Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts

At concentrations of 25 ng/ml in serum-free medium, somatomedin C (SM-C) and insulin stimulated 3H-thymidine incorporation in adult human fibroblasts 4- and 1.5-fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM-C and insulin. Furthermore, 10(-7)M dexamethasone dramatically potentiated SM-C stimulation (70-fold) and insulin stimulation (28-fold) of 3H-thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM-C and insulin. The effects of SM-C and insulin on 3H-thymidine incorporation were additive. These 3H-thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM-C and insulin had equivalent, nonadditive effects on RNA and protein synthesis and protein degradation. Half-maximal effects were seen for both peptides on all three metabolic processes at 2-5 ng/ml. In contrast to their synergism with SM-C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM-C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM-C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM-C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM-C and insulin both participate in the regulation of cell growth and metabolism in vivo.     

Cytotoxic effects of aflatoxin B1 and its association with cellular components in chicken embryo primary cultured cells.

We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Serum and insulin inhibit cell death induced by cycloheximide in the human breast cancer cell line MCF-7

Summary Continuous exposure of cells to cycloheximide (CHM) terminates in cell death. This may result from CHM’s inhibition of protein synthesis. In the present study we investigated the effect of serum and insulin on cell death induced by CHM in the human breast cancer cell line MCF-7, and correlated this effect to the inhibition of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHM (0.1 to 50 μg/ml) was shown to induce cell death in a time- and concentration-dependent manner. Including either fetal bovine serum or insulin in the culture medium inhibited this cell death in a concentration-dependent manner. Protein synthesis as measured by [³H]leucine incorporation was inhibited by the increasing concentration of CHM, However, fetal bovine serum and insulin did not alter the protein synthesis inhibition rate induced by CHM. These results indicate that inhibition of protein synthesis is not enough for cell death to proceed. Insulin or factors present in serum may stabilize some crucial cell proteins (key enzymes, cytoskeletal or membrane components) which are vital for cell life.     

Influence of cyclosporine A on growth and extracellular matrix synthesis of human fibroblasts.

Cyclosporine A (CyA) is a powerful nonsteroidal immunosuppressive agent used to prevent graft rejection of organ and bone marrow transplants. A major side effect observed can be attributed to the fibroblast and its functions: proliferation of fibroblasts and formation of fibrotic tissue in the gingiva (fibrous hyperplasia) and in the kidney are induced. The mechanism of both is still obscure. In order to elucidate whether these side effects are due to the drug acting on human fibroblasts itself or whether they are indirect ones mediated by factors released by lymphocytes, cultures of human gingiva fibroblasts were exposed to CyA under defined in vitro conditions. Incubation with CyA for 72 hours resulted in a dose-dependent stimulation of DNA synthesis, whereas glycosaminoglycan (GAG) synthesis was slightly suppressed. Long-term incubation (6 weeks) with 1 micrograms/ml CyA resulted again in stimulation of growth parameters: compared to the drug-free control, cell number increased to 168%, incorporation of 3H-thymidine into DNA to 143%, and overall protein content to 159%. Collagen and GAG synthesis were elevated to approximately 120%. When corrected for cell number or cell protein content, this represents a decline in matrix synthesis, comparable to short-term incubations. These results indicate that a direct effect of CyA on proliferation of human gingival fibroblasts is responsible for some of the observed hyperplasia.     

