts of inhibitor for varying times. Lineweaver-Burk plots indicated a competitive type of inhibition with Ki values of 37 nM for 4-hydroxy-androstenedione, 3,700 nM for testolactone, 15 nM for 1-methyl-androsta-1,4-diene-3,17-dione, and 7.5 nM for 19-azido-androstenedione. Additionally, irreversible enzyme inactivation by all four substances could be demonstrated with Kinact values of 3.64 x 10(-3), 0.57 x 10(-3), 0.34 x 10(-3), and 0.69 x 10(-3)sec-1, respectively. Perfusion of a single cotyledon of human term placenta was performed by infusing medium through catheters placed in a fetal artery and in the maternal intervillous space. Perfused medium was collected from a cannulated fetal vein and from the maternal basal plate. The medium was supplemented with [3H]androstenedione (4.2 nM) and inhibitor. The perfusates were analyzed for their [3H]estrone and estradiol content following phenolic partition and Sephadex-LH 20 chromatography. The main results were, (1) the recovery of labelled steroids increased rapidly after perfusion started and reached a plateau within 60 min, when 55 and 30% (mean values) of the infused radioactivity were recovered in the fetal and maternal perfusates, respectively, (2) similar amounts of estrone and estradiol were found in both effluates, whereas androgens (mainly androstenedione and lower amounts of 5 alpha-androstane-3,17-dione) were found nearly exclusively in the fetal perfusate, (3) formation of estrogens (estrone + estradiol) reached a plateau within 20 min of perfusion. (4) The percentage of estrogens formed was not changed by increasing androstenedione concentration in the perfusion medium unless this concentration exceeded 3.5 microM indicating limited capacity of aromatase. (5) The four aromatase inhibitors reduced estrogen formation by 50% at concentrations about 100-fold of their Ki determined in the cell-free system, (6) irreversible aromatase inhibition could not be demonstrated in the perfusion model. It was concluded that the human placenta perfusion model can be successfully used to evaluate aromatase inhibitors.     

Adenosine modulates cell growth in baby hamster kidney (BHK) cells

Adenosine is known to modulate cell growth in a variety of mammalian cells either via the activation of receptors or through metabolism. We investigated the effect of adenosine on Baby Hamster Kidney (BHK) cell growth and attempted to determine its mechanism of modulation. In wild-type BHK cells, adenosine evoked a biphasic response in which a low concentration of adenosine (1-5 microM) produced an inhibition of colony formation but at higher concentrations (up to 50 microM) this inhibition was progressively reversed. However, no biphasic response was observed in an "adenosine kinase" deficient BHK mutant, "5a", which suggests that adenosine kinase plays an important role in the modulation of growth response to adenosine. Adenosine receptors did not appear to have a role in regulating cell growth of BHK cells. Specific A1 and A2 receptor antagonists were unable to reverse the effect of adenosine on cell growth. Even though a specific A3 adenosine receptor antagonist MRS-1220 partly reversed the inhibition in colony formation at 1 microM adenosine, it also affected the transport of adenosine. Thus adenosine transport and metabolism appears to play the major role in this modulation of cell growth as 5'-amino-5'-deoxyadenosine, an adenosine kinase inhibitor, reversed the inhibition of cell growth observed at 1 microM adenosine. These results, taken together, would suggest that adenosine modulates cell growth in BHK mainly through its transport and metabolism to adenine nucleotides.     

Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures

A rapid method to obtain large amounts of tubular gland cells from chick oviduct was developed. Combined collagenase and trypsin treatment allowed within 1.5 h complete dissociation of the magnum portion of the oviduct. By differential attachment of cells, fibroblasts were separated from tubular gland- and ciliated cells. Tubular gland cells attached within 18 h to plastic Petri dishes, had large secretory granules and grew very actively. The responsiveness of cells to hormones and/or antihormone was tested by measurement of cell proliferation and specific protein synthesis. After 7 days of culture in the presence of estradiol (50 nM) or progesterone (100 nM), cell growth was increased by approximately 50 and 35% respectively. Tamoxifen (100 nM) inhibited the estradiol induced growth stimulation, but had also negative effects of its own. The anti-progesterone (in mammals) RU 486, inactive per se, did not antagonize progesterone induced growth. Ovalbumin- and conalbumin synthesis after 4-5 days of cultures under different hormonal conditions was assessed after immunoprecipitation of newly synthesized [35S]methionine labelled proteins. In the presence of estradiol (50 and 100 nM), progesterone (50 nM), and both estradiol and progesterone together (50 nM of each), ovalbumin and conalbumin synthesis was increased, when compared to control cultures without hormones, or to oviduct fibroblasts. Hormonal stimulation of ovalbumin synthesis was also shown in cell supernatant and culture medium after gel electrophoresis.     

The interaction of human blood coagulation factor VII and tissue factor: the effect of anti factor VII, anti tissue factor and diisopropylfluorophosphate.

The effect of Factor VII antibody and an antibody to the apoprotein of tissue factor has been tested on the product formed between Factor VII, tissue factor and calcium ions. The antibody to the apoprotein of tissue factor neutralized tissue factor but had no effect on the extrinsic Factor X activator activity when Factor VII had been allowed to react with tissue factor before the addition of the antibody. The Factor VII antibody neutralized Factor VII and it also blocked the Factor X activator activity when Factor VII had been incubated with tissue factor and calcium ions prior to the addition of Factor VII antibody.Diisopropylfluorophosphate (DFP) was found to neutralize native purified Factor VII and Factor VII in human plasma. This inhibition of Factor VII was very slow and required high concentrations of DFP. However, when the Factor VII had been preincubated with tissue factor and calcium ions, the neutralization of Factor VII by DFP occurred rapidly, and at much lower concentration of DFP.     

