Effect Of actinomycin-D Research Articles

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: The involvement of growth factors in drug resistance

In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2 microgram/ml), or by a short-time exposure to a high dose of ACT-D (2 micrograms/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2 micrograms/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Hydrocortisone on the Ability of Thyrotropin to Increase Deoxyribonucleic Acid Synthesis and Iodide Uptake in FRTL-5 Rat Thyroid Cells: Opposite Regulation of Adenosine 3\5'-Monophosphate Signal Action

In FRTL-5 rat thyroid cells, hydrocortisone alters two TSH-increased cAMP-mediated activities in an opposite manner. Thus, in a concentration-dependent fashion, hydrocortisone synergistically enhances TSH-increased thymidine incorporation into DNA, whereas it inhibits TSH-induced iodide uptake. The effect of hydrocortisone on TSH-increased thymidine incorporation is specific, in that it has only a minimal ability by itself to increase thymidine incorporation into DNA and slightly inhibits the activity of insulin or insulin-like growth factor-I. The effect of hydrocortisone on TSH-induced iodide uptake does not result from altered iodide efflux, but, rather, from a decrease in the maximal velocity, not the Km, of iodide influx, i.e. from a decrease in the effective number of iodide porters. The action of a cAMP analog to increase thymidine incorporation into DNA or iodide uptake was also increased or decreased, respectively, by hydrocortisone, whereas hydrocortisone did not diminish the ability of TSH to increase cAMP levels in the cells. The different effect of hydrocortisone on these two cAMP-mediated activities reflects, therefore, regulation of cAMP signal action rather than the ability of TSH to generate a cAMP signal. Twenty-four hours after TSH, actinomycin-D superinduces iodide uptake and abolishes the action of hydrocortisone to inhibit iodide uptake. It has been suggested that actinomycin-D superinduces iodide uptake in FRTL-5 cells by a posttranscriptional action, inhibition of mRNA degradation, rather than by its known transcriptional actions linked to DNA synthesis. More recent studies of the effect of actinomycin-D, given under identical circumstances, on TSH-stimulated malic enzyme mRNA levels directly validate this hypothesis. We, thus, suggest that the opposite action of hydrocortisone on the two cAMP-mediated activities may reflect positive and negative regulation of the action of cAMP at different steps in the transduction process, one being transcriptional (DNA synthesis) and the other posttranscriptional (induction of iodide porter activity).

Differential Response of Fern Leaves to Senescence Modulating Agents of Angiospermic Plants

Summary Senescence of fern leaflets in darkness is characterised by the loss of pigments. The cations, Ca+2 and Mg+2, regulate leaf senescence differently; magnesium retards the process continuously both in light and dark whereas calcium retards the process at an initial phase and subsequently stimulates the process in dark. The ions however, retard chlorophyll loss significantly in light compared to the control throughout the experimental period. The loss of chlorophyll during senescence is retarded by cycloheximide. Chloramphenicol does not exhibit any modulating effect on pigment loss. The senescence retarding effect of actinomycin-D, a response not observed in the leaves of higher plants, suggests control of fern leaf senescence at the level of transcription. Dark plus kinetin treatment of fern leaflets exhibits a significant protecting effect on chlorophyll loss compared to continuous white light (2500 lux).

Receptor binding of epidermal growth factor in cultured human choriocarcinoma cell lines: effects of actinomycin-D and methotrexate.

Binding of epidermal growth factor (EGF) to its receptor was evaluated in the four cultured choriocarcinoma cell lines BeWo, NaUCC-1, NaUCC-2, and NaUCC-3. Also, the effect of the anti-tumor drugs actinomycin-D (Act-D) and methotrexate (MTX) on the EGF receptor binding was investigated in these cell lines. Incubation of these cells with [125I]EGF at 37 degrees C resulted in a higher binding than that at 22 degrees C or at 4 degrees C. These bindings were saturable during 30- to 60-min incubation, and were specific and reversible. Scatchard analysis showed that the maximal number of receptor binding sites was 2.89 X 10(3)/cell in BeWo cells, 2.04 X 10(3)/cell in NaUCC-1 cells, 1.84 X 10(3)/cell in NaUCC-2 cells, and 1.01 X 10(3)/cell in NaUCC-3 cells. Preincubation with Act-D or MTX for 24 hr decreased the number of receptor binding sites (26%-53%) and slightly increased the receptor binding affinities. Combination of the two drugs resulted in a further diminution of EGF receptor binding sites in BeWo, NaUCC-1, and NaUCC-3 cells, respectively, but reversed the Act-D effect in NaUCC-2 cells. These results indicated that choriocarcinoma tissue is rich in EGF receptors, that the anti-tumor drugs Act-D and MTX diminish the receptor binding sites in the tissue, and suggest that MTX might induce a drug resistance to Act-D in some choriocarcinoma tissue.

