Low Concentrations Of Actinomycin Research Articles

Dynamic chromosome association with nuclear organelles in living cells.

The development of progressively sophisticated tools complemented by the integration of live cell imaging enhances our understanding of the four-dimensional (4D) nucleome, revealing elaborate molecular interactions and chromatin states. Yet, the dynamics of chromosomes in relation to nuclear organelles or to each other across cell cycle in living cells are underexplored. We have developed photoconvertible GFP H3-Dendra2 stably expressing in PC3M cells. The nuclear lamina and perinucleolar associated heterochromatin or diffuse chromosome regions were photoconverted through a single-point activation using a confocal microscope. The results demonstrated a dynamic nature for both types of chromosomes in the same cell cycle and across mitosis. While some chromosome domains were heritably associated with either nuclear lamina or nucleoli, others changed alliance to different nuclear organelles postmitotically. In addition, co-photoconverted chromosome domains often do not stay together within the same cell cycle and across mitosis, suggesting a transient nature of chromosome neighborhoods. Long-range spreading and movement of chromosomes were also observed. Interestingly, when cells were treated with a low concentration of actinomycin D that inhibits Pol I transcription through intercalating GC-rich DNA, chromosome movement was significantly blocked. Treatment with another Pol I inhibitor, metarrestin, which does not impact DNA, had little effect on the movement, suggesting that the DNA structure itself plays a role in chromosome dynamics. Furthermore, inhibition of Pol II transcription with α-amanitin also reduced the chromosome movement, demonstrating that Pol II, but not Pol I transcription, is important for chromosome dynamics in the nucleus.

AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei

BackgroundNuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. MethodsIsolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). ResultsNuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. ConclusionsThe resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. General significanceA new outlook to studying internal nuclei structure is proposed.

CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.

305: Natural and synthetic inhibitors of mTOR blunt the p53 response to low concentrations of actinomycin D but not nutlin-3

305: Natural and synthetic inhibitors of mTOR blunt the p53 response to low concentrations of actinomycin D but not nutlin-3

Differential Disruption of EWS-FLI1 Binding by DNA-Binding Agents

Fusion of the EWS gene to FLI1 produces a fusion oncoprotein that drives an aberrant gene expression program responsible for the development of Ewing sarcoma. We used a homogenous proximity assay to screen for compounds that disrupt the binding of EWS-FLI1 to its cognate DNA targets. A number of DNA-binding chemotherapeutic agents were found to non-specifically disrupt protein binding to DNA. In contrast, actinomycin D was found to preferentially disrupt EWS-FLI1 binding by comparison to p53 binding to their respective cognate DNA targets in vitro. In cell-based assays, low concentrations of actinomycin D preferentially blocked EWS-FLI1 binding to chromatin, and disrupted EWS-FLI1-mediated gene expression. Higher concentrations of actinomycin D globally repressed transcription. These results demonstrate that actinomycin D preferentially disrupts EWS-FLI1 binding to DNA at selected concentrations. Although the window between this preferential effect and global suppression is too narrow to exploit in a therapeutic manner, these results suggest that base-preferences may be exploited to find DNA-binding compounds that preferentially disrupt subclasses of transcription factors.

Enzymatic Rate Enhancements: A Review and Perspective

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.

Actinomycin D synergistically enhances the efficacy of the BH3 mimetic ABT-737 by downregulating Mcl-1 expression

Many types of cancer cells possess the ability to evade apoptosis, leading to their rapid and uncontrolled proliferation. As major regulators of apoptosis, Bcl-2 proteins serve as emerging targets for novel chemotherapeutic strategies. In this study, we examined the involvement of Bcl-2 proteins in apoptosis induced by the chemotherapeutic agent actinomycin D. A dramatic decrease in anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) mRNA and protein expression was detected upon actinomycin D treatment. Further, Mcl-l over-expression caused resistance to cell death upon treatment with actinomycin D, implicating a role for the down-regulation of Mcl-1 in actinomycin D-induced apoptosis. We also explored the therapeutic potential of actinomycin D in combination with ABT-737, an experimental agent that inhibits anti-apoptotic Bcl-2 proteins. Actinomycin D sensitized cells to ABT-737 treatment in a Bak- or Bax-dependent manner. Importantly, low concentrations of actinomycin D and ABT-737 were more effective in inducing cell death in transformed cells than their untransformed counterparts. A synergistic effect of actinomycin D and ABT-737 on cell death was observed in several human tumor cell lines. Like actinomycin D treatment, knocking down Mcl-1 expression greatly sensitized tumor cells to ABT-737, and Mcl-1 over-expression abrogated the cytotoxic effect induced by ABT-737 and actinomycin D. These results suggest that the down-regulation of Mcl-1 by actinomycin D is likely responsible for the observed synergistic effect between the two drugs. Overall, our studies provide compelling evidence that the combination of actinomycin D and ABT-737 may lead to an effective cancer treatment strategy.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Actinomycin D upregulates proapoptotic protein Puma and downregulates Bcl-2 mRNA in normal peripheral blood lymphocytes

We have examined the ability of actinomycin D to induce apoptosis in human peripheral blood lymphocytes. Run-On assays were performed to specify the primary molecular damage, reverse transcription-PCR, Western blots and flow cytometry studies were performed to ascertain which proteins of the apoptosis machinery were affected to cause actinomycin D-induced cell death. Expression of 23 apoptosis-related genes was investigated. The down-regulation of ribosomal RNA synthesis caused by actinomycin D induced a mitochondria-dependent apoptosis. Although the expression of the majority of examined genes remained indifferent against actinomycin D activity, the cellular level of p53 protein increased, subsequently upregulating both Puma mRNA and protein. Puma-mediated mitochondrial apoptosis was accompanied by nucleolin cleavage and Bcl-2 mRNA destabilization. The stability of the cellular level of Bcl-2 protein independent of a mRNA decrease suggests that protection of Bcl-2 protein against proteasomal degradation can moderate the apoptotic process. In peripheral blood lymphocytes cultured in vitro, the apoptosis induced by a low concentration of actinomycin D (10 nmol/l) is dependent on p53 and Puma activation. This apoptotic pathway is demonstrated in peripheral blood lymphocytes for the first time. A different apoptotic pathway induced in peripheral blood lymphocytes using this drug has, however, been previously revealed by other authors. The combination of cell specificity and dose-dependent effects can likely play a decisive role in apoptosis observed in peripheral blood lymphocytes after genotoxic drug application.

Bystin in human cancer cells: intracellular localization and function in ribosome biogenesis

Although bystin has been identified as a protein potentially involved in embryo implantation (a process unique to mammals) in humans, the bystin gene is evolutionarily conserved from yeast to humans. DNA microarray data indicates that bystin is overexpressed in human cancers, suggesting that it promotes cell growth. We undertook RT (reverse transcription)-PCR and immunoblotting, and confirmed that bystin mRNA and protein respectively are expressed in human cancer cell lines, including HeLa. Subcellular fractionation identified bystin protein as nuclear and cytoplasmic, and immunofluorescence showed that nuclear bystin localizes mainly in the nucleolus. Sucrose gradient ultracentrifugation of total cytoplasmic ribosomes revealed preferential association of bystin with the 40S subunit fractions. To analyse its function, bystin expression in cells was suppressed by RNAi (RNA interference). Pulse-chase analysis of ribosomal RNA processing suggested that bystin knockdown delays processing of 18S ribosomal RNA, a component of the 40S subunit. Furthermore, this knockdown significantly inhibited cell proliferation. Our findings suggest that bystin may promote cell proliferation by facilitating ribosome biogenesis, specifically in the production of the 40S subunit. Localization of bystin to the nucleolus, the site of ribosome biogenesis, was blocked by low concentrations of actinomycin D, a reagent that causes nucleolar stress. When bystin was transiently overexpressed in HeLa cells subjected to nucleolar stress, nuclear bystin was included in particles different from the nuclear stress granules induced by heat shock. In contrast, cytoplasmic bystin was barely affected by nucleolar stress. These results suggest that, while bystin may play multiple roles in mammalian cells, a conserved function is to facilitate ribosome biogenesis required for cell growth.

Ultrastructural changes of nucleoli in common wheat induced by actinomycin DPeople's Republic of China

Common wheat root tip meristematic cells were treated with low concentrations of actinomycin D (ActD), then stained whole by silver nitrate. We showed by transmission electron microscopy that the typical nucleolar structure did not form, but a granular and fibrillar network was exhibited in the nucleolar region. Our results support a correlation between nucleolar organization/assembly and the activation of RNA Polymerase I transcription. Furthermore, we speculate that the fibrillar network present in the nucleolar region of ActD treated cells may represent the basic skeletal structure required to support the nucleolus.

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2).

The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 microg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway.

The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.

Cold-stimulated increase in a regulatory subunit of cAMP-dependent protein kinase in human hepatoblastoma cells.

Although cold-stress responses in bacteria and plants have been well studied and hypothermic conditions are used in clinical treatments, there has been little investigation of cold-stress responses in human cells, and there has been no report on the involvement of signal transduction modulators in the cold-stress response in human cells. We therefore investigated alterations in the expression of genes involved in the signal transduction system and the mechanisms of cold-stimulated increases in the expression of genes in human hepatoblastoma (HepG2) cells. Using a cDNA expression array method, we found that a transcript encoding a regulatory subunit Ibeta (RIbeta) of cyclic AMP-dependent protein kinase (PKA) was increased in cold-stressed cells. Western blot analysis revealed that the amount of PKA RIbeta protein was increased by cold treatment, while that of a PKA catalytic subunit (C) was unchanged. The protein level of PKA RIbeta was increased in cells treated with low concentrations of actinomycin D, whereas that of PKA C was not, implying that the increase was caused by the suppression of transcription at low temperatures. In addition, degradation of the PKA RIbeta protein was not stimulated by cold treatment, unlike that of the PKA C protein. The results suggest that signal transduction through PKA also participates in cold-stress responses in human cells and that multiple mechanisms are involved in the increase in the level of the PKA RIbeta protein.