Vasopressin V1a receptor signaling in a rat choroid plexus cell line

A new cell line was derived from primary culture of rat choroid plexus (RCP) by immortalization with the TSOri minus adenovirus. The selected clone expressed vasopressin V1a receptors at a density of 64,000 sites per cell, and a K(d) of 7.2 nM. Addition of vasopressin to the RCP cells induced a transient calcium peak comparable to V1a receptor signalling in different expression systems. This [Ca(2+)](i) increase was dose-dependent with an EC(50) of 22 nM vasopressin. Similar [Ca(2+)](i) increase was elicited by addition of serotonin, angiotensin II, endothelin-1, and bradykinin. Heterologous desensitization of V1a receptor was observed in RCP cells exposed to the phorbol ester PMA or following stimulation of other receptors coupled to the phosphoinositide pathway. Positive immunolabelling with Factor VIII, Flt1 and CD 34 antibodies suggests that this new RCP cell line originated from endothelial cells of rat choroid plexus.     

Production of rosmarinic acid in high density perfusion cultures ofAnchusa officinalis using a high sugar medium

Summary The most direct approach to enhancing the volumetric yield of secondary metabolites in plant tissue cultures is to operate the culture under high cell density. In this study, a cell suspension ofAnchusa officinalis was cultivated using a semi-continuous perfusion technique, i.e. batch cultivation with intermittent medium exchange. Using a perfusion medium containing sucrose concentration which was two times that in the normal growth medium, the final cell density and the final product concentration were increased by more than 2-fold compared with a batch culture without medium exchange. The high cell density obtained from the semi-continuous perfusion culture can be explained by the prevention of nutrient depletion, removal of toxic by-products, as well as the control of cell size by virtue of the high sugar medium osmolarity.     

Growth arrest of epithelial cells during measles virus infection is caused by upregulation of interferon regulatory factor 1

Natural infection with measles virus (MeV) is initiated when the virus reaches epithelial cells in the respiratory tract, oropharynx, or conjunctivae. Human epithelial cells infected with MeV frequently show growth suppression. In this study, we investigated the possible mechanisms for this suppression. The bronchiolar epithelial cell A549 showed growth arrest in G(0)/G(1) following MeV infection or treatment with gamma interferon (IFN-gamma). IFN regulatory factor-1 (IRF-1) was upregulated during MeV infection, although A549 did not produce IFN-gamma. Cells of the cervical squamous cell line SiHa persistently infected with various strains of MeV displayed slower growth than uninfected SiHa cells, although the growth rates varied depending on the MeV strain. Transfection of antisense-oriented IRF-1 cDNA released the MeV-infected SiHa cells from growth suppression. Although these infected cells did not produce IFN-gamma and suppressed IFN-alpha/beta-induced Jak1 phosphorylation, Jak1 was constitutively phosphorylated. The growth rates negatively correlated with levels of both IRF-1 expression and constitutively phosphorylated Jak1. These results indicate that MeV upregulates IRF-1 in a manner that is independent of IFN but dependent on the JAK/STAT pathway. This induction of IRF-1 appears to suppress cell growth, although the extent seems to vary among MeV strains.     

Hormonal control of SBP in human hepatoma cells

Since it is currently believed that the biosynthesis of human sex steroid binding plasma protein (SBP) takes place in the liver, the secretion of this protein and its hormonal control were studied in a human hepatoma cell line. The human hepatoma-derived cell line, Hep G2, and a clone, H5A, isolated from Hep G2, were both found to secrete SBP-like protein. This protein had the same dihydrotestosterone binding parameters as plasma SBP, with a Kd ranging from 0.3 to 1 nM at 4 degrees C, and it cross-reacted with a monospecific goat anti-human SBP antiserum. In a chemically defined medium, SBP-like protein secretion was stimulated approx 2-fold by estradiol (1 microM) whereas a smaller concentration of estrogen (100 nM) has only a slight effect. A combined incubation with estradiol (100 nM) and triiodothyronine (10 nM) increased SBP-like protein secretion more than estradiol (1 microM) alone. In response to dexamethasone (100 nM) or tamoxifen (100 nM) treatment, a 3-fold increase is obtained. Therefore, these human parenchymal cells should provide a potent material for investigation of the hormonal regulation of SBP gene.     

Dolichol-sugar derivative synthesis in human breast cancer cell line (T47D). Effects of estrogens and antiestrogens

In the present paper we report evidence about the formation of polyprenyl-phosphate monosaccharides, their elongation products and the assembly of dolichyl-diphosphate-oligosaccharide to endogenous T47D clone 11 proteins upon incubation with [14C]glucose. The influence of estradiol and two nonesteroidal antiestrogens -nafoxidine and tamoxifen- was examined on the dolichol pathway in T47D cell cultures. Estradiol (1 nM) does not change the rate of synthesis of dolichyl-phosphate-sugar derivatives in contrast to nafoxidine and tamoxifen both a micromolar concentration, which induce a remarkable decrease in the formation of dolichol-sugar derivatives. In addition, T47D cells were pretreated with nafoxidine or tamoxifen during one hour, fresh medium supplemented with estradiol was added to the cells simultaneously with [14C]glucose. Results indicated that estradiol after nafoxidine induces a slight increase in the polyprenyl-sugar derivatives formation, however, estradiol after tamoxifen decreases the synthesis of these substances.