放线菌素D和环已亚胺对伊贝母胚性愈伤组织中胚性细胞团和球形胚发生及大分子代谢动态的影响

伊贝母(F.pallidiflora Schrenk)胚性愈伤组织接种于NAA 1.0mg/L+6-BA2.0 mg/L的MS培养基上,在培养10天前可产生大量单细胞到多细胞胚性细胞团,培养10至15天,逐渐形成大量球形胚。利用这样一个实验体系,在培养0、1、2、3和4天后加入放线菌素D(AMD,20μg/ml)和环己亚胺(CHM,20μg/ml),继续培养至第6天,分析大分子代谢动态和观察胚性细胞团的形成情况;培养6和10天后加入同样浓度的AMD和CHM。继续培养至第15天,分析大分子代谢动态及观察球形胚形成情况。结果表明:(1)培养0、1、2、3和4天加入AMD的分别抑制胚性细胞团的100%、63%和45%,加入CHM的抑制100%、85%和75%,培养6和10天后加入CHM抑制球形胚的100%和75%;(2)DNA、RNA和蛋白质在胚性细胞团和球形胚形成时出现两个峰值,其中RNA变化剧烈,最早出现峰值。AMD和CHM分别抑制RNA和蛋白质的合成;(3)过氧化物酶同工酶带在胚性细胞团和球形胚形成过程中顺序表达,AMD和CHM分别在转录和转译水平上对其进行规律性抑制。根据以上结果,本文对伊贝母体细胞胚胎发生的机制进行了初步讨论。     

Protein, RNA and DNA synthesis in skin fibroblast cultures from healthy donors and patients with rheumatic diseases

Synthesis of protein, RNA and DNA was studied in skin fibroblast cultures of healthy donors and patients with systemic scleroderma (SSD) and in those with rheumatoid arthritis (RA) with the use of 14C-protein hydrolyzate, 14C-uridine and 14C-thymidine, respectively. A study was also made of the stimulation of 14C-proline incorporation in protein fibroblasts upon addition to serum-free media of 5% bovine embryonic serum. The stability of RNA in fibroblasts was tested. It was shown that the rate of protein synthesis was 11 times higher in fibroblasts of RA patients and 6 times higher in those of SSD patients as compared to the rate of protein synthesis in fibroblasts of normal subjects. The rate of DNA synthesis in skin fibroblasts of RA patients was 15 times higher and in those of SSD patients 4 times higher than normal. In both RA and SSD patients, the synthesis of short-labeled RNA was 2-3 times higher than normal. The addition of embryonic serum increased 2-3 times the incorporation of 14C-proline in protein skin fibroblasts of SSD patients. It was found that all RNA in skin fibroblasts was represented by long-living molecules and that 30-40% of short-labeled RNA in skin fibroblasts of healthy donors and SSD patients underwent degradation within 1-2 hours. The data obtained indicate that fibroblasts of the two pathologies under study are characterized by considerable differences in the synthesis of DNA and the activity of the protein-synthesizing system.     

Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells

Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.     

Effect of a medium with a low serum content on protein synthesis and secretion and RNA and DNA synthesis in a skin fibroblast culture from patients with rheumatoid arthritis and systemic scleroderma

Synthesis and secretion of protein, as well as synthesis of RNA and DNA by skin fibroblasts of patients with systemic sclerodermia (SSD) and rheumatoid arthritis (RA) upon prolonged culturing of fibroblasts in the medium with low (0.5-1%) serum content differ markedly in their direction and intensity from protein, RNA and DNA synthesis by skin fibroblasts of healthy donors (HD) and by fetal fibroblasts. It has been found that skin fibroblasts of patients with RA and SSD, as well as those of HD, secrete 75-80% of protein synthesized by fibroblasts de novo upon their culturing in DMEM medium with 1% human serum. Under the same conditions, on days 2-5 of culturing, RNA synthesis in the fibroblasts of patients with RA and SSD was increased 3-4-fold, while DNA synthesis was increased 2-3-fold. Collagenolytic and caseinolytic activity in the culture medium of skin fibroblasts from HD and patients with RA reached maximal levels on days 3-5. High protein secretion was observed in DMEM serum-free medium in the presence of vitamin mixture upon culturing skin fibroblasts of patients with SSD. The results obtained show that skin fibroblasts from HD differ in their functional activity from those of patients with rheumatic disorders. It might be suggested, therefore, that the mechanism of protein secretion plays an important role in the maintenance of constant intracellular protein levels in resting cells.     

