amanitins and Carcinoma--Ehrlich-Tumor

When protein biosynthesis is inhibited by either cycloheximide of puromycine, the nucleolar RNA synthesis of Ehrlich ascites tumor cells decreases by approximately 70% within 1 h, while the removal of these protein synthesis inhibitors causes a rapid recovery of nucleolar RNA synthesis, largely within 1 h. A similar pattern of decrease and recovery of endogenous RNA polymerase activity in isolated nucleoli or in nuclei (in the presence of alpha-amanitin) may be demonstrated after addition and removal of these drugs. Analysis of the molecular species of RNA polymerase I on a phosphocellulose column indicates that only the IB form of the enzyme decreases in the nucleoli of drug-treated cells and recovers quickly after resumption of protein synthesis. The finding that the activity of the IB form enzyme remains unchanged in the whole nuclei indicates that during cessation of protein synthesis RNA polymerase IB is either released from the nucleoli into the extranucleolar compartment or becomes so loosely bound to the nucleoli that it is leached out from the nucleoli during their isolation. By using a system of assaying free, nucleolar-template bound and total RNA polymerase I activities, data supporting the above interpretation have been obtained. Namely, in isolated nuclei free enzyme activity increases with a concomitant decrease in bound enzyme activity during protein synthesis inhibition, while the total enzyme activity remains unchanged. In isolated nucleoli, both total and bound enzyme activities decreases on protein synthesis inhibition but recover quickly on its resumption. The putative bound enzyme, fractionated with the aid of actinomycin D, is exclusively IB form, whereas the unbound enzyme consists of both IA and IB forms as previously demonstrated (1). No conversion of IB form polymerase to IA form was noted on prolonged sonication in our system. The levels of ATP and GTP in the cell did not change appreciably either during cessation or resumption of protein synthesis in these cells. The data support the previous conclusion that some short-lived protein(s) is required to maintain the normal level of ribosomal RNA transcription (2) and further suggest that the protein is required to facilitate reinitiation of the transcription by RNA polymerase IB in the nucleolus.

Topics: Adenosine Triphosphate; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleolus; Cell Nucleus; Cycloheximide; Dactinomycin; Guanosine Triphosphate; Liver; Mice; Protein Biosynthesis; Puromycin; Rats; RNA; RNA Polymerase I

The effect of the denaturation of homologous and calf thymus DNA on the RNA polymerase B activity purified from rat liver and spleen and Ehrlich ascites cells, was investigated in presence of either Mn2+ or Mg2+ and in presence or absence of alpha-amanitin. On the basis of the results here reported, we suggest: 1) denatured DNA is more effective than native as template for polymerase B; 2) denatured DNA template and cations might play a role in determining the extent of the reaction alpha-amanitin-polymerase B.

Topics: Amanitins; Animals; Carcinoma, Ehrlich Tumor; DNA; DNA-Directed RNA Polymerases; Kidney; Liver; Magnesium; Manganese; Nucleic Acid Denaturation; Rats; RNA Polymerase II

Topics: Aldehydes; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Cells, Cultured; Dactinomycin; DNA-Directed RNA Polymerases; Inosine; Poly dA-dT; RNA, Neoplasm

Topics: Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Line; DNA-Directed RNA Polymerases; Enzyme Activation; Neoplasm Proteins; RNA Polymerase II

The molecular size and poly-A content of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells were measured. KCl was found to be essential for synthesis of high molecular weight RNA: when 0.4 M KCl was added to the reaction mixture, the average molecular size of the RNA formed was 14S; without KCl the average molecular size was 5S. A significant amount of poly-A sequences was found in RNA synthesized in the presence of alpha-amanitin, suggesting that RNA polymerase I and/or III may synthesized some RNA containing poly-A in isolated nuclei.

Topics: Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Cell-Free System; DNA-Directed RNA Polymerases; Male; Mice; Molecular Weight; Poly A; Potassium Chloride; RNA; RNA Polymerase I; RNA Polymerase III; RNA, Ribosomal

Topics: Agaricales; Amanita; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Mice; Oligopeptides; Phalloidine; Plant Extracts; Rats; Sarcoma, Yoshida; Water

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.

Topics: Adenocarcinoma; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Chromatography, Ion Exchange; Daunorubicin; DNA-Directed RNA Polymerases; DNA, Neoplasm; Kidney; Liver; Lymphoma, Non-Hodgkin; Magnesium; Male; Manganese; Mice; Molecular Weight; Neoplasms, Experimental; RNA, Ribosomal; Species Specificity; Spleen; Temperature; Templates, Genetic