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First published online 23 October 2007
doi: 10.1242/jcs.013730

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Depletion of topoisomerase II leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads

Jennifer M. Spence^1,*, Hui Hui Phua^1,, Walter Mills^1,, Adam J. Carpenter², Andrew C. G. Porter³ and Christine J. Farr^1,¶

¹ Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
² Haemostasis and Thrombosis Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, London W12 0NN, UK
³ Department of Haematology, Faculty of Medicine, Imperial College, London W12 0NN, UK

View larger version (77K): [in this window] [in a new window]   Fig. 1. (A) Immunofluorescence for topo II on HTETOP metaphase cells grown in the absence (topo IION) and presence (topo IIOFF) of dox for 48 hours. (B) Immunofluorescence for the SMC2 subunit of condensin on HTETOP metaphase spreads isolated from cells grown in the presence and absence of dox for 72 hours. The normal axial distribution of SMC2 is detected following depletion of topo II. Bar, 10 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 2. Topo II is not required for the structural integrity of mitotic chromosomes. Shown are representative images following the expansion and refolding assay. Stages (1) to (3) represent the first cycle of expansion and refolding, and (4) is the refolded status after cycle 2. Stage (1): colcemid-arrested metaphase before treatment; stage (2): first-round expansion in the low-salt plus EDTA TEEN buffer; stage (3): first-round contraction in the low-salt plus Mg2+ RSB buffer; stage (4): appearance after a second round of expansion and contraction. Topo-II-depleted chromosomes [both -only depleted (120 hours exposure to dox) and -plus β-depleted (120 hours exposure to dox plus 72 hours exposure to siRNA)] recover their normal starting morphology, which is a slightly thinner and more ribbon-like appearance than that displayed by the control chromosomes (Carpenter and Porter, 2004). All images are shown at the same magnification, with some regions enlarged for clarity. Bar, 10 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 3. Many metaphases in topo-II-depleted cells have one or more elongated chromosome arms stretched towards the spindle pole. (A) A control (expressing topo II and IIβ) HTETOP metaphase cell; (B,C) HTETOP topo-II-depleted metaphase cells (48 hours exposure to dox); (D,E) topo II-depleted (72 hours exposure to dox) and topo IIβ-depleted (72 hours exposure to siRNA) cells arrested in metaphase with MG132 (DNA/DAPI, blue; tubulin, or as indicated, red; CENP-C, green). Bar, 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 4. The effect of depletion of topo II on human chromosome segregation. (A-E) HTETOP topo-II-depleted (48 hours exposure to dox) segregating cells. (A) immuno-FISH showing colocalisation of segregating signals for the DNA pan-alphoid probe (green) and anti-CENP-A (red); (B) typical FISH image showing segregation of the rDNA loci (red); (C) four post-metaphase cells showing the segregation behaviour of telomeric regions, detected using a PNA anti-telomere probe (green). White arrowheads indicate telomeric DNA in chromatin bridges. White arrows point to chromatin bridges that lack detectable (TTAGGG)n signals. These regions have been enlarged for clarity. (D) in this anaphase, the centromeres (green) of both copies of chromosome 6 have segregated, while the chromosome 6 paint (red) reveals that, for one of these chromosomes, the sister chromatids are bridged. This cell also appears to contain an acentric fragment of chromosome 6. (E) Two examples of segregating cells in which chromosome 20 (green) has separated correctly (although the second cell appears to have only one copy of this chromosome), while the sister chromatids of the single X chromosome (red) form a bridge. DNA is counterstained with DAPI (blue). Bar, 10 µm. (F) Chromosome paints and centromere-specific DNA probes were used to analyse the segregation of four of the endogenous HTETOP chromosomes: 6, the X, 19 and 20. For each chromosome, the numbers of sister chromatids analysed from either untreated (topo IION) or 48 hours exposure to dox (topo IIOFF) cells ranged from 141 to 447 per experiment. Shown are the means from three independent experiments (with s.d.). The total numbers of chromatids assessed for each chromosome under topo II ON/OFF conditions were: Chr. 6: 1230 and 1183; Chr. X: 657 and 592; Chr. 19: 1146 and 1058; Chr. 20: 1298 and 1006. Segregation errors were classified as nondisjunction (ndj), lagging, bridging or others (uncharacterised, apparent 1:0, segregation events, which might reflect absence of replication, chromosome loss or hybridisation failure). The combined segregation errors for each chromosome are summarised below the graph (mean percentage and s.d.). (G) The segregation behaviour of a 2.7 Mb human X centromere-based minichromosome was analysed following its transfer (by cell fusion) into HTETOP cells. Segregation data for the endogenous X chromosome in this hybrid background are also presented (mean and s.d.). For both the mini and X chromosomes, the total number of sister chromatids examined from either untreated cells or cells exposed to dox for 48 hours was 1300 (based on data collected from three experiments for each of two independently derived hybrid cell lines). The total levels of chromatid missegregation following depletion of topo II are significantly higher, both for the whole X and for the minichromosome (Paired Student's t-test, P0.005).

View larger version (80K): [in this window] [in a new window]   Fig. 5. Immunofluorescence of topo-II-expressing and topo-II-depleted (72 hours exposure to dox) HTETOP cells. (A) Metaphases (cytospun, no colcemid) stained using an antibody against CENP-A (green); (B) partial chromosome spreads (colcemid) stained for CENP-F (green); (C) partial metaphase spreads (colcemid) stained for CENP-E (green); (D) typical metaphase cells (grown in situ, no colcemid) stained for CENP-C (green) and Aurora B (red); (E) immuno-FISH of partial chromosome spreads (colcemid) showing the typical banding pattern generated using an antibody raised against acetyl-histone H3 (amino acids 1-20) (green). The centromeric regions have been identified using a pan-alphoid DNA probe (red). The white arrowheads highlight examples of hypoacetylated centromeric and pericentromeric domains. DNA is counterstained with DAPI (blue). Bars, 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 6. The effect of depletion of topo II on anaphase PICH-coated threads. HTETOP cells expressing topo II (A), or depleted of topo II (48 hours exposure to dox) (B) were fixed and permeabilised before being co-stained for PICH (green) and CENP-B (red). Shown are cells in anaphase or undergoing aberrant cytokinesis. Arrowheads indicate examples of PICH threads that are not associated with DAPI-stained chromatin bridges. Bar, 10 µm. (C) Quantification of the number of PICH threads for anaphase cells expressing topo II (topo IION) and for cells treated for 48 hours with dox (topo IIOFF). 50 anaphase cells were analysed for each, and the stage was determined by the distance between segregating sister kinetochores.

View larger version (63K): [in this window] [in a new window]   Fig. 7. The effect of depletion of topo II on centromeric topo II cleavage activity. The HTETOP-minichromosome hybrid cells expressing topo II or depleted (72 hours exposure to dox) for topo II and/or topo IIβ (72 hours exposure to siRNA) were exposed in culture to etoposide [0 (DMSO only), 100 or 500 µM] for 60 minutes at 37° C and embedded in agarose. (A) Undigested HMW DNA was resolved by PFGE and stained using ethidium bromide. (B) After transfer, the Southern blot was probed using the X -satellite DNA DXZ1 to detect the 2.7 Mb X centromere-based minichromosome (position indicated by black arrowhead). An etoposide-specific DXZ1-hybridising fragment of 1.85 Mb (position indicated by `<') could be detected, in addition to a more general smear of hybridisation (this was more noticeable in DNA from cells expressing topo II). At the lower etoposide concentration (100 µM), a signal in the 1.85 Mb range could only be detected in cells expressing topo II (either alone, or together with the β isoform); at the higher etoposide concentration (500 µM), the 1.85 Mb signal was barely detectable after depletion of both isoforms, but could still be detected following depletion of topo II alone (although the signal was reduced relative to that of the intact minichromosome in the same sample). This suggests that both isoforms of topo II contribute to this cleavage site within the centromeric DNA. LM, limit mobility.

View larger version (27K): [in this window] [in a new window]   Fig. 8. The effect of depletion of topo II on the metaphase inter-kinetochore distance. (A) Distances between sister centromeres of human chromosome 11 detected by FISH using D11Z1 DNA as a probe were assessed using Leica Deblur software. Bar, 10 µm. (B) Measurements made of treated cells in parallel with untreated controls (treated/untreated pairs indicated by brackets) are presented as a boxplot showing the third and first quartiles, with the median indicated by a cross in each box. The maximum and minimum values are indicated by the ends of the vertical lines. Each plot is based on 40 measurements. In HTETOP cells, topo II was depleted by 72 hours exposure to dox. Cells were arrested using MG132 (10 µM for 2 hours 30 minutes), allowing distances under tension to be measured. For the dox-treated, topoII-depleted HTETOP cells, data from three independent experiments are presented. In each case, the decrease in the distance across the centromere domain under tension following depletion of topo II is significant (P=0.001, 0.0001, 0.01, respectively). To deplete topo IIβ, HTETOP cells were transiently transfected with siRNA (72 hours exposure). Colcemid-treated HTETOP cells served as a control where no spindle or tension existed. The lack of any effect from dox treatment itself was confirmed by analysis of MG132-arrested parental HT1080 cells. Clone J is a derivative of HTETOP that constitutively expresses topo II as a fusion with the C-terminus of eGFP (Carpenter and Porter, 2004). (C) Quantification of spindle lengths in HTETOP cells arrested using MG132 (10 µM for 2 hours 30 minutes) following 0 (topo IION) or 72 hours (topo IIOFF) exposure to dox. Measurements of the pole-to-pole distance, based on -tubulin and DAPI staining, were collected from three independent experiments (topo IION, n=154; topo IIOFF, n=151).