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PubMed1969

The differential sensitivity of cultured chick mesodermal cells to actinomycin D.

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.

Animals↗
PubMed1969

Cell killing by actinomycin D in relation to the growth cycle of chinese hamster cells.

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G(2) (or G(2)-mitosis) they are maximally resistant; at 1.0 microg/ml, for example, the survival in G(2) is 30-fold greater than it is in G(1). These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.

Animals↗
PubMed1968

Antibody production in vitro. II. Effects of actinomycin D and puromycin on the secondary response to sheep erythrocytes.

The effects of actinomycin D and puromycin on spleen cell suspensions from rabbits immunized to SRC's were studied. These inhibitors, in high concentration, suppressed PFC's when added initially to recently isolated cells. When such cells were incubated for several days in the presence of both antigen and inhibitor, both actinomycin D and puromycin produced an increase in PFC's after the initial suppression. This recovery effect was best seen with cells from rabbits killed 3 days after boosting with SRC's, and was usually absent when cells were taken from rabbits killed 2 days after boosting. When actinomycin D or puromycin was added after several days in culture in the presence of SRC's, surviving PFC's were found to be not only resistant to these inhibitors, but there was also an increased number of PFC's compared to similar cultures incubated without these agents. Radioautographic studies showed that PFC's stimulated by the presence of actinomycin D or puromycin were not incorporating precursors for RNA or protein synthesis. In view of the known mode of action of these inhibitors, it was postulated that they were stimulating antibody production by PFC's in vitro either by interfering with represser mechanisms or stimulating the completion of antibody molecules, perhaps by causing the release of preformed antibody chains from ribosomes. Since the presence of specific antigens in vitro were necessary for these observed stimulatory effects on PFC's, and since antigens were producing an effect on antibody production on cells which were being suppressed by these inhibitors, added initially, it was further suggested that one role of antigen in the immune response was concerned with the completion of antibody synthesis on the ribosomes, perhaps by acting as an inducer as has been suggested previously (1).

Animals↗
PubMed1993

Single-stranded phosphodiester and phosphorothioate oligonucleotides bind actinomycin D and interfere with tumor necrosis factor-induced lysis in the L929 cytotoxicity assay.

Antisense oligonucleotides may prove useful tools to elucidate the biological functions of cytokines and to address regulatory control of cytokine expression. The functional activity of cytokines generally is determined by biological assays, and a standard biological assay for TNF activity measures the cytotoxic effect of TNF on actinomycin D-sensitized L929 cells (Matthews and Neale, 1987). We observed that phosphorothioate and phosphodiester oligonucleotides, at concentrations > 100 nM in supernatants tested in this bioassay, prevented TNF-induced lysis of L929 cells. This "protective" effect was due to an interaction of the single-stranded oligonucleotides with actinomycin D as demonstrated by UV spectra of an actinomycin D-oligonucleotide solution. Substitution of cycloheximide for actinomycin D in the L929 assay eliminated the protective effect of the oligonucleotide. Our results reinforce the importance of controlling for nonspecific effects of oligonucleotides, particularly when a functional assay for protein activity is used to screen for antisense-mediated reductions in target protein expression.

Animals↗
PubMed1980

Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo.

It is well known that actinomycin D, a carcinogen, inhibits DNA-dependent RNA synthesis. The interpretation of this inhibition has been that this carcinogen binds specifically to the deoxyguanosine moiety of a DNA molecule, and thus blocks the template function. This paper presents evidence which suggests that this single mechanistic interpretation of actinomycin D action may not be adequate in eukaryotic cells. Thirty minutes after actinomycin D injection (250 microg/100 g body weight), rat liver nuclear RNA synthesis was inhibited by 81% and nucleolar RNA synthesis was inhibited by 98%. In order to determine whether this inhibition is due to an inhibition of DNA template function or of the RNA polymerase activity, the total nuclear free and engaged RNA polymerases were solubilized and the individual RNA polymerase species were partially purified by DEAE-Sephadex column chromatography. It was found that while the overall enzyme activities of RNA polymerase I and III were not affected, there was a selective inhibition of RNA polymerase II activity (42%). This result suggests that actinomycin D, like aflatoxin B1 and N-hydroxy-2-acetylaminofluorene, inhibits nuclear RNA synthesis at multiple sites; it inhibits nucleolar RNA synthesis by blocking the nucleolar DNA template function, and it inhibits messenger RNA synthesis by inhibiting at least partially the enzyme RNA polymerase II activity per se.

