Figure 1.
Replicative dependent polyploidization in HMECs.
(A) Flow cytometry analysis of DNA content of 219-7 HMECs stained with propidium iodide, at early and late culture PDs (upper panels). The percentage of cells with 4N and 8N DNA content is given. Bivariate analysis of DNA content and expression of cyclin D1 protein (lower panels), in which cyclin D1 positive cells are grouped depending on their ploidy level. Negative control, shown in the bottom right corner, was performed without the primary antibody. (B) FISH analysis of a polyploid cell nucleus showing four centromere signals corresponding to chromosome 4 (in blue) and four signals corresponding to chromosome18 (in green). DNA is counterstained with DAPI. (C) Graph illustrating ploidy evolution of 219-7 HMECs throughout the culture based on a cell per cell basis scoring of the number of centromeric signals. Average percentages from two independent experiments with standard deviations are shown.
Figure 2.
Polyploidization is reverted by hTERT immortalization.
DNA content evolution of non-transduced and hTERT-transduced HMECs at different PDs by univariate and bivariate flow cytometry. At an early PD, non-transduced 219-7 HMECs were analyzed for the presence of tetraploidy (left panels). Cells were then divided into two groups. Proliferation of non-transduced HMECs resulted in the accumulation of a 4N subpopulation (upper right panels). In contrast, proliferation of HMEC-hTERT cells revealed a sharp reduction of the 4N subpopulation (lower right panels). Figure shows plot with negative control.
Figure 3.
Polyploid HMECs arise through incomplete cytokinesis.
(A) Immunofluorescence of alpha and beta-tubulin and DAPI staining allows binucleated (white asterisks) to be distinguished from mononucleated HMECs (left image). ImmunoFISH combining alpha and beta tubulin together with centromeric specific DNA probes for chromosome 4 (in blue) and 18 (in green) revealed two tetraploid cells each with a single 4N nucleus (top right), and a tetraploid cell containing two 2N nuclei within the same cytoplasm (down right). (B) Averaged percentage of mononucleated, binucleated and multinucleated polyploid HMECs throughout the cell culture from two different experiments.
Figure 4.
Quantification of anaphase bridges and binucleates in non-transduced and hTERT-transduced HMECs.
(A) Texas Red-X phalloidin labelling of actin filaments and DNA counterstaining with DAPI of non-transduced HMECs. White asterisks highlight those binucleated cells. (B) Analysis for the presence of binucleated cells (BN; upper bars) and anaphase bridges (AB; lower bars) in non-transduced HMECs and at different PDs immediately after transducing cells with hTERT. Two independent cell transduction experiments were performed (transduction at PD36, in blue and at PD40, in green). (C) The same analysis was performed after HMEC-hTERT cells were passaged more than 8 PDs after transduction. Again, two independent transduction experiments were performed (transduction at PD26, in blue and at PD36, in green).
Figure 5.
Artificially induced anaphase bridges engender binucleated polyploid HMEC-hTERT cells.
(A) Immortalized HMECs were treated for one hour with the DNA damage-inducing agent bleomycin (HMEC-hTERT_BLEO) to induce double strand breaks. After drug washout, cells were collected at different timepoints (6, 24, 48 and 72 h after treatment). Non-treated cells (HMEC-hTERT_Control) were also collected in parallel at 0 and 72 hours. Then DNA content was analyzed by flow cytometry and the presence of binucleated cells and anaphase bridges was scored after Texas Red-X phalloidin and DAPI staining. (B) Image showing a binucleated cell (white asterisk) and two nuclei connected by a chromatin bridge (open white arrow). (C) The graph shows the evolution of the percentage of BN and AB on HMEC-hTERT_BLEO (non-patterned bars) and HMEC-hTERT_Control (stripped bars). (D) The table resumes the DNA content values (averaged from three different experiments) resulting from flow cytometry analyses. Standard deviation is shown. (E) Plots resulting from the analysis of the DNA content (Y axes) and the forward light scatter (FLS) (X axes), which allows for the identification of larger cells. Of note, upon drug treatment, a cell subpopulation with a larger area appears (blue dots). An averaged value from three independent experiments is shown. (F) HMEC-hTERT cells were treated with cytochalasin B (HMEC-hTERT_CytB), during 24 hrs before FACS sorting, to artificially obtain binucleated cells. The dot plot shows that the fraction of cells falling in the right gate (in blue) increases upon Cyt-B treatment. Averaged data and standard deviation from three independent experiments is shown.
Figure 6.
Unbroken anaphase bridges interfere with abscission in HMECs and HMEC-hTERT cells.
Non-transduced and hTERT-transduced HMECs were transfected with a H2B-GPF plasmid. (A) Time-lapse micrographies of non-transduced HMECs transiently expressing H2B-GFP. Time 0 was set as the last time point before anaphase onset. Upper images show progressively strengthened unbroken anaphase bridges that remain undisrupted connecting the two nuclei (see inset). Lower images show the formation of a binucleated cell where this process is coupled to the presence of an unbroken chromatin bridge. The scale bar represents 10 µm. (B) Three examples of HMEC-hTERT_BLEO expressing H2B-GFP with over strengthened chromatin bridges long time after anaphase onset. Scale bar represents 10 µm.