Colocalization within the Nucleolus of Two Highly Related IFN-Induced Human Nuclear Phosphoproteins with Nucleolin

We have previously reported the identification of two interferon (IFN)-induced cDNAs which code for two proteins, named 41 and 75, which have homology to a number of proteins involved in regulating gene expression. Here we establish that these cDNAs correspond to in vivo synthesized mRNAs. Expression of the 41 and 75 cDNAs, both in vitro and in vivo, generated proteins of 30 and 68 kDa, respectively. In a variety of mammalian cells, 41 and 75 were found to be located in the nucleus, with 41 being localized to the nucleolus, whereas 75, although it is mainly concentrated at the periphery of the nucleolus, is also found throughout the nucleoplasm. Treatment with interferon results in a translocation of 41 to the periphery of the nucleolus and it is in this region that the two proteins colocalize. 41 and 75 were found to colocalize with nucleolin but not with B23 or fibrillarin, three nucleolar proteins involved in ribosome synthesis. This colocalization was not affected by low concentrations of actinomycin D. In view of this and since 41 and 75 have homology to proteins involved in regulating gene expression, we suggest that, in association with nucleolin, they play a role in ribosome biogenesis.

P53-dependent ceramide response to genotoxic stress.

Both p53 and ceramide have been implicated in the regulation of growth suppression. p53 has been proposed as the "guardian of the genome" and ceramide has been suggested as a "tumor suppressor lipid. " Both molecules appear to regulate cell cycle arrest, senescence, and apoptosis. In this study, we investigated the relationship between p53 and ceramide. We found that treatment of Molt-4 cells with low concentrations of actinomycin D or gamma-irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels of p53. In these cells, p53 activation was followed by a dose- and time-dependent increase in endogenous ceramide levels which was not seen in cells lacking functional p53 and treated similarly. Similar results were seen in irradiated L929 cells whereby the p53-deficient clone was significantly more resistant to irradiation and exhibited no ceramide response. However, in p53-independent systems, such as growth suppression induced by TNF-alpha or serum deprivation, ceramide accumulated irrespective of the upregulation of p53, indicating that p53 regulates ceramide accumulation in only a subset of growth-suppressive pathways. Finally, ceramide did not increase p53 levels when used at growth-suppressive concentrations. Also, when cells lacking functional p53, either due to mutation or the expression of the E6 protein of human papilloma virus, were treated with exogenous ceramide, there was equal growth suppression, cell cycle arrest, and apoptosis as compared with cells expressing normal p53. These results indicate that p53 is unlikely to function "downstream" of ceramide. Instead, they suggest that, in situations where p53 performs a critical regulatory role, such as the response to genotoxic stress, it functions "upstream" of ceramide. These studies begin to define a relationship between these two pathways of growth inhibition.

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

Low Concentrations of Actinomycin D Potentially Cause Therapeutic Differentiation in Human Rhabdomyosarcoma Cell Line RD

Neoplastic transformation may be an alteration in the process of cell maturation that leads to an infinite capacity for proliferation. Because the cytodestruction caused by most drugs available for cancer chemotherapy is often accompanied by significant morbidity and poor response, the induction of differentiation has been proposed as an alternative approach to conventional anticancer therapy. We used human rhabdomyosarcoma cell line RD to analyze the differentiation process induced by actinomycin D, a drug of choice in the conventional treatment of rhabdomyosarcomas. Low concentrations of actinomycin D induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death. However, this potential therapeutic effect cannot be considered complete because of the presence of tumoral cells that are heterogeneous with respect to actinomycin D chemosensitivity. This heterogeneity led to the appearance of foci of resistant cells which, despite their greater degree of differentiation in comparison with the parental cell line, escaped from terminal myogenic differentiation. This subgroup of tumoral cells may be responsible for the failure of cytotoxic treatment.

Human autoantibodies against a nucleolar protein

Autoimmune sera showing prominent immunofluorescence in nucleolus were selected and analysed by immunoblotting techniques. Immunoblots using a nucleolar extract as antigen source revealed sera recognizing a 38 kDa nucleolar protein. Low concentration of Actinomycin D, which inhibits the ribosomal RNA synthesis, caused a loss of fluorescence. This suggests that the nucleolar antigen may be associated with the assembly of packaging of the ribosomes. The present nucleolar antigen has properties similar to the previously described nucleolar phosphoprotein B23 of rat cells and the recently described nucleolar protein NO38 of mouse and Xenopus cells.