Animals↗
PubMed1975

The two-dimensional characterization of amino acids in actinomycin hydrolysates.

Actinomycins, which belong to a family of chromopeptide antibiotics, consist of a hetero-tricyclic chromophore to which are attached two pentapeptide chains either identical or different in amino acid sequence. The classification of existing actinomycins and the identification of new actinomycins are dependent on the characterization and quantitation of the amino acids present in the peptide chains. A simple, fast and highly reliable two-dimensional separation technique employing electrophoresis in a formic acid/acetic acid buffer (pH 1.9) on thin layers of microcrystalline cellulose followed by thin-layer chromatography with an n-butanol:water:glacial acetic acid (50:40:10) solvent system in a direction perpendicular to the electrophoresis was developed to separate the amino acids contained in hydrolysates of the actinomycins. The separated amino acids were identified by two parameters, the chromatographic Rf value and the electrophoretic mobility calculated relative to some standard migrating compound.

Amino Acids↗
PubMed1997

Altered cleavage of DNA sequences by bleomycin and its deglycosylated derivative in the presence of actinomycin.

The antitumour antibiotics bleomycin and actinomycin are commonly used therapeutically in combination. One causes metal ion- and oxygen-dependent oxidative damage to DNA, while the other acts at the level of DNA via intercalation of its phenoxazone chromophore and probable inhibition of topoisomerases. Both drugs bind and/or cleave DNA primarily at guanine-containing sequences, which could lead to mutual interference. Using three different restriction fragments we show that binding of actinomycin to DNA causes major alterations in the sequence specificity of bleomycin.Fe-mediated cleavage, including the appearance of new cleavage sites and the suppression of others. The subtle sequence-dependence of the interference is illustrated by the different effects of actinomycin on DNA cleavage by the deglycobleomycin.Fe complex. Actinomycin sharply decreases the extent of cleavage at GpC sites by both bleomycin and deglycobleomycin whereas cleavage at GpT sites is much less affected, while novel cleavage sites are generated at GpA, ApT and, to a lesser extent, TpT steps. A dramatic increase in bleomycin.Fe cutting at GpA is barely detectable with deglycobleomycin.Fe, confirming that the carbohydrate moiety of bleomycin is important for DNA recognition. The results contribute to a better understanding of how two individually well-characterized small molecules interact simultaneoulsy with specific sequences in DNA and as such assist clarification of the principles governing drug-DNA recognition.

Antibiotics, Antineoplastic↗
PubMed1983

Binding of actinomycin D to mRNA in vivo and in vitro.

1. When rats received an intraperitoneal injection of [3H]actinomycin D, it bound to the RNA moiety of free and bound polysomes of rat liver. The labeling increased gradually up to 6 h or 6 + 6 h. The poly(A)-containing mRNA showed definite radioactivity and its specific activity was higher than that of rRNA, although the total radioactivity of rRNA was markedly higher than that of poly(A)-containing mRNAs. 2. An equilibrium dialysis method using rabbit globin mRNA showed that the binding constant of actinomycin D to globin mRNA was 0.056 x 10(6) M-1, and globin mRNA had 2 binding sites per mol for actinomycin D. 3. From the results of the present experiments and those described in the preceding paper, it is suggested that one of the mechanisms by which actinomycin D treatment in vivo and in vitro stimulates the template activity of mRNA may be binding to mRNA, which alters the conformation of mRNA.

Animals↗
PubMedDate not supplied

Actinomycin D and gemcitabine synergistically sensitize androgen-independent prostate cancer cells to Apo2L/TRAIL-mediated apoptosis.

