Synchronisation de cellules BHK 21 par carence partielle en -asparagine

The isolation of mutants requiring -asparagine from a population of BHK 21/13 cells has enabled us to synchronize the cell growth using the deprivation effect of this amino acid. In the absence of asparagine, cells cannot enter a new cycle of DNA synthesis and accumulate in G 1 phase, If asparagine is added to 24 h growth-arrested cultures, a wave of DNA synthesis is observed after a 6 h latency period, followed by a partially synchronous cell division reaching a 55% labelling index. During a 24 h period, in the presence of very small amounts of asparagine (1 to 6 × 10⁻⁶ M) a higher cell fraction accumulates at G 1 period. In these conditions the degree of synchronisation is improved, reaching an 85% labelling index, following a 24 h incubation in the presence of 6 × 10⁻⁶ M asparagine.L'isolement de mutants exigeants en -asparagine d'une population clonale de cellules BHK 21/13 nous a permis d'exploiter l'effet de la carence en cet acide aminé pour synchroniser les cellules. En l'absence d'asparagine, les cellules ne peuvent entrer dans un nouveau cycle de synthèse de DNA et s'accumulent en phase G 1. Après 24 h de carence, si l'on place les cellules ainsi quiescentes dans un milieu contenant de l'asparagine, on observe après un temps de latence de 6 h une onde de synthèse de DNA suivie d'une synchronisation partielle des divisions cellulaires. En présence de faibles quantités d'asparagine (1 à 6 × 10⁻⁶ M) un plus grand nombre de cellules s'accumulent en phase G 1 durant la période de carence de 24 h. Dans ces conditions l'enrichissement de cette phase en cellules nous a permis d'améliorer le degré de synchronisation et d'obtenir un index de marquage de 85% après une carence en 6 × 10⁻⁶ M d'asparagine.     

Effects of dexamethasone on the levels of plasma corticosteroid binding globulin in rats and monkeys.

Rat marrow cells were preincubated for 45 hours with 5.5 × 10^?4M sodium hexachloroiridate. This treatment abolished DNA synthesis whilst improving cell survival over that of controls. The synthesis of RNA, protein and glycoprotein continued and could be further increased by the addition of erythropoietin for up to 44 more hours. Heme synthesis also continued in the absence of DNA synthesis but could not be stimulated by erythropoietin.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Periodate-induced lymphocyte transformation : II. Character of response and comparison with phytohemagglutinin and pokeweed mitogen stimulation

Sodium metaperiodate is mitogenic for human peripheral lymphocytes. Evidence of stimulation can be detected as increased thymidine incorporation at 72 h after only 10 sec of exposure to the IO₄⁻. The degree of response varies with lymphocytes from different donors, but maximum stimulation for the healthy donors studied was obtained at concentrations of IO₄⁻ between 10⁻³ M and 4 × 10⁻³ M. Concentrations of 8 × 10⁻³ M and above are non-stimulatory and toxic. Exposures to optimum concentrations for 1 h or longer result in essentially no stimulation and inincreased cell death. However, a significant response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) remains. The kinetics of response over a 4 day culture period are similar for IO₄⁻, PHA and PWM. The morphology of the blast cells and the degree of response suggest that the IO₄⁻ responsive lymphocyte population may be more closely related to the PWM stimulated cells than the PHA responsive lymphocytes.     

Protective effect of vitamin E on reduction in colony formation of cultured human cells by bisulfite

Bisulfite had a severe cytotoxic effect on HeLa S-3 cells at a concentration of 10⁻⁴ M, causing complete inhibition of colony formation. A concentration of 10⁻M bisulfite had a moderate cytotoxic effect on the cells, reducing their colony-forming activity to one-third of that of untreated control cultures. Addition of vitamin E at a concentration of 10⁻⁷ M restored the colony-forming activity of 10⁻⁵ M bisulfite-treated cells to 71% of that of control cultures. Addition of 10⁻⁶ M vitamin E resulted in almost complete recovery of the colony-forming activity of bisulfite-treated cells to that of the control cultures. The counteraction of vitamin E to the cytotoxic effect of bisulfite on human cells was discussed.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Cell detachment in ouabain-sensitive and resistant ehrlich ascites cells

Ehrlich ascites tumor cells selected for resistance (OR) to the toxic effects of ouabain, a specific inhibitor of (Na⁺, K⁺)-activated ATPase, differ from wild-type ouabain-sensitive (OS) ascites cells in their detachment behavior from protein-coated glass surfaces in the absence of ouabain. In the presence of ouabain OS cells are more easily detached from cultured vessel surfaces, but ouabain is either without effect (10⁻⁴ M) or inhibits detachment of OR cells (10⁻³ M) under similar conditions. The results are discussed in terms of the possible relationship between membrane ATPases and cell behavior.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells’ (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5 mm diameter cattle follicles was cultured without treatment (control), with ODQ (10⁻⁴ M) and 10⁻⁵, 10⁻³ and 10⁻¹ M SNP with or without ODQ for 24 h. Nitrate/nitrite (NO₃⁻/N0₂⁻) concentrations were evaluated by Griess method, progesterone (P₄) and 17β-estradiol (E₂) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P < 0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10⁻⁵ M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P > 0.05), while GC cultured with 10⁻³ and 10⁻¹ M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P < 0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10⁻⁵ and 10⁻³ M SNP with or without ODQ were in the G0/G1 (80–75%) stage and in a lesser proportion (20–25%) in the S + G2/M stage of the cell cycle, while the 10⁻¹ M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10⁻⁵ M SNP increased (P < 0.05) E₂ synthesis and inhibited (P < 0.05) progesterone synthesis. The addition of ODQ reversed (P < 0.05) the stimulatory effect of 10⁻⁵ M SNP treatment on E₂, but not on P₄ synthesis (P > 0.05). These results demonstrated that E₂ synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P₄ while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

