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First published online 2 September 2003
doi: 10.1242/jcs.00740

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Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks

Andrei Ivanov^1,3,*, Mark S. Cragg^2,*, Jekaterina Erenpreisa³, Dzintars Emzinsh⁴, Henny Lukman¹ and Timothy M. Illidge^1,

¹ Cancer Research UK, Wessex Oncology Unit, Cancer Sciences Division, School of Medicine, Southampton University Hospital, Southampton SO16 6YD, UK
² Tenovus Research Laboratory, Cancer Sciences Division, School of Medicine, Southampton University Hospital, Southampton SO16 6YD, UK
³ Biomedical Research and Study Center, Latvian University, Ratsupites 1, Riga, LV-1067, Latvia
⁴ Oncology Center of Latvia, Riga, Latvia

View larger version (22K): [in a new window]   Fig. 1. Typical changes in the levels of apoptosis (A), G1 (B), G2/M (C) and polyploidy (D) observed in Namalwa (open square), W1-L2-NS (black circle) and TK6 (open triangle) cells after 10 Gy irradiation. Cells were irradiated and then samples taken regularly throughout a 14-day time course and assessed by DNA flow cytometry to determine the proportion of cells in each phase of the cell cycle. The data represent mean values with associated standard deviations taken from 6-10 experiments.

View larger version (32K): [in a new window]   Fig. 2. Mitoses observed over the time course after irradiation in two p53 mutated cell lines. (A) After 10 Gy irradiation, cell samples were taken for 4 days and assessed by western blotting for the presence of p34cdc2 and Tyr15-phosphorylated p34cdc2. The delay in G2 for Namalwa and WI-L2-NS cells shown in Fig. 1 was associated with prominent Tyr15 phosphorylation of p34cdc2 thus indicating a decrease in p34cdc2 activity. The control level of p34cdc2 activity was restored by day 3 (WI-L2-NS) or 4 (Namalwa) after irradiation. (B) The number of cells undergoing mitosis was assessed every day for 10 days by light microscopy, compared with untreated control cells and expressed as the mitotic index. These data show that adaptation of G2 arrest was followed by accumulation of WI-L2-NS (black diamonds) and Namalwa (open square) cells in mitosis. The drop in mitosis in the Namalwa cell line after 10 Gy, following the initial accumulation, is a result of mitotic failure. These data represent averages from three experiments and bars represent standard deviations.

View larger version (20K): [in a new window]   Fig. 3. Kinetics of -H2AX and Rad51 foci formation in Namalwa and WI-L2-NS cells after irradiation. (A,B) Starting from 3 minutes after 10 Gy irradiation, cell samples were taken, cytospun, fixed and stained for -H2AX and the amount of cells expressing -H2AX foci scored by immunofluorescent microscopy. (A) A histogram representing the relative frequencies of -H2AX foci per Namalwa cell, 24 hours after irradiation. The number of -H2AX foci per nucleus appeared to be dose dependent with a median number of foci of 14, 30 and 42 for 5, 10 and 15 Gy, respectively. (B) The proportion of the -H2AX foci-containing cells 24 hours post-irradiation was 100% and remained high for 2 days for Namalwa (open square) and 4 days for WI-L2-NS (black circle) cells. Then it consequently decreased reaching nearly zero for Namalwa on day 7 and 6.5% for WI-L2-NS cells on day 8. Inset: immunoblotting analysis of Namalwa whole cell lysates of -H2AX, 24 hours after irradiation. (C) The abundance of Namalwa (open aquare) and WIL2-NS (black circle) cells with Rad51 foci was detected on days 2 to 6-7 after 10 Gy irradiation. Cell samples were taken, cytospun, fixed and stained for Rad51 and the amount of cells expressing Rad51 foci scored by immunofluorescent microscopy. Insert: immunoblotting for Rad51 expression level. No difference in the overall level of Rad51 protein was found between untreated and irradiated cells. Data represent averages from at least three experiments±s.e.m.

View larger version (21K): [in a new window]   Fig. 4. Detection of DNA strand breaks by comet assay in Namalwa cells, 1 or 7 days after irradiation. After treatment and incubation for the desired time, cells were embedded in agarose and subjected to electrophoresis at pH 12.1. This alkaline electrophoresis allows the detection of single and double DNA strand breaks. The tail moment (% of migrated DNA x tail length) was measured using Scion image software and a comet assay macro. (A) 96% of non-irradiated Namalwa cells had a tail moment of between 2000 and 8000. (B) On day 1 after 10 Gy irradiation, nearly all of the cells had a tail moment greater than 10,000, providing evidence that DNA strand breaks were not repaired within 24 hours. (C) The vast majority of Namalwa polyploid cells (arrows) on day 7 post irradiation displayed tail moments similar to non-irradiated control cells. Overall, 75.2% of non-apoptotic cells on day 7 had tail moment values equal to those observed in controls. (D) Frequency histograms representing the distribution of tail moments in non-irradiated control Namalwa cells and cells on days 1 and 7 after 10 Gy irradiation.