The cytotoxic efficacy and kinetics involved in sensitization of Apo2L/TRAIL-resistant, androgen-independent prostate cancer cells to Apo2L/TRAIL or tumor necrosis factor-alpha or Fas ligand-mediated apoptosis were tested using subclinical concentrations of actinomycin D, paclitaxel, cisplatinum, gemcitabine, and radiation in CL-1, LNCaP, DU-145, and PC3 prostate cancer cell lines. CL-1 cells expressed all four Apo2L/TRAIL receptors and were resistant to Apo2L/TRAIL-mediated apoptosis (1-5,000 ng/mL) and to the sensitizers when given alone. Pretreatment with actinomycin D followed by Apo2L/TRAIL or tumor necrosis factor-alpha or anti-Fas CH-11 monoclonal antibody, but not in the reverse order, induced apoptosis in all cell lines. Synergistic sensitization in CL-1 cells was shown also with gemcitabine but not with cisplatinum, VP-16, paclitaxel, or radiation. Incubating the Apo2L/TRAIL-resistant CL-1, LNCaP, DU-145, and PC3 cell lines with 100 ng/mL actinomycin D for 4 hours followed by Apo2L/TRAIL for 24 hours resulted in 45.4 +/- 10.3%, 58.8 +/- 3.6%, 53.4 +/- 1.4%, and 84.2 +/- 8.4% apoptosis, respectively. Prolonging the sensitization time to 24 hours followed by 20 hours of incubation with Apo2L/TRAIL further enhanced the killing activity against CL-1 cells to 89 +/- 1% (delta = 60%, synergistic ratio = 3.1). This killing has a biphasic pattern that was contributed to by apoptosis (83%) and necrosis (17%) at 10 hours (peak) and 40% and 60%, respectively, at 20 hours. These results suggest that prostate cancer cells' resistance to Apo2L/TRAIL-mediated apoptosis can be reversed and synergy is achieved by sensitization of tumor cells with subclinical concentrations of actinomycin D or gemcitabine and may be useful clinically for the treatment of metastatic hormone- and drug-refractory prostate cancer.

Androgens↗
PubMed2005

Crystal and solution structures of 7-amino-actinomycin D complexes with d(TTAGBrUT), d(TTAGTT) and d(TTTAGTTT).

The formation of the complex of 7-amino-actinomycin D with potentially single-stranded DNA has been studied by X-ray crystallography in the solid state, by NMR in solution and by molecular modelling. The crystal structures of the complex with 5'-TTAG[Br(5)U]T-3' provide interesting examples of MAD phasing in which the dispersive component of the MAD signal was almost certainly enhanced by radiation damage. The trigonal and orthorhombic crystal modifications both contain antibiotic molecules and DNA strands in the form of a 2:4 complex: in the orthorhombic form there is one such complex in the asymmetric unit, while in the trigonal structure there are four. In both structures the phenoxazone ring of the first drug intercalates between a BrU-G (analogous to T-G) wobble pair and a G-T pair where the T is part of a symmetry-related molecule. The chromophore of the second actinomycin intercalates between the BrU-G and G-BrU wobble pairs of the partially paired third and fourth strands. The base stacking also involves (A*T)*T triplets and Watson-Crick A-T pairs and leads to similar complex three-dimensional networks in both structures, with looping-out of unpaired bases. Although the available NOE constraints of a solution containing the antibiotic and d(TTTAGTTT) strands in the ratio 1:1 are insufficient to determine the structure of the complex from the NMR data alone, they are consistent with the intercalation geometry observed in the crystal structure. Molecular-dynamics (MD) trajectories starting from the 1:2 complexes observed in the crystal showed that although the thymines flanking the d(AGT) core are rather flexible and the G-T pairing is not permanently preserved, both strands remain bound to the actinomycin by strong interactions between it and the guanines between which it is sandwiched. Similar strong binding (hemi-intercalation) of the actinomycin to a single guanine was observed in the MD trajectories of a 1:1 complex. The dominant interaction is between the antibiotic and guanine, but the complexes are stabilized further by promiscuous base-pairing.

Antibiotics, Antineoplastic↗
PubMed1984

Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore.

The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 1H NMR spectroscopy. At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2-amino group and the presence of a hydrogen-bond connecting the 2-amino and the 1-carbonyl groups. In 1H NMR spectra of partially 15N-enriched actinomycin D, the 1JN-H coupling constants at the 2-amino group were determined and a strong sp2 character for the 2-amino nitrogen was deduced. The strong amide character of the 2-amino group is caused by mesomerism involving the 1-carbonyl group. The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule. At lower temperatures a simultaneous and selective broadening of the alpha ring threonine and valine amide proton signals as well as of the 2-amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation. The interpretation of these results requires the presence of a non-planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non-planarity for the intercalation process and for the biological activity of the drug are discussed.