A possible involvement of type II cAMP-dependent protein kinase in the initiation of DNA synthesis by rat liver cells

Calcium-deprived T51B rat liver cells initiated DNA synthesis within 1 h after addition of calcium. The possibility of this DNA-synthetic response having been mediated through arachidonic acid metabolism (i.e., through an arachidonic acid cascade) is suggested by the facts that calcium is known to stimulate phospholipase activity which releases arachidonic acid from membrane phospholipids; that low concentrations (10⁻⁹–10⁻⁶ mole/1) of arachidonic acid itself elicited the same DNA-synthetic response from the calcium-deprived cells as calcium; and that the stimulatory actions of calcium and arachidonic acid were both blocked by the endoperoxide synthase inhibitor indomethacin.     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro

On the basis of cell shape, refractility under phase optics, spontaneous beating and nucleolar number, two cell types have been distinguished in embryonic heart cell cultures: M cells (myoblastlike) and F cells (fibroblast-like). However, by different criteria, more than two cell types have been found in the heart in vivo. In the present study, heart cell types are redefined by properties which can be used for identifying cells both in vitro and in vivo. Trypsin-dissociated cells from 7-day chick hearts were cultured for 24 h. PAS staining indicated that all M (thick, refractile) cells contain glycogen (gly⁺), while most F (spread) cells do not (gly⁻). Under the electronmicroscope, only gly⁺ cells contain myofibrils; most of these cells beat spontaneously in culture. Gly⁻ cells do not beat. Gly⁺ cells with myofibrils as well as gly⁻ cells are found in the inoculum and in the heart in vivo. The ultrastructure of gly⁺ cells in vitro and of muscle cells in the myocardium in vivo is similar. Therefore, it is concluded that gly⁺ cells in culture are derived from the myocardium (muscle) of the heart. Gly⁻ cells are apparently derived from the epicardium, the endocardium and the endothelial lining of blood vessels.     

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Inhibition of cell division by blue light

Microsporocytes of Lilium and Trillium can revert into a mitotic cycle when separated from the anther before the beginning of meiosis and cultured in vitro. Repeated short daily irradiations with visible blue light (10⁶ ergs cm⁻² sec⁻¹) inhibit the onset of mitosis; the cells remain in interphase instead. The inhibition of cell division is reversed after the end of the light treatment. Meiosis is not affected by light. Part of the premeiotic G2 phase is light sensitive. No arrest of DNA-, RNA- or protein synthesis is involved in the photoinhibition of onset of mitosis. Irradiation with blue light decreases the α- absorption band of cytochrome a₃ but not of cytochrome a. This effect on cytochrome oxidase, however, is observed in all stages of meitotic prophase as well as in G2 cells.     

The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

Human leukocyte cultures were set up with Ham's F-10 medium and stimulated with PHA-M. Treatment of the cells in G₁ from 15–20 h with 0.5 × 10⁻⁶ M Trenimon resulted in a considerable cell cycle delay, as measured by [³H]-TdR autoradiography and determination of mitotic indices. Under these conditions only few cells incorporated the tracer at the same time as most cells did in untreated cultures. However, this did not lead to a mitotic activity at the same time as obtained in controls. Most of the treated cells started their DNA synthesis and mitotic activities with a delay of around 20 h, as compared with the controls. Continuous treatment of the cells with 10⁻³ M NaF had no effect on [³H]TdR labelling or mitotic indices in otherwise untreated cultures, but led to an impressive effect on DNA synthesis in Trenimon-treated cultures, without a considerable effect on the mitotic indices. This finding could beexplained as due to a lower alkylation in cellular DNA in the presence of NaF. More cells can start with their DNA synthesis, although they are, like Trenimontreated cultures, incapable of completing it normally. Analyses of the effect of NaF on chromosomes aberrations induced by Trenimon revealed that pre-, simultaneous and post-treatments significantly enhanced the frequency of undamaged mitoses. Continuos fluoride treatment also protected the cells from Trenimon-induced damage, but the effect was not significant, possibly because of heavily damaged mitoses which appeared under these conditions. We interpret our findings as an indication of a real anti-mutagenic activity of NaF.     

Effect of ESF preparations on different types of erythroblasts

A new method of quantitative ¹⁴C-autoradiography was applied for evaluating possible effects of erythropoietin (ESF) on the DNA synthesis rate of differentiated erythroid murine bone marrow cells identified as proerythroblasts, basophilic and polychromatic erythroblasts. Eosinophilic myelocytes were used as a control cell population. ESF was prepared from the urine of a patient with chronic aplastic anemia; an inactive urinary preparation served as control. The potency of the preparations was estimated by the ⁵⁹Fe-incorporation assay. The materials to be tested were injected into polycythemic mice 4, 8 and 16 hours before in vitro short-term incubation of the bone marrow cells with ¹⁴C-thymidine and Methotrexate. Animals without test material were taken as additional controls. Autoradiographic grains were counted by an incident light microscope photometer. Eight hours after injection of ESF a significant increase in the mean ¹⁴C-thymidine incorporation was found in all three erythroid cell types when compared either with the inactive control preparation (excess incorporation 30–40%) or with the untreated control animals (excess incorporation 10–20%). It could be shown that the increase is due to an immediate action of ESF on already differentiated cells and cannot –- at least not solely –- be attributed to its action on hemopoietic stem cells. The control preparation which was inactive in terms of ⁵⁹Fe incorporation exerted a slight inhibition of DNA synthesis rate in all erythroid cells as well as in cells not committed to erythroid differentiation.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.