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A cell-based model system links chromothripsis with hyperploidy.

Mardin BR, Drainas AP, Waszak SM, Weischenfeldt J, Isokane M, Stütz AM, Raeder B, Efthymiopoulos T, Buccitelli C, Segura-Wang M, Northcott P, Pfister SM, Lichter P, Ellenberg J, Korbel JO - Mol. Syst. Biol. (2015)

Bottom Line: We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division.Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells.CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus

Characteristics of the RPE-1 TP53−/− cell linesAssays for loss of function of p53 in RPE-1 WT and the TP53−/− cell lines, C29 and C111. Cells were challenged with doxorubicin (DXR, 1.5 μM) for 1 h and then released into fresh media.The cells were released into medium containing 100 ng/ml nocodazole. Since p53 is a mediator of the DNA damage response in G2/M transition, cells with functional p53 are expected to be arrested in G2 due to massively damaged DNA (Bunz et al, 1998). When the DNA damage response is lost due to lack of p53 function, cells can enter mitosis where they are trapped with nocodazole. After 24, 48, and 72 h of incubation, cells were collected and stained with phosphohistone H3 antibody, a marker for mitotic cells. Cells were then analyzed by flow cytometry for their DNA and mitotic content. The gating for mitotic cells positive for P-H3 is exemplified in red after 72 h post-doxorubicin.The cells were released into fresh medium. Since p53 also functions during G1/S transition, cells with functional p53 are expected to be arrest in G1 while the cells lacking p53 function continue cycling. After 24, 48, and 72 h of incubation, cells were pulsed for 1 h with BrdU in order to detect ongoing DNA replication. Cells were then analyzed by flow cytometry for their DNA and S phase content. The gating for mitotic cells positive for BrdU-FITC is exemplified in red after 24 h post-DXR. Please note the decrease in BrdU-positive cells in RPE-1 WT as well as the increase in G1 cell populations. In stark contrast, TP53−/− cells have ongoing DNA replication even after massive DNA damage.Detection of senescent cells through measurement of β-galactosidase activity at pH 6, reflecting a known characteristic of senescent cells not found in dividing, quiescent or immortal cells. Cells were released into fresh media. After 72 h, the activity of β-galactosidase was assessed. Experiments were done in triplicate, and exemplary images are shown. Note that the RPE-1 WT cells were stained positive for β-galactosidase indicating senescence. In contrast, very few cells in TP53−/− cell lines were stained positive. Moreover, we frequently found dividing cells indicating a cycling population only in cells with non-functional p53.Exemplary images of the metaphase spreads of the RPE-1 WT and the TP53−/− cell lines. Cells were fixed and stained with the pan-centromere probe for probing the centromeric regions of all chromosomes, and Hoechst to mark chromosomes. Experiments were done in triplicate. The mean count distribution is plotted in Fig1C.Exemplary images of RPE-1 cell lines after soft agar assay. Transformed RPE-1 cells as well as the two TP53−/− cell lines were incubated in soft agar for 25 days and imaged afterwards. Experiments were done in triplicate. The transformed cells were able to form colonies in agar, whereas the untransformed C29 and the C111 clones were not.
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fig06ev: Characteristics of the RPE-1 TP53−/− cell linesAssays for loss of function of p53 in RPE-1 WT and the TP53−/− cell lines, C29 and C111. Cells were challenged with doxorubicin (DXR, 1.5 μM) for 1 h and then released into fresh media.The cells were released into medium containing 100 ng/ml nocodazole. Since p53 is a mediator of the DNA damage response in G2/M transition, cells with functional p53 are expected to be arrested in G2 due to massively damaged DNA (Bunz et al, 1998). When the DNA damage response is lost due to lack of p53 function, cells can enter mitosis where they are trapped with nocodazole. After 24, 48, and 72 h of incubation, cells were collected and stained with phosphohistone H3 antibody, a marker for mitotic cells. Cells were then analyzed by flow cytometry for their DNA and mitotic content. The gating for mitotic cells positive for P-H3 is exemplified in red after 72 h post-doxorubicin.The cells were released into fresh medium. Since p53 also functions during G1/S transition, cells with functional p53 are expected to be arrest in G1 while the cells lacking p53 function continue cycling. After 24, 48, and 72 h of incubation, cells were pulsed for 1 h with BrdU in order to detect ongoing DNA replication. Cells were then analyzed by flow cytometry for their DNA and S phase content. The gating for mitotic cells positive for BrdU-FITC is exemplified in red after 24 h post-DXR. Please note the decrease in BrdU-positive cells in RPE-1 WT as well as the increase in G1 cell populations. In stark contrast, TP53−/− cells have ongoing DNA replication even after massive DNA damage.Detection of senescent cells through measurement of β-galactosidase activity at pH 6, reflecting a known characteristic of senescent cells not found in dividing, quiescent or immortal cells. Cells were released into fresh media. After 72 h, the activity of β-galactosidase was assessed. Experiments were done in triplicate, and exemplary images are shown. Note that the RPE-1 WT cells were stained positive for β-galactosidase indicating senescence. In contrast, very few cells in TP53−/− cell lines were stained positive. Moreover, we frequently found dividing cells indicating a cycling population only in cells with non-functional p53.Exemplary images of the metaphase spreads of the RPE-1 WT and the TP53−/− cell lines. Cells were fixed and stained with the pan-centromere probe for probing the centromeric regions of all chromosomes, and Hoechst to mark chromosomes. Experiments were done in triplicate. The mean count distribution is plotted in Fig1C.Exemplary images of RPE-1 cell lines after soft agar assay. Transformed RPE-1 cells as well as the two TP53−/− cell lines were incubated in soft agar for 25 days and imaged afterwards. Experiments were done in triplicate. The transformed cells were able to form colonies in agar, whereas the untransformed C29 and the C111 clones were not.