The action of tomicide on macromolecular synthesis in bacterial cells]

The study of the rate of incorporation of labeled precursors for nucleic acids and protein into Staphylococcus aureus 209 P cell fraction, insoluble in trichloroacetic acid, has revealed that in the presence of tomicide in the medium in a dose of 1 MCI (600 micrograms/ml) the synthesis of DNA in inhibited rapidly and almost completely (by 90%). The inhibition of the rate of incorporation of 3H-thymidine into the cells of staphylococcal culture by tomicide directly correlates with the concentration of the preparation within the range 100-600 micrograms/ml, the inhibition of the synthesis of RNA and protein being less pronounced than the inhibition of the synthesis of DNA.     

Age Associated Variations in RNA Biosynthesis by Cells of the Periodontal Ligament: An Autoradiographic Study1

The utilization of ³H-uridine (³H-U) was used to assess age related changes in RNA biosynthesis by connective tissue cells of the periodontal ligament. One hundred forty-four BNL mice from 5 to 78 weeks old were injected with 5μ Ci/gm body weight of ³H-U and killed from 15 min to 30 days later. Autoradiographs were prepared from 5μm thick, decalcified, sagittal sections of the maxillae. Labeled periodontal connective tissue cells were grain-counted (1) below epithelial attachment, (2) below crevicular epithelium, (3) above alveolar crest, and (4) at the alveolar crest. ³H-U incorporation was apparent at all time periods (15 min to 30 days). Nuclear labeling achieved peak values 1–4 hours after ³H-U injection. Cytoplasmic values were maximum at 1–2 days. Nuclear and cytoplasmic counts were similar at 8–16 hours. From 5 to 52 weeks of age, peak values were progressively reduced, curves were broadened and shifted towards later time periods. This age decline in the rate and magnitude of incorporation of ³H-U reflects changes in the cellular needs for RNA and protein synthesis necessary for matrix production and cellular maintenance. Some age changes were seen at 26 weeks of age. In the oldest animals (78 weeks), there appeared to be an arrest in the age-decline in ³H-U utilization. This was probably associated with the inflammatory changes reported in the periodontal ligament of old animals.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts.

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ or K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate. Addition of 1.8 mEGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.     

Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis.

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+.     

苜蓿体细胞胚胎发生过程中DNA、RNA和蛋白质的合成动态

苜蓿(Medicago sativa L.)下胚轴切段产生的愈伤组织经2,4-D短时间诱导后,在无激素液体培养基中可形成大量体细胞胚胎。经2,4-D诱导后的愈伤组织在转入无激素培养基1天后,其DNA、RNA和蛋白质的合成即进入活跃合成状态,并在体细胞胚胎发育过程中保持逐步升高的趋势。在苜蓿体细胞胚胎发生过程中,有些蛋白质组分含量减少或消失,但绝大部分蛋白质组分的含量明显增加,并且有若干新蛋白的出现,其中24 KD和46 KD蛋白质为体细胞胚胎发生早期所特有。     

Protection from heat-induced protein migration and DNA repair inhibition by cycloheximide

The mechanism by which Cycloheximide (CHM) protects cells from heat induced killing has been investigated. Cycloheximide (10 micrograms/ml) added for 2 hr before and during a 3 hour heating at 43 degrees C prevented a 40% increase of heat-induced protein accumulation in the nucleus and protected cells (0.0001 vs. 0.15 surviving fraction) from heat-induced killing. Heat-induced DNA repair inhibition was also suppressed when cells were treated with CHM in the above manner. This combination of results suggests that protein accumulation in the nucleus and inhibition of DNA repair are related and these events are associated with CHM protection from heat induced cell killing.     

Antagonistic effect of magnesium chloride on the nickel chloride-induced inhibition of DNA replication in Chinese hamster ovary cells

The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl2) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni2+-induced inhibition was time- and concentration-dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl2, which was the highest noncytotoxic concentration utilized. After six hours, 500 microM and 1 mM as well as 2.5 mM NiCl2 all produced the same 50% to 60% reduction in [3H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 microM or 1 mM NiCl2 treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl2 produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl2 used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [3H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl2 for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl2 on DNA replication was prevented by the addition of magnesium chloride (MgCl2) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg2+, since the maximally effective dose of Mg2+ reduced 63Ni2+ uptake by no more than 25% while the inhibition of replication was completely reversed.     

Mode of Action of Myxin on Escherichia coli

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.