D-ribofuranosylbenzimidazole Research Articles

Association between the nucleolus and the coiled body

By means of light and electron microscopic immunocytochemistry, we have localized p80-coilin, a specific protein marker for coiled bodies, in mammalian cell lines as well as in primary rat neuron cultures. p80-coilin-stained nuclear bodies, which also contained fibrillarin, could be subsequently silver stained by a method specific for the visualization of nucleolar organizer regions. In cycling cells, most coiled bodies were not associated with nucleoli, whereas in rat neurons such an association was frequent. The treatment of cycling cells with actinomycin D or 5,6-dichloro-1-β- d-ribo furanosyl-benzimidazole led to nucleolar segregation and/or disintegration, and to an association of p80-coilin staining structures with nucleoli. p80-coilin-positive structures contained fibrillarin in both untreated and treated cells. These results support the opinion that there might be a special association between coiled bodies and nucleoli, particularly in neuronal cells.

Alternative use of a polyadenylation signal and of a downstream 3′ splice site: Effect of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole

A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site P1 instead of the major promoter-distal sites P2 and P4. 5,6-Dichlorol-β- d-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of P1. The nascent transcript is first cleaved in the branch point-3′ splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3′ end (F-RNA) and a promoter-distal RNA with a heterogeneous 5′ end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3′ end of F-RNA is about 130 nucleotides downstream from P1 and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CP1, which is the transcript ending at P1. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3′ end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.

Effect of inhibitors of the host cell RNA polymerase II on African swine fever virus multiplication

The role of the host cell RNA polymerase II in African swine fever (ASF) virus growth has been examined using inhibitors of this enzyme. The adenosine analog 5,6-dichloro-l-β- d-ribofuranosylbenzimidazole (DRB), an inhibitor of mRNA precursor synthesis in mammalian cells, strongly inhibits the production of infectious progeny virus in Vero cells, but does not significantly affect the synthesis of virus-specific macromolecules. On the other hand, virion assembly seems to proceed normally in the presence of DRB, as virus particles can be seen in electron micrographs with a morphology indistinguishable from that observed in the absence of the inhibitor. However, taking into account the inhibition of the infectivity caused by the drug, most of these particles must be defective. In contrast with this effect of DRB on ASF virus replication, the toxin a-amanitin does not inhibit the production of infectious ASF virus in Vero cells or porcine alveolar macrophages. This result indicates that the host RNA polymerase II does not transcribe viral genes and that active transcription of the cell genome is not needed for ASF virus replication.

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

Certain autoimmune sera contain antibodies against a nucleolar ribonucleoprotein particle associated with 7-2-RNA ( R. Reddy et al. (1983 ) J. Biol. Chem. 258, 1383; C. Hashimoto and J. A. Steitz (1983) J. Biol. Chem. 258, 1379). In this study, we showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB)—segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of M r 40,000 from [ 35S] methionine—labeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes.

Interferon inhibits preferentially the synthesis of proteins associated with growth stimulation of Swiss 3T3 mouse fibroblasts

The putative role of inhibition of protein synthesis within the antiproliferative effect of interferon was analyzed in serum-stimulated Swiss 3T3 mouse fibroblasts. We observed an apparent coupling between protein-synthesis inhibition during G 1 and a delayed entry into the S-phase. To reveal any specificity in the protein-synthesis inhibition, we measured the amounts of synthesis of 56 major individual proteins, by using isotope double-labelling and two-dimensional gel electrophoresis. Interferon inhibited preferentially the synthesis of proteins which were increased after serum stimulation, whereas proteins synthesized in constant or decreased amounts after serum stimulation were significantly more resistant. The effects of interferon were also compared to those of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), an inhibitor of transcription. All interferon-sensitive proteins studied were inhibited by DRB treatment, but in addition DRB also inhibited several proteins which were completely resistant to interferon. We conclude that interferon primarily inhibits protein synthesis originating from a subset of newly transcribed messenger RNAs. The mechanism(s) for inhibition of protein synthesis and the possible relationship to the antiproliferative and antiviral effects of interferon are discussed.

Serum-induced G0/G1 transition in chemically transformed 3T3 cells: Independence of protein synthesis, stable “memory”, and enhancement by cycloheximide pretreatment

Quiescent, chemically transformed (benzo-α-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (<4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable ‘memory’ that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this ‘memory’ probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8–24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

Inhibition of transcription does not affect the total amount of ubiquitinated histone 2A in chromatin

Using a polyclonal anti-ubiquitin antibody in Western blotting experiments, we detected three antibody-binding components in a HeLa cell extract: ubiquitin, a ubiquitin-histone 2A conjugate (uH2A) and a 17 kD protein, probably corresponding to an additional ubiquitin conjugate. Since ubiquitination of histone 2A (H2A) has been invoked in the transcription process, the amount of uH2A was studied after inhibition of ribosomal RNA (rRNA) synthesis with actinomycin D and of heterogeneous nuclear RNA (hnRNA) synthesis with 5,6-dichloro-l-β- d-ribofuranosylbenzimidazole (DRB). The amount of uH2A did not change, suggesting that the overall level of ubiquitination of histone 2A is not directly coupled to on-going transcription of either rRNA or hnRNA. Since the uH2A content of protein coding genes constitutes a considerable portion of total chromatin uH2A, it seems also likely that there is no major change in the degree of ubiquitination on the templates of the protein-coding genes themselves upon cessation of transcription. It is proposed that the pattern of ubiquitination of histone 2A is established on a long-term basis and that it is related to the overall organization and distribution of the chromatin material in the interphase nucleus.

High resolution autoradiographical detection of RNA in the interchromatin granules of DRB-treated cells

Isolated rat liver cells were pulse-labelled with tritiated uridine and post-incubated in the presence of an excess of unlabelled uridine and of adenosine analog DRB (5-6-dichloro-1-β- d-ribofuranosyl benzimidazole). Nuclear radioactivity was detected with high resolution autoradiography. A significant labelling of the interchromatin granules was revealed in these conditions. Pretreatments of cells with low doses of actinomycin D in order to preferentially inhibit ribosomal RNA (rRNA) synthesis prevented the labelling of the interchromatin granules during subsequent DRB treatments. These observations indicate that in DRB-treated cells, the interchromatin granules are sites of transfer or of accumulation of nucleolar RNA. Our results are discussed in connection with our knowledge of the action of DRB on RNA metabolism in mammalian cells and with recent data concerning the still enigmatic interchromatin granules which are present in the nuclei of most cells.

HeLa cell RNA and protein syntheses: Effects of long-term treatment with 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB)

The population growth rate of HeLa cells treated with 60 μM 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) in reinforced Eagle's minimum essential medium was directly related to the concentration of fetal bovine serum in the range from 5 to 30%. Control cells proliferated at a maximal rate (doubling time: 18–19 hr) at serum concentrations in this range. In 60 μM DRB-containing medium supplemented with 5% serum, the inhibition of hnRNA synthesis after 1,5, and 48 hr of treatment was equivalent to 78, 84, and 51%, respectively, whereas that of protein synthesis was equivalent to 5, 41, and 49%. In 15% serum, the inhibition of hnRNA synthesis after 1, 5, and 48 hr of treatment was equivalent to 75, 79, and 40%, whereas that of protein synthesis was equivalent to 13, 30, and 21%. Thus, after 48 hr of treatment of cells with DRB in either 5 or 15% serum, hnRNA synthesis was less inhibited than it was after 1 or 5 hr of treatment, and the level of protein synthesis in DRB-treated cells did not decrease between the 5th and 48th hr. After 48 hr of treatment of cells with DRB in 15% serum, hnRNA and protein syntheses were both somewhat less inhibited than after treatment in 5% serum. These results suggest that, on prolonged treatment, HeLa cells undergo a partial, serum-dependent adaptation to the inhibitor.

Inhibition of HeLa-S3 cell proliferation and biosynthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.

The effect of transcription inhibitors on early development of the avian embryo

The effect of transcription [actinomycin D, 5,6- dichloro-1-β- d - ribofuranosylbenzimidazole (DRB), α-amanitin] and translation inhibitors (cycloheximide, puromycin) on quail embryo development was investigated under in vitro conditions. The gastrulation process seemed to proceed normally in the presence of transcription inhibitors in the medium but the translation inhibitors stopped development and caused complete degeneration of the embryos.

Inhibition of messenger RNA synthesis by 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole without its detectable accumulation in mouse T-lymphoma cells

Two lines of evidence were obtained which indicate that inhibition of mRNA formation does not require the detectable accumulation of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), a halogenated analog of adenosine. First, the extent of inhibition by DRB of the formation of cytoplasmic poly(A) + RNA was as rapid and severe (>90% inhibition) in wild-type mouse lymphoma cells (S49) as in mutant cells (AE 1), derived from S49, which were deficient in the transport of purine and pyrimidine nucleosides. Second, the accumulation of [ 3H]DRB was measured directly and compared to the accumulation of [ 3H]adenosine. Whereas S49 cells accumulated [ 3H]adenosine in a linear manner, neither S49 nor AE 1 cells accumulated [ 3H]DRB to a significant extent. This suggests that inhibition of mRNA synthesis by DRB may (1) require the transport and intracellular accumulation of only minute amounts of DRB, or (2) result from secondary event(s) triggered by interaction of DRB with a surface membrane component.

Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole. Requirement of a free 3′-hydroxyl group, but not 2′- or 5′-hydroxyls

Five structural analogues of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), all with modified sugar moieties, have been examined for their inhibitory activities on RNA transcription in salivary glands of Chironomus tentans. The well-known ability of the parent DRB at 65 μ M concentration to selectively inhibit hnRNA/mRNA synthesis by approx. 90% was essentially abolished on methylation of the 3′-OH; but, at an overdose the analogue suppressed labeling of all RNA classes examined (hnRNA/mRNA, rRNA, 4–5 S RNA) by 70–80%. By contrast, the 2′- O-methyl derivative of DRB was almost as effective as DRB itself in blocking transcription of hnRNA/mRNA genes. Blocking of both the 2′ and 3′ hydroxyls (2′,3′- O-isopropylidene-DRB) completely abolished inhibitory activity, irrespective of the concentration employed. The 5′-deoxy-5′-chloro derivative of DRB was only slightly less effective than the parent DRB. An unusual aspect of the activities of 2′- O-methyl-DRB and 5′-deoxy-5′-chloro-DRB was their ability to stimulate synthesis of 4–5 S RNA by 25–45%. Also investigated was the influence of the various analogues on the rate of formation of [ 3H]UTP from [ 3H]uridine used as an RNA precursor. The rate of such formation of [ 3H]UTP was suppressed 2–6-fold by treatment with 2′- O-methyl or 3′- O-methyl-DRB, but was unaffected by 5′-deoxy-5′-chloro-DRB or 5,6-dichloro-1-α- d-arabinofuranosylbenzimidazole. The overall data point to the importance of a free 3′-OH in the ribose moiety of DRB for selective inhibitory activity. The α- d-arabinofuranosyl analogue, although less selective in inhibition of RNA transcription, still exhibits about 50% of the activity of DRB.

Genetic markers in mouse teratocarcinoma cells. Selection and partial characterization of mutants resistant to toyocamycin, DRB and podophyllotoxin

Stable mutants resistant to a number of cytotoxic drugs, ( i) adenosine analog toyocamycin (Toy r); ( ii) microtubule inhibitor podophyllotoxin (Pod R); and ( iii) nucleoside analog 5,6-dichloro-1-β- d-ribofuranosyl benzimidazole (Drb R), have been isolated in the mouse teratocarcinoma cell line OC15S1. Biochemical studies reveal that the genetic lesion in Toy r mutants causes a nearly total deficiency of the enzyme adenosine kinase, which phosphorylate adenosine and its analogs and plays an important role in purine nucleotides metabolism. The lesions in Pod R and Drb R classes of mutants have not yet been fully characterized. The availability of these new genetic markers in mouse teratocarcinoma cells should make it possible to examine the role of the affected functions in vivo in an intact organism.

Effect of 5,6-dichloro-1-β- d-ribofuranosyl benzimidazole on ribonucleotide metabolism and accumulation of mitochondrial RNA and low-molecular-weight cytoplasmic RNA in HeLa cells

These results provide additional information on the selective inhibition of RNA synthesis by 5,6-dichloro-1-β- d-ribofuranosyl benzimidazole (DRB). DRB only slightly inhibited the poly(A +) RNA and ribosomal RNA in the mitochondria (maximal inhibition was ~25%) but severely inhibited the poly(A +) RNA in the postmitochondrial supernatant (~95%) and the poly(A +) RNA associated with the cytoplasmic membranes (~80%). Separation of the cytoplasmic low-molecular-weight RNAs showed that DRB inhibited the 5.8 S rRNA, a product of RNA polymerase I, by ~95% while there was only a slight inhibition of the 4 S RNAs (~20%) and 5 S RNA (<5%), products of RNA polymerase III. DRB severely inhibited the appearance in the cytoplasm of 28 S rRNA (~95%) and 18 S rRNA (~80%). These results, along with other recent reports (31–34), may suggest that DRB most severely inhibits RNAs that are extensively processed and/or transcribed from genes that contain extensive intervening sequences. These experiments also indicate that the mechanism of DRB inhibition does not involve alterations in ribonucleotide metabolism. DRB did not affect the phosphorylation of any ribonucleotides to triphosphates or the cellular conversion of [ 3H]uridine to UTP. Also, the size of the UTP and ATP pools in DRB-treated cells was equal to or greater than those in control cells through a period of 240 min. Significant amounts of DRB triphosphate could not be detected in DRB-treated cells suggesting that this may not be the inhibitory form of DRB. Measurements of the specific activity of the UTP pool allowed direct measurements of the accumulation of picomoles of the individual RNAs in the presence of DRB.

