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, 108 (12), 4997-5002

Protein Farnesylation Inhibitors Cause Donut-Shaped Cell Nuclei Attributable to a Centrosome Separation Defect

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Protein Farnesylation Inhibitors Cause Donut-Shaped Cell Nuclei Attributable to a Centrosome Separation Defect

Valerie L R M Verstraeten et al. Proc Natl Acad Sci U S A.

Abstract

Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson-Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced "donut formation" and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
FTI treatment causes donut-shaped nuclei. (A) 3D surface rendering from a confocal image stack of a donut-shaped nucleus (blue) in an FTI-treated skin fibroblast cell revealing mitochondria (green) passing through the donut hole (Movie S1). (Scale bar: 25 μm.) (B) Single confocal sections through the nucleus of FTI-treated primary human skin fibroblasts stained for lamin A (B1), β-tubulin (B2), and DNA (B3), revealing a nuclear lamina lining the donut hole. (Scale bar: 25 μm.) (C) Induction of donut-shaped nuclei in a variety of cell types after treatment with 10 μM FTI for 3 d. (D) Inhibition of protein farnesylation by different doses of FTI L744832 and lovastatin for 3 d led to more donut-shaped nuclei. At low doses (≤1 μM), lovastatin did not elicit donut-shaped nuclei. When primary human skin fibroblasts were treated with 1 μM tipifarnib (R115777, indicated as R) or 1 μM lonafarnib (SCH66336, indicated as S), or with the combination of 1 μM pravastatin (Pra), 1 μM zoledronate (Zoledr), and either 1 μM tipifarnib (R) or 1 μM lonafarnib (S), a similar amount of nonfarnesylated HDJ-2 (nf-HDJ2) was observed (Lower). A larger number of donut-shaped nuclei were observed in tipifarnib-treated cells than in lonafarnib-treated cells. Combining pravastatin and zoledronate with tipifarnib or lonafarnib led to an increased number of donut-shaped nuclei. More detail is provided in Fig. S3F. *P < 0.05; ***P < 0.001.
Fig. 2.
Fig. 2.
FTI treatment causes donut-shaped nuclei in vivo. Donut-shaped nuclei are demonstrated in tissue specimens derived from gut (A, H&E) also containing blood vessels (B, stained for lamin A/C) of WT mice (A) and LMNA G608G mice expressing human progerin (B) treated with 2.25 mg of FTI per day for a minimum of 6 mo. (Inset) Close-up of the donut-shaped nucleus in the dashed rectangle. Arrow indicates donut-hole; arrowhead indicates nuclear lamina surrounding donut-hole. (Scale bar: 10 μm.) (C) Large intestine of FTI-treated WT and LMNA G608G mice had significantly increased numbers of donut-shaped nuclei compared with nontreated littermate mice. **P < 0.005; ***P < 0.001.
Fig. 3.
Fig. 3.
Donut-shaped nuclei form during mitosis attributable to a centrosome separation defect. Time-lapse video microscopy of FTI-treated human skin fibroblasts stably expressing GFP–lamin A (A; Movie S2) and HEp2 cells stably expressing GFP–histone-3 (B) showing donut-shaped nuclei arising during mitosis. (Scale bar: 10 μm.) Arrows indicate donut holes. The asterisk indicates a micronucleus attributable to incomplete chromatin segregation. (C) Serum starvation prohibited mitosis and abolished FTI-induced donut formation. (D) 3D surface rendering of a confocal image stack through an FTI-treated skin fibroblast labeled for β-tubulin (green), DNA (blue), and pericentrin (red), revealing the presence of a thick bundle of microtubules passing through the donut holes and multiple small spots of pericentrin close to and within the donut hole (Movie S3). (E) Measurements showing defective centrosome separation at metaphase and anaphase in FTI-treated HEp2 cells. (F) About 60% of FTI-treated cells had centrosomes facing the equatorial plane (inside). (G) HEp2 cells stably expressing GFP–histone-3 and labeled for pericentrin (red) in late anaphase showing centrosomes on the far side (outside) of evolving daughter nuclei in vehicle-treated cells and on the inside in FTI-treated cells. Note the centrosome in evolving donut holes in FTI-treated HEp2 (G3) and skin fibroblasts labeled for β-tubulin (green) and pericentrin (red) (G4). (H) Presence of rosette-like chromatin distribution (i.e., metaphase ring) instead of normal chromatin alignment at the