Effect of actinomycin-D on the staining of whole cells with acridine orange.

Flow cytofluorometric techniques have been used to investigate the interference of actinomycin-D with the staining of acridine orange in whole cells. The results show that reduction in fluorescence intensity will only occur at actinomycin-D concentrations greater than 10(3) ng/ml in our system, providing the drug is washed out prior to staining.

Growth, functional and structural effects of actinomycin-d, vincristine and radiation in the immature mouse kidney

Growth, functional and structural effects of actinomycin-d, vincristine and radiation in the immature mouse kidney

Interaction between Radiation and Drug Damage in Mammalian Cells

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.

Effect of actinomycin-D on the progesterone induced cytodifferentiation of the chick oviduct epithelium

The effect of actinomycin D on the ultrastructural changes induced by 5 mg of progesterone in the immature chick oviduct epithelium was studied at 0–24h after injection. The height of the epithelium increased during the experimental period. When actinomycin D was given half an hour before progesterone, it inhibited the formation of secretory granules, whereas it could not prevent the formation of the organelles involved in protein synthesis (polysomes, rough endoplasmic reticulum, Golgi apparatus) and the increase in the amount of mitochondria. On the other hand, actinomycin D enhanced the formation of progesterone induced secretory granules if it was administered after the steroid. Apical vacuoles were present within 2h after a combined treatment with progesterone and actinomycin D. Actinomycin D did not affect the changes in the plasma membrane caused by progesterone. The formation of microvilli and cilia began 7 and 12h, respectively, after progesterone. The epithelial cell plasma membranes developed a marked interdigitation, first observable 2h after progesterone administration. The intercellular spaces were markedly wider after actinomycin D, but not after progesterone only.

Dehydrogenases and isocitrate lyase activity during pollen germination inCalotropis procera

Isocitrate lyase (ICL) activity is described in the quiescent and germinating pollen ofCalotropis procera. The behaviour of the enzyme depended upon the presence or absence of exogenous substrate. The activity of this enzyme in relation to some dehydrogenases (MDH; ICDH; G-6-PDH) is compared. Effects of actinomycin-D and cycloheximide on the activity of different enzymes is described. The increase in the activity of ICL and G-6-PDH, during the activation phase, is attributed to theirde novo synthesis. The possibility of sucrose in causing repressing effects on ICL synthesis and/or activation is brought out.

Paradoxical Effects of Actinomycin-D in Rooting Hypocotyl Cuttings of Phaseolus mungo

Summary Hypocotyl cuttings of Phaseolus mungo rooted in water and most profusely in IAA. 1 mg/1 Act-D + IAA significantly increased rooting. The fluctuations of peroxidase, IAA oxidase and amylase activities appear to be associated with the production of roots through the regulation of endogenous contents of nutrition and auxin.

The effect of actinomycin-D on development and infectivity of the nematode, Nippostrongylus brasiliensis

The effect of actinomycin-D on in vitro development and infectivity was examined in Nippostrongylus brasiliensis. In control cultures N. brasiliensis developed to a stage which was comparable to the early lung stage seen in the host. This development was characterized by changes in the structure of the anterior end, growth of intestinal cells and granule formation in the excretory gland. The developmental events were inhibited or delayed by treatment with actinomycin-D (1·0 and 10·0 μg/ml). When worms were grown in the presence of actinomycin-D for varying times their capacity to infect the host was reduced or completely inhibited. These results demonstrated that actinomycin-D prevented development, including infection, of the parasitic phase of N. brasiliensis and support the hypothesis that infection is dependent on transcription.