Kinetics of decay in the expression of interferon-dependent mRNAs responsible for resistance to virus.

We used 5,6-dichloro-beta D-ribofuranosyl-benzimidazole (DRB), a selective and reversible inhibitor of mRNA production, to investigate the regulation of the pathway leading to resistance to viruses in cells treated with interferon (IF). DRB allows initiation of transcription but promotes premature termination of the nucleotide chains, so that it abolishes interferon-dependent protection against viruses. When the DRB is removed, synthesis of complete mRNAs can resume. Mouse L-929 cells were exposed to 100 microM DRB before and during a 1-hr pulse of IF followed by treatment with antibody to IF to prevent cell-to-cell spread of IF after that time. At different intervals thereafter the cells were washed and the DRB was replaced by medium; after further incubation, the cells were infected with vesicular stomatitis virus. Resistance to virus was inversely proportional to the duration of the block imposed by DRB. When the DRB was removed soon after the IF pulse, substantial protection from virus ensued, but none developed when removal of the DRB was deferred for 5-6 hr. Cells exposed to DRB for 5 hr, then pulsed with IF for 1 hr, still mounted a strong antiviral response. The data show that the ability of cells to resist viral infection decays within 5-6 hr after treatment with IF. Whether the decay is due to shutoff of transcription of mRNAs, or to their destruction or degradation, or whether regulation takes place at one or more subsequent steps in the antiviral pathway, remains to be determined.

Two mechanisms contribute to the superinduction of poly(I)·poly(C)-induced human fibroblast interferon production

The cumulative interferon yield from cultures of poly(I)·poly(C)-induced human diploid fibroblasts (FS-4) in the first 24 hr after induction is increased (superinduced) approximately 50-fold by appropriate treatment with 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) and cycloheximide. Two components have been defined in the biochemical mechanism of this paradoxical enhancement. The content of polyadenylated interferon mRNA in induced and superinduced FS-4 cells was assayed by translation in oocytes in Xenopus laevis. First, as reported earlier (P. B. Sehgal, D. S. Lyles, and I. Tamm, Virology 89, 186–198, 1978), interferon mRNA is approximately 14-fold more stable in the continuous presence of DRB (40 μ M) than in inhibitor-free controls. Second, estimates of the content of interferon mRNA during the first 3 hr of induction in control cultures and in those exposed to DRB alone or cycloheximide alone are consistent with a 3- to 4-fold higher apparent rate of synthesis of interferon mRNA in treated cultures than would be expected if cycloheximide had no effect on transcription and DRB (40 AM) inhibited transcription ∼80% (P. B. Sehgal, I. Tamm, and J. Vilbek, Virology, 70, 256–259; 542–544, 1976).

Inhibition of adenovirus replication by 5,6-Dichloro-1-,β- d-ribofuranosylbenzimidazole

The halogenated benzimidazole derivative 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) inhibits reversibly the replication of human adenovirus type 2 (Ad2) and its DNA in human KB cells. Viral DNA replication is almost completely blocked when the drug is added earlier than 4 hr postinfection in concentrations between 50 and 150 μM. Replication of viral DNA in all four size-classes (>100 S, 50–90 S, 34 S, and <20 S) is inhibited. The replication block is reversible upon withdrawal of the inhibitor. When DRB is administered at the time of maximal viral DNA replication, 16–18 hr postinfection, the inhibitor has no apparent effect on viral DNA synthesis. In the presence of 150 μM DRB, synthesis of early virus-specific RNA in the nucleus is reduced by approximately 90% and the appearance of virus-specific RNA sequences in the cytoplasm is reduced by >95%, as demonstrated by DNA-RNA filter hybridization. Thus, the block in viral DNA replication is best explained by the inhibition of the synthesis of early virus-specific RNA.

Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- d-ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [ 3H]thymidine-substituted DNA and of autoradiograms of extended fibers of [ 3H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.

The inhibition of DRB (5,6-dichloro-1-β- d-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells

5, 6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB; 75 μM) inhibited only 60–75% of nuclear heterogeneous RNA (hnRNA) synthesis in HeLa cells, while the appearance of labeled cytoplasmic poly(A)-containing messenger RNA (mRNA) was reduced by approximately 95%. DRB-sensitive and resistant transcripts spanned the entire size range of hnRNA molecules. The relative poly(A) content of DRB-resistant hnRNA molecules was the same as that of normal hnRNA. In contrast to the effects of 3′deoxyadenosine, DRB appeared to inhibit the initiation of hnRNA chains, but did not directly interfere with labeling of poly(A). We present an interpretation of these results and propose how the results might be used to gain more information about nuclear RNA metabolism.