Mentions: Model cell line: We chose the human hTERT RPE-1 (retinal pigment epithelial) cell line as a model system for characterizing de novo SR formation. This telomerase immortalized cell line exhibits a genomically stable diploid karyotype. Though not tumor derived, RPE-1 cells can be transformed with elevated levels of γ-irradiation leading to gross SR formation detectable by karyotyping. We subjected hTERT RPE-1 (herein termed “RPE-1 wild type”) and previously generated (Riches et al, 2001) RPE-1-transformed cell lines to mate-pair sequencing, which revealed the occurrence of several SRs only in the transformed lines (Appendix Fig S1A–C). We recently described a link between TP53 mutations and chromothripsis, implying that abnormal p53 function may be necessary for the induction, or tolerance, of catastrophic SRs (Rausch et al, 2012a). To establish a model amenable to study chromothripsis, we thus used zinc finger nucleases to generate an RPE-1 derivative deficient in p53. We confirmed p53 loss of function in two independent cell lines, C111 and C29 (Figs1A and EV1A–C). Interestingly, C29, but not C111, showed an increase in ploidy measured by both DNA content and chromosome counts from metaphase spreads (Figs1B and C, and EV1D), which may be explained with the previously noted tendency toward tetraploidization upon p53 inactivation (Bunz et al, 2002).


A cell-based model system links chromothripsis with hyperploidy.

Mardin BR, Drainas AP, Waszak SM, Weischenfeldt J, Isokane M, Stütz AM, Raeder B, Efthymiopoulos T, Buccitelli C, Segura-Wang M, Northcott P, Pfister SM, Lichter P, Ellenberg J, Korbel JO - Mol. Syst. Biol. (2015)

Characteristics of the RPE-1 TP53−/− cell linesAssays for loss of function of p53 in RPE-1 WT and the TP53−/− cell lines, C29 and C111. Cells were challenged with doxorubicin (DXR, 1.5 μM) for 1 h and then released into fresh media.The cells were released into medium containing 100 ng/ml nocodazole. Since p53 is a mediator of the DNA damage response in G2/M transition, cells with functional p53 are expected to be arrested in G2 due to massively damaged DNA (Bunz et al, 1998). When the DNA damage response is lost due to lack of p53 function, cells can enter mitosis where they are trapped with nocodazole. After 24, 48, and 72 h of incubation, cells were collected and stained with phosphohistone H3 antibody, a marker for mitotic cells. Cells were then analyzed by flow cytometry for their DNA and mitotic content. The gating for mitotic cells positive for P-H3 is exemplified in red after 72 h post-doxorubicin.The cells were released into fresh medium. Since p53 also functions during G1/S transition, cells with functional p53 are expected to be arrest in G1 while the cells lacking p53 function continue cycling. After 24, 48, and 72 h of incubation, cells were pulsed for 1 h with BrdU in order to detect ongoing DNA replication. Cells were then analyzed by flow cytometry for their DNA and S phase content. The gating for mitotic cells positive for BrdU-FITC is exemplified in red after 24 h post-DXR. Please note the decrease in BrdU-positive cells in RPE-1 WT as well as the increase in G1 cell populations. In stark contrast, TP53−/− cells have ongoing DNA replication even after massive DNA damage.Detection of senescent cells through measurement of β-galactosidase activity at pH 6, reflecting a known characteristic of senescent cells not found in dividing, quiescent or immortal cells. Cells were released into fresh media. After 72 h, the activity of β-galactosidase was assessed. Experiments were done in triplicate, and exemplary images are shown. Note that the RPE-1 WT cells were stained positive for β-galactosidase indicating senescence. In contrast, very few cells in TP53−/− cell lines were stained positive. Moreover, we frequently found dividing cells indicating a cycling population only in cells with non-functional p53.Exemplary images of the metaphase spreads of the RPE-1 WT and the TP53−/− cell lines. Cells were fixed and stained with the pan-centromere probe for probing the centromeric regions of all chromosomes, and Hoechst to mark chromosomes. Experiments were done in triplicate. The mean count distribution is plotted in Fig1C.Exemplary images of RPE-1 cell lines after soft agar assay. Transformed RPE-1 cells as well as the two TP53−/− cell lines were incubated in soft agar for 25 days and imaged afterwards. Experiments were done in triplicate. The transformed cells were able to form colonies in agar, whereas the untransformed C29 and the C111 clones were not.
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Related In: Results  -  Collection

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fig06ev: Characteristics of the RPE-1 TP53−/− cell linesAssays for loss of function of p53 in RPE-1 WT and the TP53−/− cell lines, C29 and C111. Cells were challenged with doxorubicin (DXR, 1.5 μM) for 1 h and then released into fresh media.The cells were released into medium containing 100 ng/ml nocodazole. Since p53 is a mediator of the DNA damage response in G2/M transition, cells with functional p53 are expected to be arrested in G2 due to massively damaged DNA (Bunz et al, 1998). When the DNA damage response is lost due to lack of p53 function, cells can enter mitosis where they are trapped with nocodazole. After 24, 48, and 72 h of incubation, cells were collected and stained with phosphohistone H3 antibody, a marker for mitotic cells. Cells were then analyzed by flow cytometry for their DNA and mitotic content. The gating for mitotic cells positive for P-H3 is exemplified in red after 72 h post-doxorubicin.The cells were released into fresh medium. Since p53 also functions during G1/S transition, cells with functional p53 are expected to be arrest in G1 while the cells lacking p53 function continue cycling. After 24, 48, and 72 h of incubation, cells were pulsed for 1 h with BrdU in order to detect ongoing DNA replication. Cells were then analyzed by flow cytometry for their DNA and S phase content. The gating for mitotic cells positive for BrdU-FITC is exemplified in red after 24 h post-DXR. Please note the decrease in BrdU-positive cells in RPE-1 WT as well as the increase in G1 cell populations. In stark contrast, TP53−/− cells have ongoing DNA replication even after massive DNA damage.Detection of senescent cells through measurement of β-galactosidase activity at pH 6, reflecting a known characteristic of senescent cells not found in dividing, quiescent or immortal cells. Cells were released into fresh media. After 72 h, the activity of β-galactosidase was assessed. Experiments were done in triplicate, and exemplary images are shown. Note that the RPE-1 WT cells were stained positive for β-galactosidase indicating senescence. In contrast, very few cells in TP53−/− cell lines were stained positive. Moreover, we frequently found dividing cells indicating a cycling population only in cells with non-functional p53.Exemplary images of the metaphase spreads of the RPE-1 WT and the TP53−/− cell lines. Cells were fixed and stained with the pan-centromere probe for probing the centromeric regions of all chromosomes, and Hoechst to mark chromosomes. Experiments were done in triplicate. The mean count distribution is plotted in Fig1C.Exemplary images of RPE-1 cell lines after soft agar assay. Transformed RPE-1 cells as well as the two TP53−/− cell lines were incubated in soft agar for 25 days and imaged afterwards. Experiments were done in triplicate. The transformed cells were able to form colonies in agar, whereas the untransformed C29 and the C111 clones were not.
Mentions: Model cell line: We chose the human hTERT RPE-1 (retinal pigment epithelial) cell line as a model system for characterizing de novo SR formation. This telomerase immortalized cell line exhibits a genomically stable diploid karyotype. Though not tumor derived, RPE-1 cells can be transformed with elevated levels of γ-irradiation leading to gross SR formation detectable by karyotyping. We subjected hTERT RPE-1 (herein termed “RPE-1 wild type”) and previously generated (Riches et al, 2001) RPE-1-transformed cell lines to mate-pair sequencing, which revealed the occurrence of several SRs only in the transformed lines (Appendix Fig S1A–C). We recently described a link between TP53 mutations and chromothripsis, implying that abnormal p53 function may be necessary for the induction, or tolerance, of catastrophic SRs (Rausch et al, 2012a). To establish a model amenable to study chromothripsis, we thus used zinc finger nucleases to generate an RPE-1 derivative deficient in p53. We confirmed p53 loss of function in two independent cell lines, C111 and C29 (Figs1A and EV1A–C). Interestingly, C29, but not C111, showed an increase in ploidy measured by both DNA content and chromosome counts from metaphase spreads (Figs1B and C, and EV1D), which may be explained with the previously noted tendency toward tetraploidization upon p53 inactivation (Bunz et al, 2002).

Bottom Line: We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division.Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells.CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus