Changes in extranucleolar transcription during actinomycin D-induced apoptosis (original) (raw)
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The Journal of Cell Biology, 1975
The reinitiation of the synthesis of major RNA species has been studied in 37 RC cells after maximal inhibition of RNA synthesis by actinomycin D (AMD). During the period of recovery from AMD, resynthesized RNA (rec-RNA) is initially composed of almost exclusively light (4-14S) heterogeneous RNA species. All normal species of RNA can be detected in the rec-RNA spectrum as early as 3 h after AMD removal. The synthesis of low molecular weight methylated RNA species increases slightly during the early period after AMD removal, while the increase of low molecular weight unmethylated species is more significant during the same period. Much of the radioactivity in the polyribosomal fraction is EDTA and puromycin sensitive. Since polysomal, puromycin-sensitive RNA is polyadenylated (as evidenced by the binding to poly-U filters), and is heterogenous in size, it belongs to the m-RNA class. The synthesis of m-RNA increases immediately after AMD removal, whereas the reinitiation of the r-RNA synthesis occurs after a lag period of about 2 h. The kinetics of recovery of the synthesis of major RNA species from AMD inhibition show a size dependency comparable to the size-related sensitivity to AMD inhibition in other cellular systems. This dependency is most clearly seen in HnRNA, the AMD sensitivity of which is measured by the length of the lag period between AMD removal and the appearance of HnRNA fractions in a sucrose density gradient. Low molecular weight HnRNA reappears first, whereas heavier fractions of HnRNA appear in the spectrum after a lag period, the length of which is in direct relation to the position of the HnRNA fraction in the gradient.
1969
For the purpose of revealing whether AMD inhibits the RNA synthesis of erythroblasts in an effective dose in vivo to eradicate erythroid cells in rabbit bone marrow, the author observed the RNA synthesis by H3-uridine incorporation in vitro and RNA level on the cells from the anemic animals taken at a certain period after a single injection of AMD in a small dose of 50 and 100μg/kg body weight. The data revealed that by such a small dose of injection of AMD the RNA synthesis of erythroid precursors, early basophilic and proerythroblast stages, was successfully suppressed without any suppressing effect on the RNA synthesis of erythroblasts in the later stages of specialization, indicating that there are at least two kinds of RNA synthesis: one seen mainly in the earlier stages of specialization and the other one seen mainly in the later stages, and they can be distinguished from each other by the AMD sensitivity.</p
Experimental Cell Research, 1975
It has previously been shown that actinomycin D at a concentration of 0.08 pg/ml preferentially inhibits nucleolar (ribosomal) RNA synthesis in monolayer cultures of Chinese. hamster cells. Treatment with 0.08 rg/ml actinomycin D of Chinese hamster cells, synchronized by mitotic selection, at the beginning of the G 1 period causes a delay in the onset of DNA synthesis while a similar treatment in the late Gl period does not affect the process. The same effect has been produced by 9 pg/ml lucanthone (miracil D), a drug selectively inhibiting ribosomal RNA synthesis. The addition of 0.08 pg/ml actinomycin D to the stationary cultures stimulated to proliferate by medium changes completely prevents cells from entering the S period, when the drug is added to the medium in the first half of the pre-replicative period. However, in the second half of the prereplicative period cells regain the ability to synthesize DNA in the presence of actinomycin D approximately to the same extent as do cells in the G 1 period directly following mitosis. Thus, the demand for a de novo ribosomal RNA (rRNA) synthesis may be considered as a specific feature of cells induced to proliferate after a period of quiescence. The data obtained are consistent with the assumption that the first part of the pre-replicative period in stimulated quiescent cells is characteristic of GO cells while later on the GO cells enter the path of G 1 cells typical for the rapidly proliferating cell populations.
Studies on the Action of the Nuclear Factor Promoting Actinomycin D-Binding Capacity of Chromatin
Differentiation, 1977
It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.
Journal of Biological Chemistry, 2004
Formation of ␥-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that ␥-H2AX foci were also formed when cells were incubated with 0.5 g/ml DNA intercalating agent actinomycin D. In untreated cells, ␥-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with ␥-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and ␥-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to ␥-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with ␥-H2AX foci. Taken together, these results suggest that histone ␥-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.
Delocalization of some small nucleolar RNPs after actinomycin D treatment to deplete early pre-rRNAs
Chromosoma, 1997
Retention of some components within the nucleolus correlates with the presence of rRNA precursors found early in the rRNA processing pathway. Specifically, after most 40S, 38S and 36S pre-rRNAs have been depleted by incubation of Xenopus kidney cells in 0.05 µg/ml actinomycin D for 4 h, only 69% U3 small nucleolar RNA (snoRNA), 68% U14 snoRNA and 72% fibrillarin are retained in the nucleolus as compared with control cells. These nucleolar components are important for processing steps in the pathway that gives rise to 18S rRNA. In contrast, U8 snoRNA, which is used for 5.8S and 28S rRNA production, is fully retained in the nucleolus after actinomycin D treatment. Therefore, U8 sno-RNA is in a different category than U3 and U14 sno-RNA and fibrillarin. It is proposed that U3 and U14 sno-RNA and fibrillarin, but not U8 snoRNA, bind to the external transcribed spacer or internal transcribed spacer 1, and when these binding sites are lost after actinomycin D treatment some of these components cannot be retained in the nucleolus. Other binding sites may also exist, which would explain why only some and not all of these components are lost from the nucleolus.
Proceedings of The National Academy of Sciences, 1962
Actinomycin D is one of a number of polypeptide antibiotics isolated in Waksman's laboratory.1' 2 Bacteriostatic effects, particularly on gram positive bacteria, and antitumor activity have been attributed to this compound.2 3 Kirk4 has demonstrated that the addition of actinomycin D (0.2 to 0.5 ,4M) to exponentially growing cultures of Staphylococcus aureus stops RNA synthesis immediately. This effect is rapidly followed by an inhibition of protein synthesis, and later by a partial inhibition of DNA synthesis. The action of this compound is not related directly to energy production since both respiration and glycolysis of inhibited cells are unaffected by concentrations up to 0.1 mM.4 Kirk also demonstrated that the combination of DNA and actinomycin D results in a spectral change of the latter compound. These observations suggest the formation of a complex between these two compounds since Kawamata and Imanishi5 found no interaction of actinomycin and RNA and the reaction appears to be relatively specific for DNA. Although Rauen et al.6 have reported complex formation between actinomycin and RNA, 100 times more RNA than DNA is required.