Interaction between radiation and drug damage in mammalian cells.II. The effect of actinomycin-D on the repair of sublethal radiation damage in plateau phase cells

The effect of actinomycin-D (AMD) on radiation damage repair was studied in plateau phase V79 Chinese hamster cells. Sublethal radiation damage repair, as demonstrated by survival fluctuations following two x-ray exposures separted by time, was observed in our plateau phase cells. Plateau phase cells exposed to 0.01-0.04 mug/ml AMD (a nontoxic regimen to 8 hours) between x-ray exposures were less able to repair sublethal damage. If plateau phase cells were plated at low dilutions into fresh medium (conditions for resuming exponential growth) immediately after the first x-ray dose, and exposed to 0.01--0.04 mug/ml AMD until the second dose, inhibition of sublethal damage repair and additional cell killing were observed particularly at 0.04 mug/ml AMD. It is suggested that radiation-drug damage interactions should be studied in plateau phase cells and in cells resuming exponential growth after plateau phase (possibly analogous to "recruitment"), as well as in exponential phase cultures.

Cuticle formation in parasitic nematodes: Ultrastructure of molting and the effects of actinomycin-D

The ultrastructure of cuticle formation and the effect of actinomycin-D on cuticle formation were examined in the second molt of Nippostrongylus brasiliensis . At the end of the first molt the rough endoplasmic reticulum of the hypodermis (epidermis) had narrow cisternae. As cuticle formation began the RER cisternae dilated and accumulated a fibrous material. These large fibrous material-containing vesicles then decreased and by the time cuticle deposition was completed they had disappeared. Actinomycin-D prevented the proliferation of RER and the formation of fibrous material-containing vesicles. Actinomycin-D induced several ultrastructural alterations in the hypodermis, the most prominent of which were large crystalline-like inclusions. Other alterations involved the configuration of RER and structure of nucleoli. These morphological aspects of cuticle formation were correlated to the molecular events that control molting.

The effects of metabolic inhibitors on the synthesis of inducible tyrosine aminotransferase in cultured hepatoma cells

The effects of actinomycin-D and 3'-deoxyadenosine (cordycepin) on the steroid-mediated induction of tyrosine aminotransferase (TAT) synthesis have been reexamined in view of recent reports that the primary inhibitory action of these compounds may affect synthesis of proteins as well as RNA. The present results confirm that cordycepin blocks the steroid-mediated induction of TAT in rat hepatoma cells (HTC), but unlike actinomycin-D, cordycepin neither increases nor maintains the levels of TAT found in HTC cells preinduced with dexamethasone. Indeed, cordycepin added to preinduced cells, either in the presence or absence of steroid, causes a prompt decline in TAT activity. These data also confirm that both actinomycin-D and cordycepin have an early inhibitory effect on protein synthesis, but the cordycepin effect is observed sooner and the extent of inhibition is greater. When actinomycin-D and cordycepin are added simultaneously to preinduced cells with the steroid removed, the actinomycin-td produced maintenance of preinduced levels of TAT persists. Also, the inhibition of protein synthesis in cultures with both inhibitors approaches that for the cells treated with actinomycin-D alone instead of cordycepin alone. These data suggest that cordycepin inhibits TAT synthesis in preinduced cells by its inhibition of protein synthesis, and this inhibitory effect of cordycepin is blocked by actinomycin-D. It is possible that actinomycin-D does this by preventing the incorporation of cordycepin into RNA. However, regardless of the correctness of this speculation, the multiple effects of cordycepin indicate that this inhibitor cannot be used either to prove or rule out the post-transcriptional model for regulation of gene expression. Also, this requirement that protein synthesis must continue in order to maintain pre-induced levels of TAT is inconsistent with the assumption that the maintenance of these induced TAT levels by actinomycin-D is due to inhibition of TAT degradation.

Additional Studies on the Regulation of Carbohydrate-Dependent Enzymes in the Jejunum: Changes in Amino Acid Pools, Protein Synthesis, and the Effect of Actinomycin-D.

Adaptive changes in rat jejunal disaccharidase levels during carbohydrate starvation and carbohydrate feeding were studied in relation to protein synthesis, amino acid pools, and cell migration. When animals were fed a sucrose-rich, normal protein diet for 24 hr after being on a casein-rich, carbohydrate-free diet for 7 days, there was 50% reduction in the size of the total amino acid pool without a significant change in the proportions of individual amino acids in the pool. The specific radioactivity of the leucine pool increased whenever carbohydrate was added to the diet and protein intake reduced. The incorporation of radioactive precursors into homogenate and brush border proteins reflected the characteristics of the pools, and was thus higher during carbohydrate feeding when the pool size was smaller. The rate of cell migration was unaffected by diet. Disaccharidase activities fell during prolonged carbohydrate starvation. When animals were treated with actinomycin-D, ribonucleic acid synthesis measured 18 hr later was reduced 50%, and cell migration was arrested. The drug did not inhibit the sucrose-stimulated rise in homogenate sucrase activity, although it did abolish the expected increase in fructokinase activity. With actinomycin-D treatment, brush border sucrase activity did not parallel the increases seen in the homogenate, but rather fell markedly. The data demonstrate that dietary carbohydrate increases jejunal disaccharidase activity without elevations in the rate of protein synthesis and that the effect is also independent of ribonucleic acid synthesis and cell migration. By contrast, jejunal fructokinase activity is probably regulated at the level of transcription.

