Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Nicotinamide adenine dinucleotide (NAD) stimulation of DNA synthesis by human bone marrow cells.

Addition of 20 to 200 μg/ml of Nicotinamide Adenine Dinucleotide (NAD) to short term cultures of normal human bone marrow cells increases DNA synthesis only if human serum is present. Although NAD derivatives; reduced (NADH) and phosphorylated (NADP and NADPH) are equally effective, the components of NAD, Nicotinamide (NA) and Adenine (Ad) have no effect or reduce DNA synthesis at high concentrations. When malignant cells are tested; acute myeloblastic leukemia cell division is unaffected or reduced by NAD, NA or Ad.     

Eimeria tenella: stimulation of DNA synthesis in infected cultured animal cells.

An autoradiographic study was conducted to determine the influence of the intracellular coccidian parasite of chickens, Eimeria tenella, on the incorporation of tritium-labeled thymidine in kidney cell cultures. Evidence was obtained for a parasite-induced increase in thymidine incorporation in host cell cultures which is too great to be attributed to unscheduled DNA synthesis. The stimulatory effect became significant (P < 0.05) at 24 hr after inoculation and further increased at 48 and 72 hr postinoculation. Both parasitized and unparasitized cells in the infected cultures showed similar increases in thymidine incorporation. Furthermore, the increased incorporation of thymidine in the infected cultures was found to be independent of both the percentage of parasitized cells and development of the parasite.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Regulation of cyclic-AMP synthesis in amphibian melanotrope cells through catecholamine and GABA receptors

Catecholamines and GABA are neurotransmitters involved in the regulation of release of pro-opiomelanocortin (POMC) derived peptides from the neurointermediate lobe of Xenopus laevis. The present study concerns the relation of these neurotransmitters to the adenylate cyclase system of the melanotrope cell. During in vitro incubation of isolated melanotrope cells it was found that dopamine, adrenaline and LY 171555 induced inhibition of forskolin-stimulated cAMP production and concomitantly inhibited MSH release. Activation of the GABAb receptors by baclofen also induced inhibition of cAMP production and alpha MSH secretion. Activation of the GABAa receptors evoked stimulation of cAMP production, while alpha MSH release was slightly inhibited, indicating that the GABAa mechanism may prove to be complex. A dual regulation through two subtypes of this receptor might be involved, one stimulating release through the adenylate cyclase system, while the other would inhibit secretion.     

In vitro interactions of murine peritoneal macrophages and sarcoma cells. II. Induction of DNA synthesis in macrophages

Unstimulated peritoneal macrophages grown in vitro for 5 days do not incorporate thymidine. Sarcoma (AA) cells introduced to the culture on the fifth day die and disintegrate whereas the macrophages become markedly stimulated and autoradiography shows that they are triggered to synthesize DNA. In syngeneic cocultures up to 28% of the macrophages incorporate thymidine after 5 days of coculture.     

Lectin-mediated stimulation of DNA synthesis in cultures of embryonic neural retina cells

Plant lectins and other agents which are mitogenic for lymphocytes and fibroblasts were tested for their effects on DNA synthesis in primary monolayer cultures of neural retina cells from 10-day chick embryos. Concanavalin A (ConA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), and anti-retina cell antiserum significantly stimulated [³H]TdR incorporation; the maximum increase was reached 15 h after exposure of the cultures to these agents. Cells stimulated by ConA to synthesize DNA subsequently divided. The divalent succinyl derivative of ConA had a considerably lesser effect than the native tetramer, suggesting that cross-linking of cell surface components may be an important aspect of the changes that lead to the stimulation of DNA synthesis in these cells.Using [¹²⁵I]ConA, the average number of ConA-binding sites per 10-day retina cell was estimated to be 1.7 × 10⁶ (under the culture conditions employed); binding of the lectin to 25–50% of these sites was sufficient to elicit the maximal stimulation of DNA synthesis. Continuous association of the lectin with the cell surface for up to 8 h was essential for the maximal effect, since removal of the lectin from the cell surface (with α-methyl mannose) prior to this time reduced or prevented the stimulation of DNA synthesis.The stimulation by ConA of DNA synthesis in these cultures was dependent on the cell density and was reduced or absent at lower than optimal densities. Examination of this effect suggested that the frequency of intercellular contacts or specific cell associations play a role in the responsiveness of these cells to stimulation of DNA synthesis by ConA.     

