amanitins and Fibrosarcoma

The transition of a stationary cell to a motile cell is associated with the breakdown of the cytoskeleton and expression of enzymes responsible for the degradation of the extracellular matrix. Reverse transcription PCR was used to address the question of whether disruption of the microfilament and microtubule systems is a signal for the increased expression of the urokinase plasminogen activator receptor (uPAR), a key molecule involved in extracellular matrix degradation. Disruption of the cytoskeleton of human fibroblasts with cytochalasin B (1 x 10(-5) M or 1 x 10(-7) M), colchicine (1 x 10(-5) M or 5 x 10(-7) M), or nocodazole (1 x 10(-6) M) resulted in major increases in the levels of uPAR mRNA with smaller increases in uPAR protein levels. Both cycloheximide and alpha-amanitin inhibited the increase in uPAR mRNA levels, demonstrating the requirement for the de novo synthesis of a short-lived protein for transcriptional activation. In contrast to normal fibroblasts, treatment of the metastatic cell line HT1080 with either cytochalasin B or colchicine produced little change in the high levels of uPAR mRNA expressed in these cells. These data suggest that cytoskeletal disruption may contribute to cell migration by signaling an increase in uPAR synthesis.

Topics: Amanitins; Base Sequence; Cell Size; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin B; Cytoskeleton; Fibroblasts; Fibrosarcoma; Gene Expression Regulation; Humans; Molecular Sequence Data; Nocodazole; Polymerase Chain Reaction; Protein Synthesis Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Transcriptional Activation; Tumor Cells, Cultured

A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to alpha-amanitin at concentrations up to 10 micrograms/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled alpha-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50- to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to alpha-amanitin is due to an alteration of RNA polymerase II.

Topics: Amanitins; Cell Line; DNA-Directed RNA Polymerases; Fibrosarcoma; Humans; Mutation; RNA Polymerase II

Nuclear RNA polymerase activity was studied in homotransplanted rat glial tumors where the primary tumor was produced by transplacental injection of ethylnitrosourea. Alpha amanitin, cycloheximide, and rifampicin were tested as inhibitors of this activity. Alpha amanitin significantly inhibited RNA polymerase activity in all tumors. This indicated that the major nuclear RNA polymerase activity seen in vitro in the tumor nuclei was RNA polymerase II. This is similar to the activity seen in normal glial nuclei. Cycloheximide and rifampicin which have no effect on RNA polymerase activity in normal glial nuclei inhibited about 20% of the polymerase activity in three of the tumors. The size and multiplicity of the nucleoli in these tumor cells suggests that RNA polymerase I could account for the activity which is inhibited by cycloheximide.

Topics: Amanitins; Animals; Autoradiography; Brain Neoplasms; Cell Nucleolus; Cell Nucleus; Cycloheximide; DNA-Directed RNA Polymerases; DNA, Neoplasm; Endoplasmic Reticulum; Ethylnitrosourea; Fibrosarcoma; Glioma; Guanosine Triphosphate; Neoplasm Transplantation; Neoplasms, Experimental; Neurofibroma; Rats; Rifampin; Transplantation, Homologous