First published online 29 March 2005
doi: 10.1242/jcs.02306
Journal of Cell Science 118, 1757-1767 (2005)
Published by The Company of Biologists 2005
Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation
H. I. Abdel-Halim1,2,
A. T. Natarajan1,
L. H. F. Mullenders1,* and
J. J. W. A. Boei1
1 Department of Toxicogenetics, Leiden University Medical Center, PO Box 9503, 2300 RA Leiden, The Netherlands
2 Department of Zoology, Faculty of Sciences, Suez Canal University, Ismailia, Egypt
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Fig. 1. MMC-induced chromatid interchanges. Examples of MMC-induced chromatid interchanges as observed in human lymphocytes using FISH combining whole chromosome and band-specific probes. (A) Metaphase spread containing a quadriradial chromatid exchange between chromosome 9 (red) and an unpainted chromosome (blue) with the exchange breakpoint at the paracentromeric heterochromatic band of chromosome 9 (green). Two unaffected chromosomes 8 (green, with red paracentromeric euchromatic band) are also present. (B) The most commonly observed quadriradial between the homologues of chromosome 9 (red) with both exchange breakpoints within the heterochromatic bands (green). (C) An exchange involving the heterochromatic band (green) of chromosome 1 (red) and an unpainted chromosome. (D) The heterochromatic bands of chromosomes 1 (green) and 9 (red) are involved in a chromatid exchange. The black and white images of the DAPI counterstained chromosomes were included to visualize the exchanges at higher contrast.
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Fig. 6. PCC analysis of MMC-treated human lymphocytes. (A-E) Sections of PCC spreads of MMC-treated G1 human lymphocytes after in situ hybridization using whole chromosome-specific probes for chromosome 8 (green) and 9 (red) together with band-specific probes 8p11.2 and 9q12-13 in reversed colors. Colocalization of the chromosome 9 homologues was observed at the position of the heterochromatic bands (A) or at other part of chromosome 9 sometimes leading to a partial alignment (B,C). Also, chromosome breakage within the green-painted heterochromatic band (D) or in an arm of chromosome 9 (E) was frequently observed. (F) The induced colocalization of homologous chromosomes 8 or 9, or breaks in these chromosomes in G1 human lymphocytes after treatment with 4 µM MMC for 1 hour (background frequencies are deduced). The induced colocalization and breaks were significantly different (P<0.01, Wilson's method) between chromosomes 8 and 9.
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Fig. 3. Interphase FISH with band-specific probes for chromosome 8 and 9 on human fibroblasts. (A) G-banded chromosomes 8 and 9, showing the position and size of the bands covered by the applied chromosome band-specific probes 8p11.2 (green) and 9q12-13 (red). (B) Example of confluent human fibroblasts after hybridization. Criteria for manual analysis were made according to the number of hybridization signals in the nucleus. Discrimination was made between cells with: two separate hybridization signals for each probe (left) and nuclei in which signals of homologous chromosomes were so close together that only one single, usually larger and brighter, hybridization signal (middle) or two touching signals (right) could be observed.
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Fig. 5. Position of chromosome bands 8p11.2 and 9q12-13 within confluent fibroblasts. (A) The position of hybridization signals observed in 200 nuclei from control and MMC-treated confluent human fibroblasts are plotted in one quadrant of the nucleus. The outer curve represents the contours of an average sized nucleus. The inner curve corresponds to one quadrant of a circle with a radius equal to the median of the radial distances of the hybridization signals. Therefore, 50% of hybridization signals are located inside this circle. (B) The inter-homologue distance distribution of chromosome bands 8p11.2 and 9q12-13 was determined by analyzing 200 nuclei from control and MMC-treated confluent human fibroblasts. Cells with ascending inter-homologue distances are plotted against the inter-homologue distances.
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Fig. 2. Distribution of MMC-induced chromatid interchanges over the genome. Frequencies of chromatid interchanges observed in human lymphocytes after treatment in G1 with 4 µM MMC for 1 hour. (A) The genomic frequency (estimated by analysis of 200 Giemsa-stained metaphase cells) as well as the specific frequencies for chromosomes 1, 2, 8 and 9 (analysis of 900 cells after FISH painting) are plotted. Chisquare statistics showed a highly significant (P<0.001) difference among the four chromosomes used taking into account their DNA contents. No chromatid interchanges were observed in 900 mock-treated cells. (B) The frequencies of chromatid interchanges observed for chromosome 1 and 9 are subdivided into two categories–namely, those between the painted homologues (1-1 or 9-9) and between two non-homologous chromosomes (1-other or 9-other).
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Fig. 4. Colocalization of hybridization signals of homologous chromosome bands in human fibroblasts. The percentage of cells with one hybridization signal for the homologues of either chromosome 8 or 9 was analyzed manually. (A) The analysis was performed on confluent cells treated with 4 µM MMC for 1 hour and fixed either immediately (recovery time 0 hours) or after a recovery time of 20 hours. Solvent controls were included for both time points. Each bar represents the average percentage obtained from at least three independent experiments. In each experiment 500 cells were analyzed. The error bars are standard deviations (s.d.) of the mean values. A significantly increased colocalization was evident for chromosome 9 in the treated cells (P<0.05, Student t-test) and no difference was observed between 0 hour and 20 hour recovery. (B) The analysis was performed on cycling cells treated with 4 µM MMC for 1 hour, 24 hours after they were released from confluency by subcultivation to reduced cell density. The cells were fixed directly after the treatment. Since BrdU was present during treatment, BrdU-positive cells must have been treated in the S-phase of their cell cycle. 500 BrdU-positive and -negative cells were analyzed. A significant increase of the number of cells containing one hybridization signal for chromosome 9 was observed in MMC-treated cells (P<0.05, Wilson's method for difference in proportions).
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Fig. 7. Colocalization of chromosome 9 homologues in wild-type and DNA repair-deficient fibroblasts. Percentage of cells with one hybridization signal for chromosome 9p12-13 band-specific probe observed in wild-type human fibroblasts (VH-25) and fibroblast cell lines derived from XPA and XPF patients. Confluent cells were treated for 1 hour with 4 µM MMC and fixed directly afterwards. Analysis was preformed manually in 500 cells per experiment. The average percentage of at least three experiments is presented together with the standard deviation of the mean values. A significant increase (P<0.05, Student's t-test) in the number of cells with one hybridization signal was observed for wild-type and XPA fibroblasts. In XPF cells there was no significant change; the data were taken from cells of two different XPF patients.
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Fig. 8. Chromatid interchanges and breaks induced by MMC in wild-type and XPF lymphoblastoid cells. (A) Genomic frequencies for chromatid interchanges as observed in wild-type and XPF lymphoblastoid cells. Exponentially growing cells were treated with 4 µM MMC for 1 hour at different recovery times. Data from at least two experiments were analyzed. The difference between wild-type and XPF cells is significant at 28 hour and 40 hour time points (P<0.05, Student t-test). (B) Frequencies of chromatid exchanges and breaks observed for chromosome 9 after a recovery of 28 hours. A significant difference was observed only for exchanges (P<0.01, Wilson's method).
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© The Company of Biologists Ltd 2005
