Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.     

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D

Summary The effect of cytochalasin B (CB) and cytochalasin D (CD) on the endocytotic uptake of horseradish peroxidase (HRP) by intestinal absorptive cells was investigated by morphometric methods. The results showed that CD inhibited endocytosis considerably, and without any detrimental side-effects. CB had hardly any effect on the endocytosis of HRP, but caused a significant decrease in the number of apical vesicles and tubules involved in the transport of cell-coat glycoproteins from the Golgi apparatus to the brush border.Electron-microscopic autoradiographic analysis of the effect of CD showed that although endocytosis is inhibited significantly by the drug, the amount of radiolabelled cell-coat material entering the lysosome-like bodies was unaltered compared with control cultures. These observations support our hypothesis that the cell-coat glycoproteins of the absorptive cells enter the lysosome-like bodies by a crinophagic rather than by an exocytotic-endocytotic mechanism.     

DNA synthesis in permeabilized karyoplasts from cytochalasin B-enucleated mouse L cells

DNA synthesis was examined in karyoplasts permeabilized by hypotonic buffer or lysolecithin. DNA synthesis was not affected by the omission of a single deoxyribonucleated triphosphate but was greatly reduced in the absence of three of the deoxyribonucleotide triphosphates. The rate of DNA synthesis was linear for 40 min and continued for up to 2–3 h. The DNA synthesized in the permeabilized karyoplasts was a continuation of semi-conservative replication occurring in the intact cell.     

Radiation-stimulated DNA synthesis in cultured mammalian cells

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H³-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G₁ and G₂ cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.     

The stimulation of DNA synthesis by cytochalasin B in proliferative epidermal and dermal cells

Cytochalasin B influences a variety of cellular events that are associated with the contractile microfilament system and the formation of binucleate cells. Along with the formation of binucleate cells, cytochalasin B also causes an acceleration of cells from G1 to S in the cell cycle. By pulsing the cytochalasin B for 30 minutes and allowing for a previously established lag time (17.5 hours) a stimulation of thymidine incorporation into DNA of proliferative epidermal and dermal cells was found in both control and stripped epidermis. Autoradiographic analysis confirmed that the stimulation was due to an increased number of basal cells accelerated from G1 to S phase. A minimal number of binucleate basal cells, 1 in 300, was observed, which suggests that the stimulated synthesis is independent of binucleate cell formation. The amount of stimulation is maximum with cytochalasin B concentration pulse between 5gamma and 30gamma/ml. The results suggest a possible link in coupling cell membrane and surface events with subsequent increased cell nuclei synthetic activity.     

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unscheduled DNA synthesis in cultured ataxia telangiectasia fibroblast-like cells

The capacity of cultured fibroblast-like cells from 5 ataxia telangiectasia (AT) patients to repair γ-ray-induced damage formed in DNA under aerobic conditions was measured by unscheduled DNA synthesis (UDS). The AT cultures exhibited generally reduced UDS compared to the normal cultures for γ-ray doses of 4–12 kR. Reduced UDS in the AT cultures was observed for repair-incubation periods at which UDS was not saturated (0.5 and 1 h), and for periods characterized by maximal amounts of UDS in both the normal and AT cultures (2 and 4 h). The results suggest a class of lesions repairable in the normal cells by UDS within 1 h many persist in the AT cells for 4 h or longer.     

Variants of asynchronous DNA synthesis and mitoses in multinucleate cells induced by cytochalasin B

Cases of asynchronous progression with separate nuclei of S-period and initial mitotic stages in multinucleate cells were discovered in Chinese hamster cell cultures during a prolonged action of cytochalasin B (7 days) and after its stopping (7 days of cell cultivation without drug). The interphase asynchrony under experimental conditions vary in value corresponding to the level of interphase asynchrony in spontaneous multinucleate cells in control cultures. So, the interphase asynchrony in cytochalasin B-induced multinucleate cells is suggested not to be connected with the drug action. Fusion of heterophase cells and a high level of proliferation activity of multinucleate cells seem to be the main reason of interphase asynchrony both in control cultures and in experimental conditions. Unlike the interphase asynchrony, the appearance of the mitotic asynchrony in multinucleate cells is shown to be connected with the action of cytochalasin B. The high level of the mitotic asynchrony remains after the stopping of drug action. A conclusion is made that mitotic asynchrony of nuclei, along with multipolar mitosis and cytokinesis inhibition, is one more display of the cytotoxic action of cytochalasin B on mitosis.     

Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.     

The effect of cytochalasin D on protein synthesis in Xenopus laevis oocytes

Defolliculated fully grown oocytes of Xenopus laevis were treated with cytochalasin D (10 micrograms/ml) and their protein synthesis was studied by labelling with S-35 methionine. This treatment brought about an alteration in pigment pattern as well as a reduction in amino acid uptake by the oocytes. However, the radioactive amino acid taken by cytochalasin-treated oocytes was incorporated into protein in the same proportion as in untreated oocytes. These results suggested that subcortical pigment distribution and amino acid uptake in fully grown oocytes were microfilament-dependent processes, whereas protein synthesis in the oocyte was not.     

Translesion DNA synthesis across non-DNA segments in cultured human cells

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require poleta. In vitro primer extension assays showed that purified poleta, polkappa and poliota were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only poleta and polkappa could fully bypass it. Poleta and poliota, but not polkappa, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells.     

Effect of polycations on prostaglandin synthesis in cultured glomerular mesangial cells

The presence of uniform negative charges on the surface of cultured rat glomerular mesangial cells was demonstrated by an ultrastructural marker, cationized ferritin. Interaction between cell surface negative charges and protamine sulfate, stimulated the synthesis of prostaglandins E₂, F_{2 α}, 6-keto-PGF_1α and thromboxane B₂ (TXB₂) in a dose-dependent manner, reaching a maximum response at protamine concentration of 50 μg/ml. The effect of protamine sulfate was reversed by 25 units/ml heparin. The polyanions, l-glutamic and l-aspartic acids, reversed the protamine effect in a dose-dependent manner. Excess substrate, arachidonic acid, masked the protamine sulfate-stimulated PGE₂ synthesis by mesangial cells. The effect of protamine sulfate on PGE₂ synthesis was rapid, peaked in 5 min and was independent of extracellular Ca²⁺. A synthetic cation, poly(l-lysine) hydrobromide, exerted a similar effect on cellular PGE₂ synthesis in mesangial cells. The effect of poly(l-lysine) was dependent on the molecular mass of the cationic species employed and was maximum at 17 to 90 kDa. The use of large molecular mass polymers of l-lysine (175 and 565 kDa) resulted in a decline in PGE₂ synthesis. These observation indicate that, in mesangial cells, changes in cell membrane electrical charge are linked to enhanced biosynthetic activity and eicosanoid synthesis.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

The effect of methylnitronitrosoguanidine on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in asynchronous and synchronous cultured cells

Methylnitronitrosoguanidine (MNNG) is reported to inhibit DNA synthesis in intact human cells, in the cells from patients with ataxia telangiectasia (AT) or the cells from two rodent species. DNA synthesis in different cell lines exhibits varying sensitivity to MNNG inhibitory effect. 4-5-fold higher concentrations of MNNG are required for 50% inhibition of DNA synthesis in AT cells or in field vole cells as compared with the concentration required for human cells or Chinese hamster. The different compactness of two chromatin fractions might possibly result in lower sensitivity of DNA synthesis in heterochromatin to MNNG-induced inhibition as compared with the sensitivity of euchromatin. The genetic expression of AT defect on the cellular level is supposed to be connected with changes in supramolecular packaging of chromatin in interphase nuclei.     

Influence of cytochalasin d-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.     

Effect of cytochalasin D on secretion by rat pancreatic acini

Using the model of isolated acini the effect of cytochalasin D (CD) on rat pancreatic secretion in vitro was studied. The influence of CD (0.01-10 micrograms/ml = 0.02-19.7 microM) on amylase secretion and 3H-pulse-labelled protein release was measured under two sets of conditions: (a) basal, and (b) stimulated by 77pM caerulein. Basal secretion was not altered, but stimulated secretion of amylase and 3H-labelled proteins were similarly inhibited by up to 45%. It is concluded that CD affects only exocytosis of zymogen granules and not intracellular transport.