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Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus

Multinucleated OIS Cells Originate from Aberrant Mitosis(A) MNCs in human nevi. Dermal nevus-containing section of human skin stained with DAPI (panels 1 and 2) or for melan A (Mel A; panels 3 and 4). Panel 2 is a magnified insert with multinucleated nevus cells (arrows). Panel 4 shows a magnification of melan-A-positive multinucleated nevus cells (top) and a section of overlaying epidermis with mononucleated melanocytes (bottom). Scale bars, 500 μm for panels 1 and 3, 50 μm for panel 2, and 100 μm for panel 4. 17% of nevus cells (out of 334) and 0% epidermis melanocytes (out of 365) are multinucleated in this specimen.(B) Multinucleated senescent 12-day-induced ERRAS cell, stained for microtubules (left, red on overlay) and DAPI (middle, blue on overlay). Scale bars, 30 μm.(C) 15-day ERRAS induction (Ras) increases the percentage of MNC with two nuclei, more than two nuclei, and lobular nuclei. Data indicate means ± SEM from three independently derived ERRAS cell populations. ∗p = 0.021; ∗∗p = 0.005 (paired Student’s t test). Cntr, control.(D) Origin of multinucleation in GFP-Lamin A-expressing ERRAS cells observed throughout 12 days of Ras induction and classified as originating from mitotic failure (light gray), cell fusion (medium gray), and interphase fragmentation (fragm, dark gray). n = 23 multinucleation events (see also Figure S1I).(E and F) In (E), cytokinesis failure leading to binucleation is shown. (F) Prolonged mitotic arrest followed by slippage, generating a highly multinucleated cell. For (E) and (F), bright-field image (top) and corresponding GFP fluorescence (bottom) are shown at selected times (in hours:minutes, or hh:mm). Arrows indicate cells undergoing multinucleation.See also Figure S1 and Movies S1, S2, S3, S4, S5, S6, and S7.
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fig1: Multinucleated OIS Cells Originate from Aberrant Mitosis(A) MNCs in human nevi. Dermal nevus-containing section of human skin stained with DAPI (panels 1 and 2) or for melan A (Mel A; panels 3 and 4). Panel 2 is a magnified insert with multinucleated nevus cells (arrows). Panel 4 shows a magnification of melan-A-positive multinucleated nevus cells (top) and a section of overlaying epidermis with mononucleated melanocytes (bottom). Scale bars, 500 μm for panels 1 and 3, 50 μm for panel 2, and 100 μm for panel 4. 17% of nevus cells (out of 334) and 0% epidermis melanocytes (out of 365) are multinucleated in this specimen.(B) Multinucleated senescent 12-day-induced ERRAS cell, stained for microtubules (left, red on overlay) and DAPI (middle, blue on overlay). Scale bars, 30 μm.(C) 15-day ERRAS induction (Ras) increases the percentage of MNC with two nuclei, more than two nuclei, and lobular nuclei. Data indicate means ± SEM from three independently derived ERRAS cell populations. ∗p = 0.021; ∗∗p = 0.005 (paired Student’s t test). Cntr, control.(D) Origin of multinucleation in GFP-Lamin A-expressing ERRAS cells observed throughout 12 days of Ras induction and classified as originating from mitotic failure (light gray), cell fusion (medium gray), and interphase fragmentation (fragm, dark gray). n = 23 multinucleation events (see also Figure S1I).(E and F) In (E), cytokinesis failure leading to binucleation is shown. (F) Prolonged mitotic arrest followed by slippage, generating a highly multinucleated cell. For (E) and (F), bright-field image (top) and corresponding GFP fluorescence (bottom) are shown at selected times (in hours:minutes, or hh:mm). Arrows indicate cells undergoing multinucleation.See also Figure S1 and Movies S1, S2, S3, S4, S5, S6, and S7.

Mentions: To confirm previous reports of multinucleate senescent melanocytes in benign human nevi, we stained nevi with DAPI to detect DNA. This clearly revealed melan-A-positive nevus cells with multiple nuclei, while an overlaying epidermis contained only mononucleate melanocytes (Figure 1A). To investigate the origin of multinucleation in OIS, we generated primary human fibroblasts (IMR90) expressing tamoxifen-activatable oncogenic H-RasV12 fused to the estrogen receptor (ER) ligand-binding domain (Reuter and Khavari, 2006) (Figure S1A), hereinafter referred to as ERRAS cells. In this model, H-Ras signaling is readily induced with tamoxifen (hereinafter referred to as activated Ras or induced ERRAS cells), while uninduced cells serve as a control. As reported previously (Barradas et al., 2009; Lin et al., 1998; Reuter and Khavari, 2006; Young et al., 2009), activation of oncogenic H-RasV12 induced downstream MEK signaling (Figure S1A) and, after a transient proliferation burst, led to a gradual decrease in DNA synthesis (Figure S1B). Within 2 weeks, cell growth ceased, and cells displayed characteristic markers of senescence, such as senescence-activated β-galactosidase (SA-β-gal) and senescence-associated heterochromatic foci (SAHF) (Figures S1C and S1D) as previously described (Dimri et al., 1995; Narita et al., 2003). This was accompanied by statistically significant 2.5- and 6.2-fold increases in proportion of MNCs with two and more than two nuclei, respectively (Figures 1B, 1C, and S1E). Thus, this in vitro system recapitulates the multinucleation phenotype observed in OIS in vivo.


Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Multinucleated OIS Cells Originate from Aberrant Mitosis(A) MNCs in human nevi. Dermal nevus-containing section of human skin stained with DAPI (panels 1 and 2) or for melan A (Mel A; panels 3 and 4). Panel 2 is a magnified insert with multinucleated nevus cells (arrows). Panel 4 shows a magnification of melan-A-positive multinucleated nevus cells (top) and a section of overlaying epidermis with mononucleated melanocytes (bottom). Scale bars, 500 μm for panels 1 and 3, 50 μm for panel 2, and 100 μm for panel 4. 17% of nevus cells (out of 334) and 0% epidermis melanocytes (out of 365) are multinucleated in this specimen.(B) Multinucleated senescent 12-day-induced ERRAS cell, stained for microtubules (left, red on overlay) and DAPI (middle, blue on overlay). Scale bars, 30 μm.(C) 15-day ERRAS induction (Ras) increases the percentage of MNC with two nuclei, more than two nuclei, and lobular nuclei. Data indicate means ± SEM from three independently derived ERRAS cell populations. ∗p = 0.021; ∗∗p = 0.005 (paired Student’s t test). Cntr, control.(D) Origin of multinucleation in GFP-Lamin A-expressing ERRAS cells observed throughout 12 days of Ras induction and classified as originating from mitotic failure (light gray), cell fusion (medium gray), and interphase fragmentation (fragm, dark gray). n = 23 multinucleation events (see also Figure S1I).(E and F) In (E), cytokinesis failure leading to binucleation is shown. (F) Prolonged mitotic arrest followed by slippage, generating a highly multinucleated cell. For (E) and (F), bright-field image (top) and corresponding GFP fluorescence (bottom) are shown at selected times (in hours:minutes, or hh:mm). Arrows indicate cells undergoing multinucleation.See also Figure S1 and Movies S1, S2, S3, S4, S5, S6, and S7.
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fig1: Multinucleated OIS Cells Originate from Aberrant Mitosis(A) MNCs in human nevi. Dermal nevus-containing section of human skin stained with DAPI (panels 1 and 2) or for melan A (Mel A; panels 3 and 4). Panel 2 is a magnified insert with multinucleated nevus cells (arrows). Panel 4 shows a magnification of melan-A-positive multinucleated nevus cells (top) and a section of overlaying epidermis with mononucleated melanocytes (bottom). Scale bars, 500 μm for panels 1 and 3, 50 μm for panel 2, and 100 μm for panel 4. 17% of nevus cells (out of 334) and 0% epidermis melanocytes (out of 365) are multinucleated in this specimen.(B) Multinucleated senescent 12-day-induced ERRAS cell, stained for microtubules (left, red on overlay) and DAPI (middle, blue on overlay). Scale bars, 30 μm.(C) 15-day ERRAS induction (Ras) increases the percentage of MNC with two nuclei, more than two nuclei, and lobular nuclei. Data indicate means ± SEM from three independently derived ERRAS cell populations. ∗p = 0.021; ∗∗p = 0.005 (paired Student’s t test). Cntr, control.(D) Origin of multinucleation in GFP-Lamin A-expressing ERRAS cells observed throughout 12 days of Ras induction and classified as originating from mitotic failure (light gray), cell fusion (medium gray), and interphase fragmentation (fragm, dark gray). n = 23 multinucleation events (see also Figure S1I).(E and F) In (E), cytokinesis failure leading to binucleation is shown. (F) Prolonged mitotic arrest followed by slippage, generating a highly multinucleated cell. For (E) and (F), bright-field image (top) and corresponding GFP fluorescence (bottom) are shown at selected times (in hours:minutes, or hh:mm). Arrows indicate cells undergoing multinucleation.See also Figure S1 and Movies S1, S2, S3, S4, S5, S6, and S7.
Mentions: To confirm previous reports of multinucleate senescent melanocytes in benign human nevi, we stained nevi with DAPI to detect DNA. This clearly revealed melan-A-positive nevus cells with multiple nuclei, while an overlaying epidermis contained only mononucleate melanocytes (Figure 1A). To investigate the origin of multinucleation in OIS, we generated primary human fibroblasts (IMR90) expressing tamoxifen-activatable oncogenic H-RasV12 fused to the estrogen receptor (ER) ligand-binding domain (Reuter and Khavari, 2006) (Figure S1A), hereinafter referred to as ERRAS cells. In this model, H-Ras signaling is readily induced with tamoxifen (hereinafter referred to as activated Ras or induced ERRAS cells), while uninduced cells serve as a control. As reported previously (Barradas et al., 2009; Lin et al., 1998; Reuter and Khavari, 2006; Young et al., 2009), activation of oncogenic H-RasV12 induced downstream MEK signaling (Figure S1A) and, after a transient proliferation burst, led to a gradual decrease in DNA synthesis (Figure S1B). Within 2 weeks, cell growth ceased, and cells displayed characteristic markers of senescence, such as senescence-activated β-galactosidase (SA-β-gal) and senescence-associated heterochromatic foci (SAHF) (Figures S1C and S1D) as previously described (Dimri et al., 1995; Narita et al., 2003). This was accompanied by statistically significant 2.5- and 6.2-fold increases in proportion of MNCs with two and more than two nuclei, respectively (Figures 1B, 1C, and S1E). Thus, this in vitro system recapitulates the multinucleation phenotype observed in OIS in vivo.

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus