Protein Synthesis In Human Fibroblasts Research Articles

Suitability of different cytotoxicity assays for screening combination effects of environmental chemicals in human fibroblasts

Cytotoxicity screening assays measuring survival, growth, colony forming ability, DNA and protein synthesis in human fibroblasts were tested for their suitability to determine combination effects. Thereby, the dose-response curves of a hydrophilic substance A alone and after pretreatment with a membrane damaging substance B were compared. Substances B were applied at concentrations which did not induce toxic effects in the assays (noec). Synergistic combination effects were demonstrated by reduction of the EC 20, value of substances A in the combination in comparison to substances A alone. The following substance pairs (substance B/substance A = membrane damaging/hydrophilic) were tested: n-dodecylbenzenesulfonic acid/2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol/2,4-dichlorophenoxyacetic acid, 1,1,2,2-tetrachloroethane/4-chloroaniline, pentachlorophenol/CrCl 3. While survival, growth, and DNA synthesis assays were suitable methods for detecting synergistic combination effects, the growth assay was the most sensitive. Here, all four substance pairs showed synergistic combination effects.

Ginseng extract inhibits protein degradation and stimulates protein synthesis in human fibroblasts

Aqueous extracts of Panax ginseng inhibit intracellular protein degradation in confluent cultures of IMR-90 human diploid fibroblasts. The magnitude of the inhibition is similar to that observed with insulin and polypeptide growth factors. Furthermore, the inhibition of proteolysis by ginseng, like that produced by insulin and growth factors, is selective in that it applies to long-lived proteins but not to short-lived proteins. Ginseng also stimulates protein synthesis in human fibroblasts indicating that components of ginseng extract are capable of acting directly on human cells to promote protein accumulation.

Arming α2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts

A conjugate containing α 2-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol α 2-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25·10 −9M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3·10 −6M) unconjugated A chain was required, and 180-times less ricin (4.6·10 −11M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of α 2-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for α 2-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.

Analogs of (2‘-5‘)oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells.

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.

The role of cavitation in the in vitro stimulation of protein synthesis in human fibroblasts by ultrasound

The synthesis of protein by human embryonic fibroblasts in vitro was stimulated by irradiation with ultrasound at a frequency of 3 MHz, a spatial and temporal peak intensity of 0.5 W.cm −2, pulsed at a mark: space ratio of 2:8 ms for 5 min at ambient pressure. This stimulation was suppressed by the application of a positive pressure of two atmospheres during irradiation, although increasing the pressure in the absence of ultrasound had no significant effect on the rate of protein synthesis in control cells. Dilatation of the rough endoplasmic reticulum and plasma membrane changes, found in the majority of cells after treatment with ultrasound at ambient pressure, were also suppressed in cells irradiated at elevated pressure. Since it is unlikely that increasing the pressure could modify any ultrasonic effect other than cavitation, these results suggest that cavitation is involved in the mechanism of stimulation.

Control of protein synthesis in human fibroblasts by intracellular potassium.

Abstract Intracellular potassium ion (K+) in cultured human fibroblasts (HF cells) was maintained at reduced steady-state levels by incubating cells in various ouabain concentrations. Small decreases in cell K+ had no effect on protein synthesis and cell growth, but when cell K+ fell below 60–80% of control levels, the rate of protein synthesis decreased in proportion to further reductions in K+. DNA synthesis was also inhibited, presumably because of its dependence on protein synthesis. On the other hand, RNA synthesis remained uninhibited over a wide range of K+ concentrations, an effect characteristic of many specific inhibitors of protein synthesis. In ouabain-treated cells neither levels of ATP nor transport of amino acids was limiting for protein synthesis. Loss of activity of messenger or other species of RNA was not responsible for inhibition of protein synthesis, since in the presence of actinomycin D, the rate of protein synthesis could be decreased or increased solely by adjusting cell K+. Release from ouabain inhibition restored K+ levels, macromolecular synthesis, and cell growth, but there was no resulting synchrony of cell division. In cell populations partially synchronized by serum starvation and refeeding protein synthesis was sensitive to reduction in K+ levels throughout the cell cycle. Our quantitative results show that cell K+ levels, when sufficiently reduced, can determine the rate of protein synthesis and hence the rate of cell growth.