Inhibition Of Nucleolar RNA Synthesis Research Articles

Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo

Previous studies suggested multiple sites of action of aflatoxin B1 (AFB1) in vivo to inhibit rat liver nuclear RNA synthesis--it impairs nucleolar DNA template function and inhibits RNA polymerase II activity. We have previously shown that AFB1 activated in vitro inhibits nucleolar RNA synthesis. The question is whether AFB1 can inhibit RNA polymerase II under these in vitro conditions. Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6 mg AFB1 and liver nuclei were isolated 2 h later. When the total nuclear free RNA polymerases were extracted and assayed in the absence and presence of alpha-amanitin (3.2 micrograms/ml), we found that only alpha-amanitin-sensitive (i.e., RNA polymerase II) activity was inhibited (97%). DEAE-Sephadex chromatography confirmed this result. When total nuclear free RNA polymerases were incubated with AFB1 activated in vitro under conditions producing 70% inhibition of nucleolar RNA synthesis, no inhibition was observed for either alpha-amanitin-sensitive or -resistant activities. Similar results were obtained with low and high (28 and 167 micrograms/ml) concentrations of AFB1. This was further confirmed using highly purified RNA polymerase II. We conclude that AFB1 inhibition of RNA polymerase II activity in vivo is not a result of direct interaction of AFB1 to the enzyme.

The effects of zinc chloride on the RNP structures in HEp-2 cells: Accumulation of perichromatin granules

The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

Further studies on satellite nucleoli in rat and mouse hepatocytes

To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immunostaining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.

Nuclear alterations induced by cadmium chloride and l-canavanine in HeLa S3 cells. Accumulation of perichromatin granules

The effects of L-canavanine and cadmium on the ribonucleoprotein constituents of HeLa S3 cells have been analyzed. Both chemicals induce a similar pattern of alterations in different RNP structures as well as in both RNA and protein synthesis. Pulse and chase autoradiographic experiments reveal that both canavanine and cadmium induce a preferential inhibition of nucleolar RNA synthesis and a slowdown in the transport or processing of nucleolar and extranucleolar RNA. Nucleoli become round and compact. Accumulation of perichromatin granules and fibrils occurs, there is a depletion of interchromatin fibrils, and nuclear formations appear which seem to be involved in the morphogenesis of perichromatin granules accumulated during the treatments. The appearance of clusters of 29- to 35-nm granules might be related with a deficient assembling of constituents of perichromatin granules. The effects of different inhibitors of the transcriptional processes on the accumulation of perichromatin granules suggest that these granules represent a particular subpopulation of hnRNP.

Functional significance of perichromatin granule accumulation induced by cadmium chloride in isolated rat liver cells

The addition of cadmium chloride (CdCl 2) to the culture medium of isolated rat liver cells induced rapid nuclear lesions. As observed by biochemical analysis and high resolution autoradiography after labeling the cells with [5- 3H]uridine, CdCl 2 induced a preferential inhibition of nucleolar RNA synthesis and a slowing down of the processing of both extranucleolar polyadenylated and nucleolar non-polyadenylated RNA molecules synthesized before the action of CdCl 2. Inhibition of rRNA synthesis was accompanied by a condensation of nucleoli. Disturbance of hnRNA and rRNA processing induced accumulation of large numbers of perichromatin granules contiguous to and within extranucleolar and nucleolar condensed chromatin, respectively, with concomitant loss of interchromatin fibrils. Autoradiographs after 15 min labeling followed by 3 h chase in the presence of CdCl 2 revealed that all of the incorporated uridine remained over the perichromatin fibrils and granules at the border of the condensed chromatin. These observations support our hypothesis that perichromatin granules represent storage forms of recently synthesized RNA, and that there may be at least two types (hnRNA and rRNA) of perichromatin granules.

Inhibitory Effects of Nucleoside Triphosphates on Nucleolar RNA Synthesis<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.

Transport of RNA from nucleus to cytoplasm following mitogenic stimulation of human lymphocytes.

An improved method affording clean separation of nuclei from cytoplasm was utilized to study the kinetics of appearance of [3H]uridine-labelled polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)−) RNA in the cytoplasm of human lymphocytes stimulated with the mitogen concanavalin A (Con A). While our previous and present studies utilizing lymphocyte preparations purified on Ficoll-Hypaque gradients had shown that the earliest observable increase in labelled total cell RNA occurred after 12 h of exposure to Con A, we were here able to detect highly significant increases in levels of uridine-labelled poly(A)− RNA in the cytoplasm of lymphocytes which had been exposed to mitogen for 6 h; these cells had been pulsed with [3H]uridine for 2 h prior to harvest. This suggested that the early effects of mitogen may be to increase processing and transport of RNA rather than to increase the production of gene transcripts. At least 13 h of exposure to Con A was required before labelled poly(A)+ RNA appeared in the cytoplasmic extracts of stimulated cells and it did not appear in those of resting cells. The lag between addition of isotope and appearance of labelled RNA in cytoplasm was inversely related to the duration of incubation of the cells with mitogen. However, once the label appeared in the cytoplasm, its rate of appearance and its final level were similar at both 12 and 36 h of incubation with Con A. These kinetic data also suggest that mitogen acts mainly to accelerate the processing and transport of RNA at least during the first 12 h of mitogenic stimulation. A concentration (0.05 μg/ml) of actinomycin D which is known to inhibit specifically the transcription of nucleolar RNA in a variety of cell types was found to affect the transcription of poly(A)+ RNA in human lymphocytes; in some experiments, specific inhibition of nucleolar RNA synthesis was observed at 0.005 μg actinomycin D per millilitre.

Diacridines, bifunctional intercalators—IV.: Effect on nucleolar RNA synthesis

The inhibition of nucleolar RNA synthesis in HeLa cells by acridines as well as by a series of 9-NH 2 diacridines, connected through their 9-NH 2 groups by hydrocarbon chains from (CH 2) 2 to (CH 2) 12, and by the polyamine chains spermidine and spermine, has been investigated. Nucleolar RNA synthesis in HeLa cells is most sensitive to the longer chain diacridines, indicating that double intercalation may be essential for their action. These data are in agreement with the action in vitro of these compounds. However, if the degree of inhibition of nucleolar rRNA synthesis is related to the nuclear uptake, the single intercalators appear to be more effective inhibitors.

Studies on nucleoli of maturing human erythroblasts

Human erythroblasts which are prereticulocyte maturation stages of red blood cells were studied by light microscopic cytochemistry and electron microscopy to provide more information on the ultrastructure of the micronucleoli which are terminal stages of nucleolar changes found during maturation of these cells. As indicated by light microscopy of smeared cells, micronucleoli were virtually the only types of nucleoli present in the last stages of maturing erythroblasts, i.e., polychromatic and orthochromatic (late polychromatic) erythroblasts. Accordingly, they were not portions of the periphery of other nucleoli. Inasmuch as most of the micronucleoli exhibited characteristic segregation of nucleolar fibrillar and granular components they presumably are producing little if any preribosomal RNA, since such segregation generally reflects inhibition of nucleolar RNA synthesis.

Repopulation of postmitotic nucleoli by preformed RNA. II. Ultrastructure.

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.

Effects of anthramycin and actinomycin on RNA synthesis patterns in L1210 cells.

AbstractAnthramycin and actinomycin D, two different types of DNA‐binding antibiotics, were compared for their effects on RNA synthesis in suspension cultures of mouse leukemia L1210 cells. RNA was labeled with radioactive uridine and selective effects on the synthesis of different classes of RNA were studied by sucrose gradient sedimentation of RNA purified from whole cells or from nucleolar and nucleoplasmic fractions.Two major differences were noted. (1) Whereas actinomycin produced two phases in the inhibition of uridine incorporation, the rapid phase being complete within a few minutes, anthramycin produced only a slow progressive inhibition. (2) Whereas actinomycin selectively inhibits nucleolar 45s RNA synthesis, anthramycin inhibits this RNA equally to the inhibition of the same size RNA in the nucleoplasm.Both antibiotics caused a shift towards lower molecular weight (slower sedimentation) in the distribution of nucleoplasmic RNA molecules synthesized in the presence of drug. When the two antibiotics were compared at concentrations producing equal extents of inhibition of nucleoplasmic RNA synthesis, anthryamycin produced the greater shift. The shift in sedimentation anthramycin produced the greater shift. The shift in sedimentation was not due to a slowing of RNA chain growth rate, since the change in sedimentation persisted when uridine incorporation time was increased so as to compensate for the reduction in RNA synthesis rate.The selective inhibition of nucleolar RNA synthesis by actinomycin could be due to possible differences in the properties of the different RNA polymerases, or to differences in the initiation rates for transcription. The absence of selectivity in the case of anthramycin might be related to the near irreversibility of its binding to DNA. The shift towards lower molecular weight of the RNA synthesized is compatible with (but does not constitute strong evidence for) premature termination of RNA chains.

The reversibility of inhibition of RNA and DNA synthesis induced by aflatoxin in rat liver. A tentative explanation for carcinogenic mechanism.

AbstractAflatoxin is a potent hepatocarcinogen. Biochemically the short‐term action which seems to be the most important is an inhibition of nucleic acid synthesis. Injection of low doses (from 500 to 1,000 μg/kg) made it possible to modulate the different inhibitions induced by aflatoxin.The following events are successively observed: first, inhibition of nucleolar RNA synthesis, then inhibition of replication and, finally, inhibition of total nuclear transcription. With such aflatoxin doses these phenomena are reversible, and the different syntheses recommence in the following order: after 24 h the total nuclear RNA synthesis; next, nucleolar RNA synthesis and lastly, after 48 h, DNA synthesis.Therefore there is a period of at least 24 h during which RNA is synthesized and DNA is not.These results may be compared to those obtained by other authors working in vitro and showing aflatoxin‐DNA binding. They support a hypothesis concerning the carcinogenesis mechanism proceeding by deletion at the genetic level.