View larger version (78K): [in a new window]   Fig. 5. Double labelling to show concurrent -H2AX and Rad51 immunofluorescence after irradiation. Following irradiation of Namalwa cells, cell samples were taken, cytospun, fixed and stained for -H2AX and Rad51 and assessed by confocal immunofluorescent microscopy. (A) -H2AX and Rad51 foci in a Namalwa cell 4 hours after 10 Gy irradiation; red channel represents Texas Red-labelled -H2AX, green channel represents FITC-labelled Rad51 foci in the same cell. (B) Namalwa cells 3 days post irradiation. The -H2AX labelling pattern (red) has changed to confluent higher order -H2AX structures. Note the increased -H2AX immunoreactivity in smaller, presumably 4C cells (arrows) and low/absent -H2AX signal in the large Rad51-positive Namalwa endopolyploid cell (asterisks). (C) Namalwa cells, day 4 post-irradiation. The amount of Rad51 foci (green)-positive cells has increased in comparison with day 1. Clustering of the label and formation of higher-order Rad51 structures (arrow) are observed during this time period. Two adjacent or contiguous Rad51 foci was a consistent feature of endopolyploid cells (insertion). DNA was counterstained with 7-AAD. Scale bars: 20 µm.

View larger version (81K): [in a new window]   Fig. 6. Proliferative potential of Namalwa endopolyploid cells after irradiation. (A) Two channel cyclin B1-FITC/PI flow cytometry was performed on Namalwa cells for the first 4 days after irradiation. These data revealed an accumulation of cyclin B1 in G2 arrested 4C cells on day 1 after irradiation. However, on day 4 the amount of cyclin B1-positive cells decreased to below the control values for the 4C fraction, but comprised 41% of the 8C fraction. (B) Cyclin B1 and -H2AX double staining was performed on fixed Namalwa cells 3 days post-irradiation and assessed by immunofluorescent microscopy. These studies revealed the localisation of cyclin B1 (green) in the cytoplasm of large polyploid cell nuclei, indicating that many of these cells are maintained in a G2-like state. -H2AX (red) was excluded from a proportion of these cells (arrows). Rare polyploid cells undergoing mitosis (M) were -H2AX negative. Ao indicates an apoptotic cell with aggregated -H2AX label. DNA was counterstained with DAPI (blue). (C) Identical cyclin B1 and -H2AX double staining was performed on untreated Namalwa cells. (D) DNA image cytometry for multipolar anaphase on day 4 after 10 Gy irradiation. Cytospun cells were fixed, processed and the DNA stained with Toluidine Blue to allow stoichiometric measurement of DNA content by light microscopy and image analysis. Integral optical density (IOD) values, presented for each pole, are relevant to 2C DNA content in control G1 cells. Metaphases (arrow) had double IOD values corresponding to 4C. (E) A frame from a time-lapse video showing the division of a tetraploid Namalwa cell (arrow) on day 7 after irradiation (bottom); division of a diploid untreated 4C cell in culture was taken as a control (top). Scale bars: 40 µm.

View larger version (31K): [in a new window]   Fig. 7. Protection of polyploid cells from apoptosis. After irradiation, cell samples were taken and assessed by two-channel FACS analysis for Annexin V and PI. Subsequently, cells were characterised according to their DNA content and the percentage of Annexin V-positive cells in each fraction compared. (A) The comparison of the proportion of Annexin V-positive Namalwa (A) and WI-L2-NS (B) cells in the 2C-4C (black squares) and >4C (black circles) ploidy fractions on days 2-4 after irradiation. For both Namalwa (P<0.01) and WI-L2-NS (P<0.02) cells, significant differences were detected (Student's t-test). Data represent mean values from three to five experiments±s.e.m. (C) In unirradiated control cells, 85% of Annexin V-positive cells showed PI fluorescence related to 2C-4C cells. (D) The proportion of Annexin V-positive cells was assessed on day 4 after 10 Gy in relation to the actual ploidy value. As can be seen, irradiation was gradually decreasing with each step of ploidy increase, suggesting the protection of endopolyploid cells or their positive selection by apoptosis, accompanying each round of DNA replication. (E) After irradiation, Namalwa cell samples were taken on day 3, stained and assessed by immunofluorescent microscopy for Annexin V (FITC, green) and Rad51 (Alexa Fluor 633; red). Annexin-V-positive apoptotic cells are Rad51-foci-negative and vice versa. DNA is counterstained with DAPI (blue). Scale bar: 40 µm.