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Infection and Immunity Jun 1992In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte...
In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads. LALF bound LPS in a dose-dependent manner as assessed by the precipitation of LPS-LALF complexes with 50% saturated ammonium sulfate. We also studied the ability of LALF to neutralize LPS. LPS preincubated with LALF was less mitogenic for murine splenocytes, was less pyrogenic in the rabbit fever assay, was less lethal in mice which had been sensitized to LPS with actinomycin D, and induced less fever, neutropenia, and pulmonary hypertension when infused into sheep. Our findings extend prior studies which suggested that LALF binds to and neutralizes LPS from multiple strains of gram-negative bacteria.
Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Arthropod Proteins; Dactinomycin; Invertebrate Hormones; Lipopolysaccharides; Male; Rabbits; Sheep
PubMed: 1587618
DOI: 10.1128/iai.60.6.2506-2513.1992 -
Molecular Cancer Jan 2016Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current...
BACKGROUND
Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis.
METHODS
Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation.
RESULTS
Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA.
CONCLUSIONS
The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.
Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Female; Hydroxamic Acids; Imidazoles; Mice; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-myc; Time Factors; Tumor Suppressor Protein p53; Vorinostat
PubMed: 26728659
DOI: 10.1186/s12943-015-0489-8 -
High-throughput screen of natural product extracts in a yeast model of polyglutamine proteotoxicity.Chemical Biology & Drug Design Apr 2014Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high-throughput...
Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high-throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103-expressing yeast cells in 384-well plates (Z' ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50-500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat-shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.
Topics: Animals; Biological Products; Dactinomycin; Gene Expression Regulation; High-Throughput Screening Assays; Humans; Models, Biological; PC12 Cells; Peptides; Protein Aggregation, Pathological; Rats; Saccharomyces cerevisiae
PubMed: 24636344
DOI: 10.1111/cbdd.12259 -
Cell Proliferation Dec 1999The expression of heat-shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress-induced injury. However, existing data about the...
The expression of heat-shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress-induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL-4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL-4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.
Topics: Animals; Apoptosis; Dactinomycin; Flow Cytometry; Heat-Shock Proteins; Hot Temperature; Lymphoma; Mice; Tumor Cells, Cultured
PubMed: 10646688
DOI: 10.1111/j.1365-2184.1999.tb01354.x -
British Journal of Cancer Dec 2013Side effects of chemotherapy are a major impediment in the treatment of cancer. Cyclotherapy is an emerging therapeutic strategy for protecting normal cells from the... (Review)
Review
Side effects of chemotherapy are a major impediment in the treatment of cancer. Cyclotherapy is an emerging therapeutic strategy for protecting normal cells from the side effects of chemotherapy. Low, non-genotoxic doses of known p53 activators can be used to induce p53-dependent cell cycle arrest in normal cells bearing wild-type p53. This cytostatic effect of p53 can protect normal cells from the toxicity of S- or M-phase poisons. Here, we have reviewed existing cyclotherapy regimens using two well-known p53 activators, nutlin-3 and actinomycin D. We have highlighted an exemplar clinical perspective for cyclotherapy in breast cancer. The recent development of novel stapled peptides as activators of p53 without the corresponding cytotoxicity holds great promise for cyclotherapy to enhance the therapeutic window of existing chemotherapy drugs.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Dactinomycin; Female; Genome; Humans; Imidazoles; Phosphorylation; Piperazines; Tumor Suppressor Protein p53
PubMed: 24231949
DOI: 10.1038/bjc.2013.702 -
The Biochemical Journal Nov 19731. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total... (Comparative Study)
Comparative Study
1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent K(m) and V(max.) values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent K(m) values were very similar in both organs. From the estimated V(max.,) it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time-response and a dose-response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose-response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver.
Topics: Animals; Aspartate Carbamoyltransferase; Carbon Radioisotopes; Carboxy-Lyases; Cricetinae; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Kinetics; Leucine; Liver; Lung; Orotic Acid; Pentosyltransferases; Phosphotransferases; Protein Biosynthesis; Pyrimidine Nucleotides; Pyrimidines; RNA; Ribonucleotides; Uridine
PubMed: 4780700
DOI: 10.1042/bj1360781 -
Journal of Virology May 1977RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient...
RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.
Topics: Animals; Caniformia; Cell Line; Dactinomycin; Molecular Weight; Picornaviridae; Poly A; RNA, Viral
PubMed: 559106
DOI: 10.1128/JVI.22.2.572-576.1977 -
In Vivo (Athens, Greece) 2017We have previously reported a procedure for isolating and culturing biliary epithelial cells (BECs). The aim of this study was to reconsider the method for obtaining...