equatorial plane in FTI-treated HEp2 cells. (I) Treatment of skin fibroblasts with 100 μM monastrol for 20 h increased donut-shaped nuclei. (J) 3D surface rendering of an FTI-treated skin fibroblast cell line at metaphase, revealing rosette-like chromatin distribution (blue) and a monopolar spindle with microtubules (β-tubulin, green) arising from one centrosome (pericentrin, red, arrow). Skin fibroblasts treated with 10 μM FTI for 3 d (K) or 100 μM monastrol for 20 h (L) labeled for pericentrin (red), β-tubulin (green), and DNA (blue) showing the mitotic spindle arising from the center of the rosette-like chromatin ring. (Scale bar: 10 μm.) ***P < 0.001.
Fig. 4.
Fig. 4.
FTI-induced loss of pericentrin causes formation of donut-shaped nuclei and binucleation. (A) Reduced expression of the large (378 kDa) and small (250 kDa) pericentrin (PCNT) isoforms in FTI-treated primary human skin fibroblasts. MW, molecular weight. Quantification of 378-kDa (B) and 250-kDa (C) PCNT expression levels normalized to β-tubulin and compared with primary skin fibroblasts treated with vehicle alone. (D) RNA interference directed against PCNT resulted in reduced levels of the 378-kDa PCNT product in HeLa cells. Knockdown of PCNT significantly increased binucleation (E) and donut formation (F). (G) Blocking proteasome-mediated degradation reduced donut formation in FTI-treated primary skin fibroblasts. PI, protease inhibitor. (H) Donut formation in MEFs lacking lamin B1 (Lmnb1Δ/Δ) or lamin B2 (Lmnb2−/−) and WT controls. (I) Formation of donut-shaped nuclei in HeLa cells after RNAi directed against lamin B1, indicating that lamin B1 is required for FTI-induced donut formation. (J) Percentage of cells with donut-shaped nuclei after expression of a nonfarnesylated version of lamin B1 (LaB1-SAIM) or WT lamin B1. Expression of LaB1-SAIM and, to a lesser extent, lamin B1 increased donut-shaped nuclei even without FTI treatment. (K) Localization of GFP-labeled WT lamin B1 (Upper) or LaB1-SAIM (Lower) in late mitosis showing that nonfarnesylated lamin B1 is mislocalized to the nucleoplasm (more detail is provided in Fig. S6). (Scale bar: 10 μm.) *P < 0.05; **P < 0.005; ***P < 0.001.
Fig. 5.
Fig. 5.
FTI treatment results in donut-shaped nuclei and binucleated cells that divide abnormally. FTI-treated primary human skin fibroblasts showing donut-shaped nuclei in binucleated cells in phase-contrast images (A) and in single confocal sections after staining for lamin A (B1), vimentin (B2), and DNA (B3, Hoechst). (B4) Merged image. (Scale bar: 10 μm.) (C) Distribution of donut-shaped nuclei between mononucleated and binucleated cells. (D) FTI treatment caused increased numbers of binucleated cells in two primary human skin fibroblasts (Fibro) lines and in HEp2 cells. (E) Large fraction of binucleated cells contained donut-shaped nuclei. (F) Photoconversion of Dendra2–histone-4 from green to red to track individual nuclei over time. (G) Follow-up study of cell fate for 4 d in FTI-treated skin fibroblasts after photoconversion of mononucleated and binucleated cells with and without donut-shaped nuclei showing a lower probability of cell division in binucleated cells compared with mononucleated cells, regardless of the presence of donut-shaped nuclei. (H and I) Time-lapse video microscopy studies of HEp2 cells showing that ~50% of cells with donut-shaped nuclei divide abnormally, often yielding new donut-shaped nuclei (I, Upper; 55%) or an abnormal number of daughter nuclei (I, Lower; 21%). Arrows indicate daughter nuclei. (Scale bar: 10 μm.) (J) Single confocal sections showing defective karyokinesis in an FTI-treated skin fibroblast after labeling for lamin A (J1), vimentin (J2), and DNA (J3, Hoechst). (Scale bar: 25 μm.) (K) Inactive X-chromosomes in female FTI-treated human skin fibroblasts labeled for the histone macroH2A that preferentially binds to the inactive X-chromosome (arrows). (Scale bar: 10 μm.) (L) Quantification of primary female human skin fibroblasts containing more than one inactive X-chromosome in response to FTI treatment, revealing higher levels of aneuploidy in cells with donut-shaped nuclei. Costaining of macroH2A with Ki67 was used to identify noncycling cells. *P < 0.05; ***P < 0.001.

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