Inhibition of Rna and Protein Synthesis in Granular Cells by Actinomycin-D and Puromycin

The effects of actinomycin-D and puromycin on RNA and protein synthesis were studied in newborn rat epidermis. These antibiotics inhibited RNA and protein synthesis and resulted in ultrastructural changes of keratohyalin granules and of other granular cell constituents: Actinomycin-D produced Medusa bead-like keratohyalin granules and puromycin prevented enlargement of keratohyalin granules by interfering with the insertion of tonofibrils and the addition of dense deposits. This work suggests that the dense deposits observed on keratohyalin granules may originate in the nucleus.

The effect of actinomycin-D on regeneration time inTubularia.

Actinomycin-D was administered to regenerating stem segments ofTubularia spectabilis to ascertain if the synthesis of RNA is necessary for the completion of hydranth development. Preliminary experiments have indicated that this drug suppresses a burst of H3-uridine uptake into the extractable RNA of these regenerates.Wound healing was unaffected by doses of this drug which significantly retarded subsequent hydranth differentiation.The degree of regenerative suppression in this animal increases as a direct function of the administered concentration of actinomycin-D.The period of greatest sensitivity to actinomycin-D occurs early in the regenerative period: hours 6-17 following hydranth amputation. Wittman (1969) has demonstrated a puromyein-sensitive peak of C14-leucine uptake into proteins synthesized at hour 25 or beyond. The authors conclude that the control of hydranth differentiation in regeneratingTubularia resides at the transcriptional level of gene action.

Unterschiedliche Reaktion primärer und permanenter Affennierenzellen auf Actinomycin-D-Einwirkung / Different Reaction of Primary and Continuous Cell Lines of Monkey Kidneys to Actinomycin-D

Primary cercopithecus aethiops kidney cells and continuous cell lines of monkey kidneys CV-1 and BSC-1, react differently to Actinomycin-D. The effect of Actinomycin-D was measured by the incorporation of 3H-Uridine after cells were treated similarly with the inhibitor. Both continuous cell lines exhibited, in the same way, a stronger reaction to Actinomycin-D than did the primary cells.

Effect of actinomycind on the uptake of radioiodine by thyroidal and extrathyroidal tissues—II

Actinomycin D, in vivo, in mice markedly reduces the sodium, potassium and iodide ion excretion indicating an impaired renal clearance. Histopathological examination of kidney of mice injected with the drug reveal distinct degenerative changes in the distal tubule region. The degree of damage to the kidney parallels the amount of the drug injected. The rates of the emptying time of iodide from the stomach to the intestine and excretion through the kidney are significantly lowered in the experimental animals. The increased thyroidal radioiodine uptake after 5 hr is due to a longer availability of the radioiodine caused by the impaired renal clearance and a delay in emptying time of stomach. Histological findings do not show any evidence of stimulation of thyroid gland.

Actinomycin-D: effects on memory at different times after training.

INHIBITORS of macromolecular synthesis have been used extensively for studies of memory storage. The amnesic effects of two inhibitors of protein synthesis, acetoxy-cycloheximide and cycloheximide, have been attributed to specific interference with the synthesis of protein required for formation of long-term memory1–4. The effects of actinomycin-D, an inhibitor of DNA-dependent RNA synthesis, have been particularly difficult to interpret because doses sufficient to produce marked inhibition of cerebral RNA synthesis produce fairly rapid onset of irreversible systemic toxicity5,6. Even much smaller doses produce localized chromatolysis7 and may lead to death. Studies with rodents injected before learning show that marked inhibition of RNA synthesis does not interfere with retention for a few hours after injection5,6,8, but the marked toxicity of this drug has limited its usefulness in studies of the participation of cerebral RNA synthesis in long term memory.