Amides↗
PubMedDate not supplied

Infection with Leishmania infantum Inhibits actinomycin D-induced apoptosis of human monocytic cell line U-937.

Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D-induced apoptosis of the human monocytic cell line U-937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite-free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis-inducing agent. Actinomycin D-induced apoptosis in U-937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U-937 cells via a mechanism involving inhibition of caspase-3 activation. Furthermore, protein kinase C delta (PKC delta) cleavage was increased in actinomycin D-treated U-937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC-mediated pathways by LPG can be implicated in the enhanced survival of the parasites. These results support the claim that promastigotes of L. infantum, as well as its surface molecule, LPG, which is in part released in the culture medium, inhibit macrophage apoptosis, thus allowing intracellular parasite survival and replication.

Animals↗
PubMed1971

Sea urchin embryos are permeable to actinomycin.

When eggs and cleaving embryos of the sea urchin are exposed to [(3)H]actinomycin D, they become radioactive, and autoradiograms show that the radioactivity is inside the cells. At midcleavage, nuclei are more radioactive than cytoplasm. Extraction and chromatography of the intracellular labeled compounds identify them as actinomycin D and a water-soluable derivative. Conversion does not take place outside the cells. Treatment of embryos for 90 minutes with actinomycin D inhibits synthesis of RNA by more than 90 percent, leaving unaffected turnover of the pCpCpA terminals in transfer RNA. These data justify earlier interpretations of actinomycin experiments with embryos and justify use of the drug as a tool in the analysis of gene expression.

Animals↗
PubMed1971

Spore refractility in variants of Bacillus cereus treated with actinomycin D.

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)1, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. (45)Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of (45)Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.

Bacillus cereus↗
PubMed1966

Inhibition of inducible liver enzymes by endotoxin and actinomycin D.

Berry, L. Joe (Bryn Mawr College, Bryn Mawr, Pa.), Dorothy S. Smythe, and Louise S. Colwell. Inhibition of inducible liver enzymes by endotoxin and actinomycin D. J. Bacteriol. 92:107-115. 1966.-Bacterial endotoxin at the ld(50) level lowers liver tryptophan pyrrolase in mice, it prevents for 16 to 20 hr the induction of the enzyme by a concurrent injection of cortisone, it lowers significantly but does not prevent substrate induction, and it reduces the enzymatic activity promptly and significantly when administered during the course of hormonal induction. The ld(50) amount of actinomycin D has a similar effect on tryptophan pyrrolase, except that its inhibition of induction by cortisone persists for a longer period of time. Endotoxin in the intact mouse induces tyrosine-alpha-ketoglutarate transaminase almost as well as cortisone, but not in the adrenalectomized animal, a fact that suggests induction of this enzyme is due to release of endogenous adrenal hormones. Actinomycin D, on the other hand, has an effect on this transaminase similar to that on tryptophan pyrrolase. The site of action of endotoxin and actinomycin D would appear to be similar for one of the two enzymes studied and different for the other, a relationship that requires a specificity difficult to imagine for a material as complex as endotoxin.

Adrenalectomy↗
PubMed1968

Effect of structural and stereochemical methylproline isomers on actinomycin biosynthesis.

The inhibitory effect of methylprolines on actinomycin biosynthesis by Streptomyces antibioticus was studied; the order of effectiveness was 3- >4- >5-methyl-dl-proline. Cis-3-methyl-dl-proline was 14 times more active than the trans isomer. It was also found that 4- and, possibly, 5-methylproline were incorporated into the actinomycin molecule. When 4-methylproline was present, three new actinomycins, representing 50 to 60% of the antibiotic mixture, were synthesized. Growth of the organism may be stimulated at concentrations (0.1 to 1.0 mug per ml) of 3-methylproline that inhibit antibiotic formation, thus providing additional evidence for a different mechanism of actinomycin synthesis from that of protein synthesis. Azetidine-2-carboxylic acid, piperdine-2-carboxylic acid, and hydroxyproline (but not sarcosine) reversed the inhibition due to 3-methylproline.

Chromatography, Paper↗
PubMed1974

Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D.

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.

Cell Line↗