aphidicolin and Glioma

When C6 cells in culture were exposed at 37 degrees C to 1 microM colchicine or to 1 microM colcemid, a tubulin-binding antimitotic alkaloid, levels of alphaB crystallin in cells began to increase after about 10 h, reaching a maximum of more than 1 microg/mg protein after 24 h. The level of alphaB crystallin returned to near the control level within two subsequent days of culture in the normal medium. Northern blot analysis showed that the accumulation of alphaB crystallin was preceded by an increase in the level of the mRNA for alphaB crystallin. Nuclear run-off transcription assays showed that colchicine induced new synthesis of mRNA for alphaB crystallin. Immunofluorescence staining revealed that alphaB crystallin accumulated in the peripheral areas of cells, as did the depolymerized tubulin, after several hours of treatment with colcemid, and then it gradually became more conspicuous in the cytoplasm. Vinblastine and nocodazole, which also promote the disassembly of microtubules by binding to tubulins, also induced the synthesis of alphaB crystallin. Furthermore, induction of alphaB crystallin by these drugs was observed in quiescent cells that had been cultured in serum-free medium. However, taxol, a microtubule-stabilizing antimitotic agent, did not stimulate the synthesis of alphaB crystallin, but rather, it suppressed the induction of synthesis of alphaB crystallin by the microtubule-disrupting drugs. Induction of alphaB crystallin by colchicine or by other drugs that promote the disassembly of microtubules was sensitive to staurosporine, an inhibitor of protein kinases, and the induction was completely suppressed in the presence of 10 nM staurosporine. These results suggest that the expression of alphaB crystallin is stimulated, via phosphorylation reactions that are sensitive to staurosporine, when the depolymerization of microtubules is enhanced.

Topics: Animals; Aphidicolin; Colchicine; Crystallins; Culture Media, Serum-Free; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Glioma; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Microtubules; Mitosis; Paclitaxel; Protein Kinase Inhibitors; Rats; Staurosporine; Tubulin; Tumor Cells, Cultured

Iron (Fe) is known to be necessary for cellular proliferation. Previous studies have suggested that neuroblastoma cells appear to be relatively sensitive to growth inhibition by a specific Fe chelator, deferrioxamine (DFO), in vitro. Also, DFO has been recently used for the treatment of neuroblastoma patients. In this paper we demonstrate that neuroblastoma cell proliferation in vitro is extremely sensitive to inhibition by DFO as compared to another cell line with almost identical growth kinetics. Neuroblastoma cells treated with DFO adapt appropriately to Fe chelation as measured by marked upregulation of transferrin receptor mRNA, increased functional transferrin receptor, and decreased cellular ferritin concentration. Further studies that quantitated cellular incorporation of 59Fe from added transferrin-59Fe in the presence of DFO indicated that neuroblastoma cells were more sensitive to inhibition of Fe incorporation by the chelator as compared to the other cell line. Neuroblastoma cells treated with DFO showed a consistent arrest in the G1 phase of the cell cycle. For cells taken from the "resting" state this block occurred before the vast majority of cells had entered S or G2-M phases of the cell cycle. Further evidence that neuroblastoma cells were arrested before the G1-S interface was provided when cells inhibited by DFO and released into aphidicolin exhibit arrest at the G1-S interface, whereas release from aphidicolin into DFO resulted in entry into S phase. Also, DFO-treated cells exhibited a decrease in both p34cdc2 immunoreactive protein as well as kinase activity. The results of these latter studies strongly indicate evidence for a Fe requirement for malignant cell proliferation before the onset of DNA synthesis. Our results also provide a basis for further studies that will better define a therapeutic approach to patients with neuroblastoma utilizing DFO treatment.

Topics: Aphidicolin; Cell Count; Cell Division; Deferoxamine; Drug Screening Assays, Antitumor; Ferritins; G1 Phase; Glioma; Humans; Iron; Neuroblastoma; Receptors, Transferrin; S Phase; Transferrin; Tumor Cells, Cultured

The purpose of the present study was to examine the distribution pattern of electron-dense acridine orange (AO) chromatin interaction products in rat glioma C6 cells at different phases of the cell cycle. For synchronization in the early S-phase the cells in logarithmic growth were treated with 3 micrograms/ml aphidicolin, a specific inhibitor of DNA polymerase alpha and then cultured in normal medium. For synchronization in the M-phase the cells cultured with aphidicolin and then returned to normal medium were treated with 0.05 micrograms/ml colcemid. Histoautoradiographic analysis of the C6 cells using the pulse chase method demonstrated approximately 16 h of cell cycle time and about 6.5 h of S-phase. Ultracytochemically, AO chromatin interaction products were found in all phases of the cell cycle except for the mitotic phase, namely in G1, S, and G2. The highest percentage of AO chromatin interaction products was observed in the early S-phase and the lowest in the G2 phase. The mean number of AO chromatin interaction products per nuclear area increased in the course of S-phase parallel with an increase of 3H-uridine uptake during the S-phase. The results show a characteristic distribution pattern of AO label specific for each of the four stages of the cell cycle, however, the significance of the coincident RNA synthetic activity remains to be elucidated.

Topics: Acridine Orange; Animals; Aphidicolin; Autoradiography; Cell Cycle; Chromatin; Demecolcine; Diterpenes; DNA, Neoplasm; Glioma; Microscopy, Electron; Rats; Staining and Labeling; Tumor Cells, Cultured