BACKGROUND
We have previously reported a procedure for isolating and culturing biliary epithelial cells (BECs). The aim of this study was to reconsider the method for obtaining pure BECs using the mouse gallbladder.
MATERIALS AND METHODS
Cells that were obtained from the gallbladder alone were sorted by fluorescence-activated cell sorting (FACS) for purifying based on the expression of the epithelial cell adhesion molecule (EpCAM). The viability rate was measured based on the negative expression of 7-aminoactinomycin D (7-AAD).
RESULTS
More than 75% of cells from the gallbladder were determined to be pure BECs. An analysis of the EpCAM revealed that 73.3% of the cells were 7-AAD-negative. Finally, the 0.82×10 pure BECs that survived were obtained and seeded on a collagen gel plate. However, these pure BECs showed almost no proliferation.
CONCLUSION
Pure BECs could be accumulated using FACS. However, the number of BECs was insufficient for the culturing process.
Topics: Animals; Cell Count; Cell Separation; Cell Survival; Cells, Cultured; Dactinomycin; Epithelial Cell Adhesion Molecule; Epithelial Cells; Flow Cytometry; Gallbladder; Male; Mice, Inbred C57BL; Microscopy, Phase-Contrast; Reproducibility of Results
PubMed: 28358696
DOI: 10.21873/invivo.11041 -
The Journal of Biological Chemistry May 1984Rates of ferritin accumulation in the L-6 line of rat skeletal myoblasts cultured in the presence of ferric nitrilotriacetate (FeNTA) were measured and found to vary...
Rates of ferritin accumulation in the L-6 line of rat skeletal myoblasts cultured in the presence of ferric nitrilotriacetate (FeNTA) were measured and found to vary with the extracellular concentration of FeNTA as predicted from dose response experiments. The rate of ferritin accumulation is constant for up to 92 h in these cells after addition of iron, with the exception of the first few hours of synthesis in which the rate is approximately twice that observed at later times. Experiments in which the specific activity of newly synthesized ferritin was calculated implied that the rate of ferritin degradation might be higher in this earlier period as well; pulse-chase experiments confirmed this hypothesis. Ferritin synthesis is thought to be induced by iron in the absence of RNA synthesis. Accordingly, actinomycin D was shown not to inhibit the synthesis of ferritin in response to FeNTA. Rather, a pronounced stimulation of the synthetic rate was observed. Finally, desferrioxamine was shown both to decrease the rate of ferritin synthesis and increase its rate of degradation. Possible mechanisms for these phenomena are discussed.
Topics: Animals; Cell Line; Dactinomycin; Deferoxamine; Ferritins; Iron; Kinetics; Muscles; Rats
PubMed: 6715360
DOI: No ID Found -
Proceedings of the National Academy of... Jul 1985The induction of heat shock proteins in three species of Leishmania, L. tropica, L. enrietti, and L. donovani is reported. When cultures of promastigotes are shifted...
The induction of heat shock proteins in three species of Leishmania, L. tropica, L. enrietti, and L. donovani is reported. When cultures of promastigotes are shifted from 26 degrees C to 37 degrees C or 40 degrees C, the synthesis of proteins with apparent molecular weights of 88,000, 74,000, and 54,000 is stimulated. Actinomycin D added just prior to the shift prevented the appearance of these proteins but had no effect when present 30 min after the transfer onward, suggesting that the regulation of leishmanial heat shock proteins occurs at the transcriptional level. Exposure of L. tropica promastigotes to sodium arsenite elicits the synthesis of three major and four minor polypeptides. Their apparent molecular weights are, respectively, 94,000, 78,000, and 56,000 and 70,000, 45,000, 22,000, and 18,000. The response of Leishmania organisms to heat shock and to sodium arsenite is similar to that of other organisms, but some of the proteins identified as stress proteins in the parasite differ in size. The heat shock proteins might play a role in cytodifferentiation during the life cycle of the parasite and also in cellular adaptation to higher temperatures.
Topics: Animals; Arsenic; Arsenites; Dactinomycin; Electrophoresis, Polyacrylamide Gel; Heat-Shock Proteins; Leishmania; Molecular Weight; Sodium Compounds; Temperature; Transcription, Genetic
PubMed: 3859870
DOI: 10.1073/pnas.82.13.4414