Effects of pulsed electric fields on DNA of human lymphocytes

The effects of pulsed electric fields of low frequency (50 Hz) on DNA of human lymphocytes were investigated. The influence of additional external factors, such as hydrogen peroxide (H₂O₂) and γ-irradiation, as well as the repair efficiency in these lymphocytes, was also evaluated. The comet assay, a very sensitive and rapid method for detecting DNA damage at the single cells level was the method used. A significant amount of damage was observed after exposure to the electric fields, compared to the controls. After 2 h incubation at 37^°C, a proportion of damage was repaired. H₂O₂ and γ-irradiation increased the damage to lymphocytes exposed to pulsed electric fields according to the dose used, while the amount of the repair was proportional to the damage.     

Electroendocytosis: stimulation of adsorptive and fluid-phase uptake by pulsed low electric fields

We present a novel approach for stimulating uptake via endocytic pathways by exposing cells to a train of pulsed low electric fields (LEF) in the range of 2.5-20 V/cm. Electric field treatment of COS 5-7 and HaCaT cells in the presence of BSA-FITC augments the adsorption of the probe to plasma membranes with subsequent enhanced internalization. The uptake of BSA-FITC is maximal when the cells are exposed to LEF in the presence of the probe while uptake of a fluid-phase marker, propidium iodide (PI), is more effective when the probe is added immediately after termination of a 1-min exposure. LEF-stimulated uptake decays with a half-life of about 3 and 1 min for and BSA-FITC and PI, respectively. The uptake is inefficient at 4 degrees C but increases with temperature. The uptake proceeds via cell membrane vesiculation, showing a high extent of colocalization of BSA-FITC with plasma membrane vesicles labeled with a phospholipid fluorescent analogue. Unlike constitutive endocytosis where the BSA-FITC is exposed to acidic pH, in LEF-induced uptake the probe is exposed to the more alkaline pH of the cytosol. The staining kinetics of nuclear targets by PI reflects the release of the probe from the LEF-induced vesicles into the cytosol 1-3 h after exposure. The LEF-induced adsorptive pathway was approximately 2.5 more effective than the LEF-induced fluid-phase one. The observed 5- to 6-fold increase of BSA-FITC uptake induced by LEF may be partially attributed to a clathrin-dependent route (up to 25%), whereas the rest of the uptake may be assigned to macropinocytotic and clathrin/caveolin independent pathways or to a novel, yet unidentified, route driven by LEF. This study provides a basis for a general approach towards the efficient incorporation of a variety of molecules such as antibodies, enzymes or genes into cells.     

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation

TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.     

Induction of transformation and DNA synthesis after microinjection of raf proteins.

Full-length and N-terminal deletion mutants of human c-raf-1 cDNA were cloned into Escherichia coli expression plasmids. Bacterially expressed c-raf proteins were purified by anion-exchange, gel filtration, and affinity chromatography. Microinjection of mutant c-raf proteins into G0-arrested NIH 3T3 cells induced DNA synthesis and morphological transformation, whereas microinjection of full-length c-raf had no effect. The amino terminus of the raf protein has an important negative regulatory influence; alteration of this region resulted in increased kinase activity and oncogenicity.     

beta-estradiol stimulation of DNA synthesis requires different PKC isoforms in HepG2 and MCF7 cells.

The role exerted by protein kinase C (PKC) on estrogen-induced DNA synthesis has been investigated in hepatic and mammary gland cells, HepG2 and MCF7. 17-beta-estradiol stimulated DNA synthesis in HepG2 and MCF7 cells, maximal effect occurring at 10 nM. DNA synthesis stimulation was prevented by anti-estrogen ICI 182,780 and by inhibitor of PKC, Ro 31-8220. The rapid estradiol effects in MCF7 cells were determined by following the inositol trisphosphate (IP(3)) production and PKC-alpha membrane translocation. After estradiol treatment the increase of IP(3) production, prevented by anti-estrogen or by phospholipase C (PLC) inhibitor (neomycin), was present in MCF7 cells. In MDA cells, devoid of estrogen receptor, no effect was observed. The PKC-alpha presence on the membranes appeared unchanged in MCF7 cells. The PLC inhibitors, neomycin and U73,122, and PKC-alpha down regulator, phorbol 12-myristate 13-acetate (PMA), were able to prevent estradiol-induced DNA synthesis in hepatoma cells, but ineffective in mammary cells; wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3-K), blocked DNA synthesis in both cell lines. These data show that beta-estradiol, via an estrogen receptor-mediated mechanism, activates more signal transduction pathways, and consequently different PKC isoforms in two responsive cell lines. In both cell lines PI3-K/PKC pathway is functional to the estrogen regulation of DNA synthesis, whereas in HepG2 cells the parallel involvement of the PLC/PKC-alpha pathway is present. The reported results indicate that the DNA synthesis stimulation by beta-estradiol requires the estrogen receptor and utilises one or more activated pathways in dependence on the cell equipment.