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Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis

Institut de Biologie Structurale J-P Ebel (CEA-CNRS), 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France

View larger version (21K): [in a new window]   Fig. 1. Random cycling REF-52 cells do not recover from hydroxyurea or aphidicolin block after 30 hours of exposure. Random cycling REF-52 cells were treated with aphidicolin (10 µM) or hydroxyurea (2 mM) for the indicated times, then released for 24 hours into nocodazole (0.25 µg/ml), and finally subjected to FACscan analysis. Failure of cells exposed to drug for 30 hours or more to replicate upon release is reflected by the absence of cells arrested in 4N by nocodazole.

View larger version (18K): [in a new window]   Fig. 2. Failure to replicate following prolonged exposure to hydroxyurea or aphidicolin is not dependent on p53 or pRB function. T-antigen-transformed REF-52 cells (TAG) were subjected to analysis as for REF-52 (Fig. 1). FACScan analysis of random cycling TAG cells treated with aphidicolin (10 µM) or hydroxyurea (2 mM) for the indicated times, then released for 24 hours into nocodazole (0.25 µg/ml), shows failure to progress in the cell cycle after 30 hours of treatment.

View larger version (21K): [in a new window]   Fig. 3. REF-52 cells released from G0 state require longer drug exposure than random cycling cells to permanently arrest. REF-52 cells were synchronized in G0, as described in Materials and Methods, treated with aphidicolin (10 µM) or hydroxyurea (2 mM) for the indicated times, and then released for 24 hours into nocodazole (0.25 µg/ml). FACScan analysis shows failure to replicate after at least 45 hours of S phase arrest.

View larger version (15K): [in a new window]   Fig. 4. REF-52 cells remain arrested for at least 7 days following release from prolonged treatment with aphidicolin or hydroxyurea. (A) FACScan analysis of G0 synchronized REF-52 cells treated with aphidicolin (10 µM) for the indicated times and then released for 24 hours or for 7 days in nocodazole (0.25 µg/ml). Cells do not replicate and progress to mitosis during this period of time, as shown by the absence of 4N population. (B) Cell counts confirm the absence of cell proliferation after aphidicolin treatment for times equal to or greater than 45 hours. G0 synchronized REF-52 cells treated with aphidicolin for the indicated times were either harvested (time 0) and counted or were released into drug-free medium. Cell counts were then taken every 24 hours over 4 days in the released population. For counting, cells that had initially been plated at equal density were trypsinized, resuspended in 1 ml PBS and the number of cells present in 20 µl was determined using a Thoma cell counting chamber. Each value is an average of at least 8 counts, and error bars represent the corresponding standard deviations.

View larger version (14K): [in a new window]   Fig. 5. Human non-transformed IMR-90 fibroblasts permanently arrest following prolonged S phase arrest, as indicated by FACScan analysis of random cycling cells treated with aphidicolin (10 µM) or hydroxyurea (2 mM) for the indicated times and then released for 24 hours into nocodazole (0.25 µg/ml). In parallel with results from REF-52, IMR-90 cells released from G0 arrest require longer than random cycling cells to permanently arrest (data not shown).

View larger version (25K): [in a new window]   Fig. 6. REF-52 cells induced to permanently arrest by aphidicolin contain markers and activity consistent with S phase arrest. (A) Protein expression levels. REF-52 cells released from G0 arrest were treated with aphidicolin (10 µM) for the indicated times, then harvested. To determine expression levels of various cell cycle proteins, samples were subjected to SDS-PAGE, and then exposed to the appropriate antibodies for western blots. (B) Cyclin-dependent kinase activity. The kinase activities of Cdk2 and Cdc2 were determined in similar cell extracts, following specific immuneprecipitation of the enzymes, using histone H1 as substrate. Autoradiographs of 32P incorporation from [-32P]ATP are shown. (C) Release of MCM3 and MCM4 from the nuclear matrix fraction during prolonged arrest in S phase. MCM proteins were analyzed for their localization to the nuclear matrix when treated with aphidicolin. For analysis of prolonged S phase block, G0 synchronized REF-52 cells were treated with aphidicolin for the times indicated. Immunoblots of MCM3 and MCM4 proteins in chromatin-bound and soluble fractions are shown. The soluble fractions contain both cytoplasmic and soluble nuclear proteins. Cdc25A, which remains constant in the two fractions, was used as a loading control. (D) The progressive release of MCM3 from the nuclear matrix fraction to the soluble fraction during normal S phase progression is shown as a control. For this analysis, REF-52 cells were synchronized at the G1/S boundary by 30 hours treatment with aphidicolin, released into drug-free medium, and then extracts were prepared every 2 hours during S phase recovery.

View larger version (25K): [in a new window]   Fig. 7. DNA replication is suppressed in vivo following prolonged aphidicolin exposure, but extracted nuclei remain competent for replication. (A) In vivo analysis of DNA replication. Aphidicolin (10 µM)-treated REF-52 cells were released for 30 minutes in drug-free medium containing 10 µM BrdU and the percentages of replicating cells double-labeled for BrdU and for propidium iodide were determined by confocal immunofluorescence microscopy. An average of 1000 cells were counted in each of at least three samples at each time point. Values represent the percentages of BrdU-positive and -negative cells. Error bars represent the corresponding standard deviations. (B) Quantitative analysis of in vitro replicated nuclei derived from REF-52 cells and counts derived from microscopic imaging, as in part C. Following 15 hours of exposure to aphidicolin almost no nuclei (less than 1%) are positive. After 30 hours and 60 hours of exposure prior to harvest of nuclei, positive nuclei are 52% and 54% of the total, respectively. This experiment was replicated three times with similar results. An average of 450 nuclei were counted for each condition. (C) Visualization of in vitro DNA replication. REF-52 cells were released from G0 in the presence of aphidicolin (10 µM) and nuclei were harvested at the times indicated. Isolated nuclei were then subjected to in vitro DNA replication protocol as described in Materials and Methods. Fields of nuclei are shown following the replication assay, imaged for presence of biotin-16-dUTP and counterstained with propidium iodide. In the merged images green nuclei are biotin-16-dUTP positive and red nuclei are positive for only propidium iodide.